Progress in Brain Research Volume 29 Brain Barrier Systems

P R O G R E S S IN B R A I N R E S E A R C H V O L U M E 29

BRAIN BARRIER SYSTEMS

PROGRESS I N BRAIN RESEARCH

ADVlSORY BOARD W. Bargmann

H. T. Chang E. De Robertis J. C. Eccles J. D. French H. Hyden

J. Ariens Kappers S. A. Sarkisov

J. P. Schade F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra

Los Angeles Goteborg Amsterdam Moscow

Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 29

BRAIN B A R R I E R SYSTEMS

EDITED

ABEL LAJTHA New York State Research Institute for Neuroc1ienii.str.v and Drur Addiction, and Department of Bioclieniistry, College of’ Physicians arid Surgeons, Colrtiiihiu University, New York

AND

DONALD H. FORD Department of’ Anatoniy, State University of’ New York, Downstate Medical Center, Brooklyn, New York

ELSEVIER P U B L I S H I N G C O M P A N Y A M S T E R D A M / LONDON / N E W Y O R K

1968

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0 1968 BY ELSEVIER PUBLISHING COMPANY, AMSTERDAM

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List of Contributors

H. M. ADAM,Department of Pharmacology, University of Edinburgh Medical School, Teviot Place, Edinburgh (Scotland). CHARLES F. BARLOW,Department of Neurology, Harvard Medical School, Boston, Mass. (U.S.A.). CLAUDE F. BAXTER, Neurochemistry Laboratories, Veterans Administration Hospital, Sepulveda, California (U.S.A.). LOUISBAKAY,Division of Neurosurgery, State University of New York at Buffalo, School of Medicine, 462 Grider Street, Buffalo, New York (U.S.A.). R. BLASDFRG,New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York (U.S.A.). MILTONBRICHTMAN,Laboratory of Neuroanatomical Sciences, National Institute of Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). S. CLAYMAN, McGill University Cancer Research Unit, McIntyre Medical Sciences Building, 3655 Drummond Street, Montreal (Canada). MIROBRZIN,Institute of Pathophysiology University of Ljubljana, Ljubljana (Yugoslavia). R. V. COXON, University Laboratory of Physiology, Oxford (England). CHRISTIAN CRONE,Department of Physiology, University of Copenhagen, Institute of Medicine, Juliane Maries Vej 28, Copenhagen (Denmark). T. Z. CSLKY,Department of Pharmacology, University of Kentucky, College of Medicine, Lexington, Kentucky (U.S.A.). R. W. P. CUTLER, Neurology Service of the Children’s Hospital Medical Center, Peter Bent Brigham Hospital. 300 Longwood Avenue, Boston, Mass. (U.S.A.). D. H. DEUL,N.V. Philips-Duphar, Weesp (The Netherlands). J. DOBBING, Department of Physiology, London Hospital Medical College, London (England). PHILIPDUFFY,Department of Pathology, Division of Neuropathology, College of Physicians and Surgeons, Colombia University, New York, N.Y. (U.S.A.). K . A. C. ELLIOTT, Department of Biochemistry and the Montreal Neurological Institute, McGill University, Montreal (Canada). J. FOIXH-PI,Department of Biochemistry, Harvard Medical School and McLean Hospital, Boston, Massachusetts (U.S.A.). D. H. FORD,Department of Anatomy, State University of New York, Downstate Medical Center,:450 Clarkson Avenue, Brooklyn, New York (t!.S.A.). C. M. FRENCH, Department of Physiology, The London Hospital Medical College, Turner Street, Londonl( England).

VI

LIST O F C O N T R I B U T O R S

STANLEY GINSBURG, Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, N.Y. (U.S.A.). LEONARD GRAZIANI, Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, N.Y. (U.S.A.). JANNY A. HAISMA, Biochemistry Department, Institute of Psychiatry, State University, Groningen (The Netherlands). P. L. IPATA, lstituto di Chimica Biologica dell’Universit8 di Pisa (Italy). J. JONGKIND, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). J. ARlENs KAPPERS, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). ROBERTKATZMAN,Saul R. Korey, Department of Neurology, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, N.Y. (U.S.A.). IGOR KLATZO,Section on Clinical Neuropathology, National Institute of Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). HAROLD KoENiC;, Department of Neurology and Psychiatry, Northwestern University Medical School and Neurology Service, VA Research Hospital, 333 East Huron Street, Chicago, 111. (U.S.A.). ABELLAJTHA, New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York (U.S.A.). GiuLio LEw, Istituto Superiore di Smith, Viale Regina Elena 299, Rome (Italy). A. LOWENTHAL, Fondation Born-Bung pour la Recherche, Department of Neurochemistry, Berchem-Antwerpen (Belgium). P. MANDEL, Centre de Neurochimie, Centre National de la Recherche Scientifique, Strasbourg (France). H. MCILWAIN, Department of Biochemistry, Institute of Psychiatry, The Maudsley Hospital, Denmark Hill, London, S.E. 5 (England). FREDERICK MINARD,Abbott Laboratories, Scientific Division, North Chicago, Ill. (U.S.A.). K. D. NEAME,Department of Physiology, University of Liverpool, Liverpool 3 (England). G . D. PAPPAS,Department of Anatomy, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, N.Y. (U.S.A.). H. PAPPIUS, Department of Neurology and Neurosurgery, McGill University and Montreal Neurological Hospital, 3801 University Street, Montreal (Canada). E. PASCOE,Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). G . QUADBECK, Tnstitut fur Pathochemie und allgemeine Neurochemie, Pathologisches Blnstitut der Universitat Heidelberg, Berliner Strasse 5, Heidelberg (Germany). D. P. RALL,Department of Experimental Therapeutics, National Cancer Institute, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.).

LIST OF CONTRIBUTORS

VII

B. M. RIGOR,Department of Pharmacology, University of Kentucky College of Medicine, Lexington, Kentucky (U.S.A.). S. ROBERTS, Department of Biological Chemistry and the Brain Research Institute, UCLA Center for the Health Sciences, Los Angeles, California 90024 (U.S.A.). CARLOA. Rossi, lstituto di Chimica Biologica, Dell’Universita di Pisa, Pisa (Italy). J. P. SCHADE, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). P. G . SCHOLEFIELD, McGill University Cancer Research Unit, Mclntyre Medical Sciences Building, 3655 Drummond Street, Montreal (Canada). 0. STEINWALL, Department of Neurology, University of Goteborg, Goteborg (Sweden). VIRGINIATENNYSON, Department of Pathology, Division of Neuropathology, College of Physicians and Surgeons, Colombia University, New York, N.Y. (U.S.A.). WALLACE W. TOURTELOTTE, Department of Neurology, University Hospital, University of Michigan Medical Center, Ann Arbor, Michigan (U.S.A.). D. B. TOWER,Laboratory of Neurochemistry, National Institute for Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). J. F. L. VAN BREEMEN, Biochemistry Department, Institute of Psychiatry, State University, Groningen (The Netherlands). C. J. VAN DEN BERG, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). Department of Physiology, University of Montreal, Montreal, Nico M. VAN GELDER, Quebec (Canada). DIXONM. WOODBURY, Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.). B. D. WYKE,Neurological Laboratory, Department of Applied Physiology, Royal College of Surgeons of England, Lincoln’s Inn Fields, London-WC 2 (England).

Vlll

Other volumes in this series:

Volume I : 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 by W. Bargmann and J. P. Schade Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schade Volume 5 : Lectures on the Diencephalon Edited by W . Bargmann and J. P. Schadk Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. SchadC Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova Volume 8: Biogenic Aiuines Edited by Harold E. Himwich and Williamina A. Himwich Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich Volume 10: The Stritctitre und Function of the Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. Schadt Volume 11 : 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. Schade Volume 13 : Mechanisms of Neural Regeneration Edited by M. Singer and J. P. Schadd Volume 14: Degeneration Pafterns in the Nervous Sysfem Edited by M. Singer and J. P. Schade

IX Volume 15: Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrca Volume I6 : Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schade Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J. P. SchadC Volume 18 : Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. SchadC Volume 19: Experimental Epilepsy by A. Kreindler Volume 20 : Pharmacology and Physiology of the Reticular Formation Edited by A. V. Valdman Volume 21A : Correlative Neurosciences. Part A : Fundamental Mechanisms Edited by T. Tokizane and J. P. Schade Volume 21 B: Correlative Neurosciences. Part B: Clinical Studies Edited by T. Tokizane and J. P. SchadC Volume 22: Brain reflexes Edited bij E. A. Asratyan Volume 23 : Sensory Mechanisms Edited by Y.Zotterman Volunie 24: Carbon Monoxide Poisoning Edited by H . Bour and I. McA. Ledingham Volume 25: The Cerebelluni Edited by C. A. Fox and R. S. Snider Volume 26: Developmental Neurology Edited by C.G.Bernhard Volume 27: Slructure and Function of the Limbic System Edited by W.Ross Adey and T. Tokizane Volume 28: Anticholinergic Drugs Edited by P. B. Bradley and M. Fink Volume 30: Cerebral Circulation Edited by W.Luyendijk

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Preface

I n recent years, many important developments have occurred in many research disciplines that are directly related to brain permeability. In morphological fields, the fine resolution provided by electron microscopy has resulted in new concepts of membrane structure. Electron microscopy, combined with autoradiography, have contributed significant information concerning dynamic events occurring in and around such membranes. Permeability measurements with drugs have led to new theories of the physical and chemical requirements which determine the penetration of drugs into the central nervous system. Measurements of permeability during development and in later more mature stages of life have resulted in theories that relate brain permeability and function and, as in senescence, brain permeability and pathology. Recent work with normal metabolites in the brain has shown complex active transport mechanisms to be present in the walls of the various cell types making up the nervous system. Cerebral transport phenomena have been shown to have a considerable degree of selectivity and specificity and seem highly significant in controlling the mechanisms of brain metabolism. Inasmuch as these membranes and transport systems sometimes appear to restrict entry of materials into the brain, it is apparent that they represent, in part at least, what has been termed the “Blood-Brain Barrier”. Thus, the brain-barrier system may determine which metabolites can gain access to the brain, may determine the level of these metabolites available to various brain parts and brain cells, and may determine the rate of supply and the rate of elimination. It has also been suggested that specific transport processes may be interfered with in pathological states (i.e. amino acidurias, and phenylketonuria), and therefore that alterations of permeability can be involved in altered mental function. It is obvious from this very brief survey that great advances have been made recently on a number of fronts, in such areas as anatomy, physiology, neurochemistry, and pharmacology. Despite these advances, however, it is not uncommon to attend meetings and learn that the failure of almost any compound to enter the brain is due to the Blood-Brain Barrier, or that the only amino acid capable of penetrating the brain is glutamine. Most textbooks of neuroanatomy treat the concept of “Brain-Barrier” as being too complicated for discussion or provide some very structural rigid concept for restricting entry of most compounds. Although a number of conferences have been planned to consider these important advances in cerebral permeability, barriers or transport, none has taken place in recent years. Thus it was the purpose of the conference held in Amsterdam from September 26 to 30, entitled “Brain Barrier Systems” to gather together leading investigators from America and Europe who have been working on the various anatomico-bio-

XI1

PREFACE

chemical-physiologic aspects of the complex membrane systems in the brain, and attempt to define as concisely as possible our state of knowledge today about these “barriers”. It is most fortunate that so many of the outstanding contributors to this field could come and participate at the conference. Many more investigators with important contributions are missing from the volume, because many of the authors had previous commitments and so were, to our regret, unable to participate. Whiletheir absence is certainly felt, it was fortunate that it was possible to have contributions on most of the important aspects of the problem. The conference, therefore, could show the relationship and interdependence of the disciplines concerned with barrier phenomena, and it was highly successful in clarifying the nature of the investigations still required to permit us to fully understand how these “membranes” serve to maintain, or influence, normal brain function. In planning this conference, we were delighted to obtain the cooperation of the Netherlands Central Institute for Brain Research, which has taken care of all the local arrangements. We were indeed fortunate to be able to gather in the stimulating atmosphere provided by this castle (De Hooge Vuursche), which by its nature led us to many fruitful hours of discussion after the close of the formal meetings. Unfortunately, it was not feasible to hide microphones along the paths in the beautiful garden and in the woods surrounding the conference to record all the free discussions that went on till late in the evening hours. We are also indebted to the Office of Naval Research for their interest, both intellectual and financial, in the support of this conference. Additional financial support was provided by the drug houses of E. R. Squibb and Sons, Organon, Abbott Laboratories, and the Warner Lambert Research Laboratories. Without the assistance of the Brain Institute, the Office of Naval Research, and the research directors of the above drug companies, all the best intentions and hope for having a conference dealing with the importance of the various Brain-Barriers in the neurobiologic system would have long ago foundered. Thus, it is proper that we should express our thanks for their interest. Finally, thanks are due to all the participants for their enthusiastic participation, for the excellent contributions, and for the stimulating discussions, all of which made the conference such a success and makes the present book an excellent summary of the problems. DONALD H. FORD AEELLAJTHA

Contents

List of Contributors Preface . . . . . .

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

V XI

STKUCTUKAL CONSIDERAIIONS The composition of nervous membranes J.Folch-Pi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 The intracerebrdi movement of proteins injected into blood and cerebrospinal fluid of mice Milton W. Brightinan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Electron microscopic cytochemistry and rnicrogasometric analysis of cholinesterase in the nervous system Virginia M. Tennyson, Miro Brzin and Philip Duffy . . . . . . . . . . . . . . . . . 41 The fine structuie of the choroid plexus: Adult and developmental stages Virginia M. Tennyson and George D. Pappas . . . . . . . . . . . . . . . . . . . . 63 Transport and effects of cationic dyes and tetrazolium salts in the central nervous system Harold Koenig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 The evidence that ganglioside, a metal, and chemical energy are involved in the serotonin household of synaptic vesicles from brain D. H . Deul, Janny A. Haisma and J. F. L. van Breemen . . . . . . . . . . . . . . . . 125

CEREHKOSPINAL FLUID Cerebrospinal fluid transport R.V.Coxon. . . . . . . . . . . . . . . . . . . . . The choroid plexus as a glucose barrier T. Z. Csaky and B. M. Rigor. . . . . . . . . . . . . . Transport through the ependymal linings David P. Rail . . . . . . . . . . . . . . . . . . . .

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

135

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

147

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

159

SUBSTRATES FOR BRAIN METABOLISM Mechanisms of metabolite transport in various tissues P. G . Scholelield and S. Clayman . . . . . . . . . . . . . . . . . . . . . . . . . . 173 A comparison of the transport system for amino acids in brain, intestine, kidney and tumour K. D. Neame. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Transport as control mechanism of cerebral metabolite levels Abel Lajtha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Regional differences in cerebral amino acid transport Giulio Levi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219

CONTENTS

XIV

Influence of elevated circulating levels of amino acidson cerebral concentrations and utilization of amino acids Sidney Roberts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Specificity of cerebral amino acid transport: A kinetic analysis Ronald G . Blasberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 A possible enzyme barrier for y-aminobutyric acid in the central nervous system Nico M. van Gelder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

ION MOVEMENTS Ion movements in isolated preparations from the mammalian brain 273 Henry Mcllwain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cation exchange in blood, brain and CSF Robert Katzman, Leonard Graziani and Stanley Ginsburg . . . . . . . . . . . . . . .283 Distribution of nonelectrolytes in the brain as affected by alterations in cerebrospinal fluid secret ion Dixon M. Woodbury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

FACTORS INFLUENCING BARRIER FUNCTION Changes in barrier effect in pathological states Louis Bakay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Clinical importance of alterations in barrier G . Quadbeck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Drug influence on the barrier G.Quadbeck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Transport inhibition phenomena in unilateral chemical injury of blood-brain barrier Oskar Steinwall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 Changes in blood-brain permeability during pharmacologically induced convulsions Robert W. P. Cutler, Antonio V. Lorenzo and Charles F. Barlow . . . . . . . . . . . . 367 The effect of hypothermia on electric impedance and penetration of substances from the CSF into the periventricular brain tissue Igor Klatzo, Choh-Luh Li, Don M. Long, Anthony F. Rak, Miroslaw J. Mossakowsky, Levon 0. Parker and Louis E. Rasmussen . . . . . . . . . . . . . . . . . . . . . . 385 Changes in brain accumulation of amino acids and adenine associated with changes in the physiologic state Donald H. Ford . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 The development of the blood-brain barrier John Dobbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Intrinsic amino acid levels and the blood-brain barrier Claude F. Baxter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429

.

SPACESIN

THE

CENTRAL NERVOUS SYSTEM

Introduction to session on brain spaces K. A. C. Elliott. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Spaces in brain tissue in vitro and in vivo Hanna M. Pappius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 Delineation of fluid compartmentation in cerebral tissues Donald B. Tower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Thiocyanate in the brain and the size of the extracellular space C. M. French . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .485

xv

CONTENTS

Some spaces and barriers in postmortem multiple sclerosis Wallace W. Tourtellotte and Julius A . Parker . . . . . . . . . . . . . . . . . . . Inhibition of sheep brain 5-nucleotidase by nucleoside triphosphates P. L . lpata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions A . Lajtha and D . Ford . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

493

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527

.. Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

535 539

547

Structural Considerations

The Composition of Nervous Membranes J. F O L C H - P I McLean Hospital, Belniont, and Department of Biochemistry, Harvard Medical School, Boston, Mass. ( U S A . )

The literature on the chemistry of membranes is much too vast to allow a meaningful review within the space and time available for this presentation. Therefore, instead of attempting such an impossible task, the subject matter of this presentation will be limited to the discussion of four groups of compounds which occur mainly, if not exclusively, in the nervous system and all of which are clearly identifiable as membrane constituents. These compounds are gangliosides, proteolipids, polyphosphoinositides and neurokeratin. Gangliosides are neuronal components; proteolipids, polyphosphoinositides and neurokeratin are myelin components. Since even our information on these compounds - and specially the sum total of our uncertainties about them - is much too vast for adequate presentation in the time available, the following comments will bear mainly on those aspects of their chemistry that are specially pertinent to their function as membrane constituents. GANCLIOSIDES

In 1941, Klenk isolated from brain a new aniino acid to which he gave the name of iieuruminic acid. In 1942, he described a group of new brain glycolipids that were

characterized by the presence of neuraminic acid and which were otherwise constituted by a lipid moiety, presumably a ceramide, and a carbohydrate moiety presumably consisting of one or more monosaccharides. He named them gangliosides because their distribution in the tissue suggested that they were components of ganglion cells. Subsequent work by Klenk and other workers established that neuraminic acid was present in gangliosides usually as an N-acetyl derivative, and that it was identical with siulic acid which had been isolated by Blix from submaxillary mucin a few years previously (Klenk, 1936); that the carbohydrate moiety of gangliosides contained hexosamine(s) in addition to neutral sugars; and, finally, especially after the introduction of thin layer chromatography, that gangliosides comprised a large number of closely related chemical compounds. The chemistry of gangliosides has been the object of recent authoritative reviews, to which the reader is referred for a detailed discussion of the subject (Svennerholm, 1964; Ledeen, 1966). In summary, gangliosides are complex glycolipids consisting, Referenrrs p. 13-14

2

J. F O L C H - P I

according to Kuhn and Wiegant (1963), of a lipid moiety in the form of a ceramide, and of a carbohydrate moiety in the form of a tetrasaccharide, as follows: galactose (1 -3)N-acetylgalactosamine( 1-4)gaIactose( I -4)glucose( 1- 1)ceramide

To this backbone are attached, I , 2 or 3 sialic acid residues constituting respectively mono-, di- or trisialogangliosides. Variations in the carbohydrate moiety have been reported and at present upward to 12 different gangliosides have been recognized. In addition, the ceramide moiety, although consisting mainly of sphingosine and stearic acid, contains also higher and lower homologs of sphingosine, and a host of different fatty acids, thus multiplying several times the number of individual gangliosides that occur in nature. In parallel with this chemical work, it was observed that, although gangliosides were extracted from brain tissue with conventional lipid solvents, they were easily soluble in water as undialyzable solutes. In aqueous solutions they appeared to be monodisperse (Folch et al., 1951), with an apparent molecular weight which was first computed, from ultracentifuge data, as being 250 000 and which by other methods of measurement employing different parameters has been given values ranging from 180 000 to 400 000. This observation led to the assumption that a physically homogeneous high molecular weight compound was being dealt with, and to it was given the name of strandin (Folch et al., 1951). With advances in the chemistry of gangliosides it became apparent that strandin was a polymeric form of gangliosides, and even a critical micellar concentration of 0.015 % was suggested (Howard and Burton, 1964). Since it had been observed that preparations of gangliosides contained small amounts of polypeptides (Folch et a / . , 1951; Folch and Lees, 1959) or proteins, it has been also suggested that these presumed protein contaminants might play a part in determining the remarkable homogeneity of the micellar solutions of strandin (Rosenberg and Chargaff, 1956). That gangliosides are, at least in part, membrane components appears to be a reasonable assumption on the basis of their distribution in the nervous system, of their rate of accumulation during brain development (Folch, 1955), of histochemical evidence (Diezel, 1959, and of their distribution among subcellular fractions of brain tissue (Wolfe, 1961 ; Wherrett and Mcllwain, 1962; Seminario et al., 1964; Burton et al., 1964; Eichberg et al., 1964; Spence et a/., 1964). In addition, a consideration of some of the properties of gangliosides clearly points to them as being exceptionally well designed as membrane constituents: the presence of the carboxyl group of sialic acid which permits binding with organic and inorganic cations, the presence of the lipophilic groups of the ceramide and of the hydrophilic groups of the carbohydrate moiety which permit interaction with many different substances including proteins, lipids, and many small molecule substances, and the ability to form micelles of fairly uniform size. Indeed, it is not surprising that gangliosides have been implicated by many workers in different membrane functions : cation transport (Mcllwain, 1962), acetylcholine release at the presynaptic membrane, synaptic inhibition, receptor function for serotonin (Burton et al., 1964), for tetanus toxin (Van Heyningen, 1963), which parallel the well established function of sialic acid as a viral receptor in red

COMPOSITION O F NERVOUS MEMBRANES

3

blood cells, just to mention a few highlights in a considerable literature dealing with possible functions of gangliosides. We will close this brief survey by discussing the interaction of gangliosides with sodium, potassium, calcium and magnesium, and an apparent effect of the presence or absence of polypeptide on the behavior of the resulting complexes. As expected, all four cations combine with gangliosides, presumably by simple electrostatic bonds. These combinations are reversible, and each cation can displace the others from combination with the ganglioside, the divalent cations being more effective than the monovalent. However, the calcium-ganglioside complex is much less polar than either the free ganglioside or the complexes of ganglioside with the other cations (Quarles and Folch-Pi, 1965). Thus, when these salts of gangliosides are dissolved in the biphasic system chloroform : methanol water 8 : 4 : 3, v/v/v, free ganglioside, and its sodium, potassium or magnesium salts remain in the upper (polar) phase. On the other hand, the calcium complex will remain in the upper phase at low and at high concentrations of calcium ions, but at intermediate concentrations of these ions, it will partition into the lower, least polar phase. This effect of calcium appears to require the presence of small amounts of other lipids, sulfatides being especially effective in this action. In addition, the presence of polypeptide will tend to produce an accumulation of calcium gangliosidate at the interphase. All these interactions illustrate the dramatic changes that may occur in the physical properties of gangliosides, hence on their possible behavior as membrane constituents, and they also point to a possible crucial influence of the presence of small amounts of polypeptides on ganglioside properties. The effect of polypeptides does not appear to be a general protein property. Since the concentrations of calcium that effect the change in polarity of the ganglioside fall in part within the physiological range of concentrations of calcium, it is clear that the observations on the model employed may have implications for the behavior of gangliosides in vivo.

+

Proteolipids, polyphosphoinositides and neurokeratin These three groups of substances are closely related biochemically and anatomically. As will be detailed below, these are myelin constituents and, since myelin itself is formed by the infolding of the plasma membrane of the satellite glial cells around the axons, it is obvious that myelin components are membrane components by definition. In addition, polyphosphoinositides are constituents of both proteolipids and neurokeratin and proteolipids and neurokeratin appear to be very closely related. Since polyphosphoinositides are components of both proteolipid and neurokeratin, it might be pertinent to review highly their history. In 1941 Folch and Woolley (1942) reported the occurrence of inositol as a constituent of brain lipids. Subsequent work resulted in the isolation of an inositol-rich lipid fraction (Folch, 1949) which appeared to have as constituents, inositol diphosphate, glycerol and fatty acids in integral molar ratios and to which the name diphosphoinositide was given (DPI). Later work, using chromatographic techniques, showed, that besides diphosphoinositide, there was a triphosphoinositide (Dittmer and Dawson, 1961 ; Brockerhoff and Ballou, 1961), References p. 13-14

4

J. F O L C H - P I

and that, in fact, the latter might well be the most abundant of the two, DPI possibly being derived by partial dephosphorylation of TPI. Proteolipids - The name proteolipid was introduced in 1951 by Folch and Lees to designate substances consisting of a protein moiety and a lipid moiety and characterized by a complete insolubility in water and solubility in some organic solvents, especially in chloroform : methanol mixtures. The name is intended to emphasize that proteolipids are lipoproteins which behave like lipids. The original observation that led to the discovery of proteolipids was that chloroform : methanol extracts of brain, presumably freed of nonlipid material by water washing, contained protein material (Folch and Lees, 195I). This protein material remained in chloroform through successive water washings, i.e., it was not only soluble in chloroform but insoluble in water. The protein material could be obtained by simply taking to dryness the extract, and extracting the residue with chloroform : methanol. Apparently, in the course of drying the protein underwent some rearrangement that resulted in the loss of its original solubility in chloroform : methanol. As a consequence, the protein remained as an insoluble residue. It contained 14 % N, 1.75 % S and, after acid hydrolysis, 91 % of its nitrogen could be recovered as free amino acids. Its amino acid composition revealed a preponderance of monoamino-mono-carboxylic acids, a high concentration of methionine and cysteine (or cystine) and a relatively small concentration of acidic and of basic amino acids. The material was resistant to the action of trypsin, pepsin, papain and erepsin. Later, it was found to be hydrolyzable by pronase. Distribution of proteolipids. - Although especially abundant in nervous tissue, proteolipids are also found in a wide variety of animal and vegetable tissues. Bovine tissues contain the following amounts of proteolipid protein (mg/g tissue weight) : heart, 3.5; kidney 2.0; liver, 1.6; lung, 0.95; uterus, 0.6; biceps, 0.4. In spinach chloroplasts they represent 2-4 % of dry weight (Zill and Harmon, 1961). These values are only indicative because the yields obtained may have been incomplete. In the nervous system, proteolipids are found at highest concentration in white matter (20-25 mg/g wet tissue) and at about 1/5 this concentration in gray matter. They are present in peripheral nerve at only 1/20 to 1/80 the concentration in white matter (Folch et al., 1958), which may well indicate a qualitative difference between peripheral and central myelin. They are absent from fetal brain and their appearance and progressive accumulation is concurrent with myelination (Folch, 1955). In a study of 28 different anatomical areas of the human nervous system, Amaducci (1962) has observed marked and consistent differences from one anatomical area to another. He has shown that the highest concentration of proteolipids occurs in central white matter, with 1/5 to 1/10 as much in gray matter, and only 1/20 to 1/80 as much in peripheral nerve. Within this general pattern the concentration of proteolipids decreases progressively from cerebral white matter to spinal cord white matter, with cerebellar white matter showing an intermediate value. In spinal cord itself, the concentration of proteolipids appears to decrease in the anterolateral columns

COMPOSITION OF NERVOUS MEMBRANES

5

from rostra1 to caudal levels, while no such gradient is found in the posterior columns. The anterior and posterior spinal roots contain proteolipids at substantially lower concentrations than are found in spinal cord white matter, but this amount is still several times that present in peripheral nerves. The concentration of proteolipids found in gray matter from various areas shows no clear pattern. On the basis of all these observations it had been assumed that, in the central nervous system, proteolipids were in part myelin components. Later work on isolated myelin has established that this is the case and that proteolipids are the main protein found in myelin (Autilio, 1966). In gray matter and in non-neural tissues, especially in heart, proteolipids have been traced to mitochondria at least in part. Ptirifcation of ,i.hite matter proteolipids - Hitherto proteolipids have been obtained from tissue only by extraction with chloroform : methanol. From the extracts thus obtained proteolipids have been prepared in various states of relative purity by the “fluff” method, by emulsion-centrifugation, by dialysis, or by chromatography. The “fluff” method (Folch and Lees, 1951), the one originally used for the preparation of proteolipids, is based on the tendency of proteolipids to concentrate at interfaces. The chloroform : methanol extract is allowed to equilibrate with at least five-fold its volume of water; a biphasic system consisting of a chloroformic phase and an overlying water-methanol phase is eventually obtained. The proteolipids are in part concentrated at the interface as a fluff and, in part, in the chloroformic phase. By further handling, proteolipids A and B are obtained from the fluff, and proteolipid C from the chloroformic phase. These preparations contain from 20 to 70 % protein, and are, otherwise, purely operational concentrates of proteolipids. The emulsion-centrifugation procedure (Folch e t a ] . , 1959) is based on the difference of density between free lipids and proteolipids. A washed chloroform-methanol extract of white matter is taken to dryness in vacuum. The resulting residue is emulsified in 30-fold its weight of water, and the emulsion is centrifuged at 4600 g for 1 h. The supernatant is decanted, the residue suspended in the same amount of water as before and the new suspension centrifuged as before. The whole cycle is repeated twice more. The third and fourth supernatants are water clear or only slightly opalescent. The residue from the fourth centrifugation is again suspended in the same volume of water as before, and the new suspension centrifuged at 200 g for 10 min. The supernatant is decanted. The residue is soluble in chloroform containing small amounts of methanol and water. It is a crude proteolipid preparation that contains approximately 30-40 % protein, 40 % phosphatides, and 12-15 % each of cerebrosides (including sulfatides) and cholesterol. This crude preparation can be purified further by extracting in succession, twice with 70-fold its weight of ethyl ether, and twice with 80-fold its weight of ethanol. The final residue represents a total preparation of white matter proteolipids with the composition given in Table I. Preparatiori ofproteolipids by dialysis - Since proteolipids are high molecular weight compounds, they can be separated from free lipids by dialysis in organic solvents. Murakami et a/. (1962) used dialysis in the purification of brain heart proteolipids Rrfcrmrrs p. 13-14

TABLE I A V E R A G E C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D F R A C T I O N S P R E P A R E D BY D I F F E R E N T M E T H O D S

(Unless otherwise noted, all components are expressed at % of the respective fraction)

Procedure and Fraction

Fluff method Proteol. A Proteol. B Proteol. C Emulsion-Centrifugation Crude Concentrated Dialysis Chromatography I I1 111 Water-soluble proteolipid protein

Yield, fresh tissue

Proreolipid protein

Phosphatides

Cerebrosides

Cholesterol

E 1 em/'% ar 280 mp

(wig)

( 7);

( %)

I %I

( %)

20 10 10

12-20

5-15

3540

30

50 20

55-65

25-30

traces traces traces

a 5 0 25-30 20

35-45 55-65 70

40 25 20-25

5-1 3

7 1 0.2

6 - 8 9 -11 10.5- 13.5

4

95 95 95

2- 5 2 2

-

-

13-14 13-14 13-14

99-100

1

-

1622

4 7

3- I 2- 5

-

COMPOSITION O F NERVOUS MEMBRANES

3.0

I II 'I

2.5

g

t

I

2.0

01

+ U t

7 5 2 5 4 5 J: 7 0 3 0 6

80 20 3

8515l\!

2

I

'

60409

7

/I

I I I

1.5

v,

z

LL]

n

a

1.0

0 k-

CL

0.5

0

5

10

15

20

25

30

TUBE NUMBER (each 5 m l )

Fig. 1 . Chromatography of proteolipids obtained by the emulsion-centrifugation method on a silicic acid column. A 10 mm inner diameter column packed with 4 g silicic acid was used. It was loaded 1 2 8 0 mp = 8.1. The ratios on the upper line express the prowith 69 mg proteolipids Elcm portions of chloroform, methanol and water of the eluting mixture. - Optical density at 280 mp; _ _ _ _ amount of P.

and Thompson et al. (1963) have applied it to the purification of myelin proteins. In the case of brain white matter, the proteolipid and free lipid mixture obtained from a washed lipid extract, or partially purified proteolipid preparations are dissolved in chloroform-methanol 2 : I and the solution placed in a cellophane dialysis tubing previously washed with water and with chloroform-methanol, and dialyzed against the solvent mixture. The system is shaken gently, the diffusate is changed daily and the dialysis allowed to proceed until the diffusate is free of solutes. Usually 7 days suffice. The composition of such preparations is given in Table I.

Chromatography of proteolipids - Matsumoto et al. ( 1 964) have chromatographed the concentrated proteolipid preparations prepared by the emulsion centrifugation procedure, on silicic acid columns. The details of such a chromatographic run is given in Fig. 1. It shows that the first two peaks obtained are free lipids, with little or no protein, and that they are followed by three peaks consisting mainly of protein. It is noteworthy that the last protein peak can only be eluted by chloroform-methanol 1 : 1 containing HCI. This last fraction shows solubility properties different from those of the starting preparation in the sense that in the biphasic system chloroform-methanolwater 8 : 4 : 3 (v/v/v), the original proteolipid is found quantitatively in the chloroformic phase, whereas the proteolipid recovered from the last chromatographic Rrferencrs p. 13-14

8

J. F O L C H - P I

fraction has a definite partition between the two phases, the methanolic-water phase containing about 1/5 as much proteolipid as the chloroformic phase. Properties and composition of proteolipids - All proteolipid preparations described above are soluble in chloroform or in mixtures ofchloroform with methanol and water. They are completely insoluble in water and in aqueous solutions and in the biphasic system chloroform-methanol-water 8 : 4 : 3 (v/v/v), they will concentrate quantitatively in the chloroform phase. All the proteolipid preparations have been found to be resistant to the action of pepsin, trypsin, papain and erepsin. This resistance is not due to the presence of lipids, because it is found in the water-soluble proteolipid protein ( v i ) which is free of lipids, and in the insoluble denatured proteolipid protein described below. The only enzyme that attacks proteolipids is pronase, although the extent of this susceptibility has not been determined exactly. The chemical composition of the various proteolipids is given in Table I. Composition given for proteolipids A, B and C is merely indicative because both yield and composition vary widely according to the exact conditions followed in preparation. The other methods of preparation yield more consistent products. An important fact illustrated by this table is that the amount of lipids in proteolipids may vary from 60 % (in crude emulsion-centrifugation proteolipid) to less than 5 % in the three chromatographic fractions without any change in general solubility properties. Proteolipid protein - It has been isolated as an insoluble material by drying from solutions in biphasic systems (Brockerhoff and Ballou, 1961) or by exposure toalkaline pH’s at certain ionic strengths (Webster and Folch, 1961). At pH 8 or 9, proteolipids can split, with liberation of free lipids and, of protein, as an insoluble material, provided the medium contains ions at sufficient concentration. At pH 8.8 between ionic strengths 0.001 and 1 .O the proportion of proteolipid split is proportional to the logarithm of the ionic concentrations; this fact suggests that the mechanism of splitting is by ionic competition. These insoluble proteolipid proteins still contain small amounts of lipids. The lipid content can be reduced by extraction with hot chloroform : methanol; the lipid most firmly bound to the protein appears to be a polyphosphoinositide mainly triphosphoinositide. It can be removed only with chloroform : methanol acidified with HC1 to 0.04 N concentration (Pritchard and Folch-Pi, 1963). The amino acid composition of the different proteolipids has been estimated repeatedly by different methods and in different laboratories with wholly concordant results. These are that the amino acid patterns of the different white matter proteolipid preparations are identical or so similar as to be indistinguishable from each other. Table I1 gives the amino acid composition of preparations obtained by emulsion centrifugation of the chromatographic parallel fractions obtained from them, and the water soluble proteolipid (v.i.). For more meaningful comparison, serine, the concentration of which varies with the amount of phosphatidyl serine present, has been oomputed uniformly at 6 % of total amino acids on a molar basis; methionine and

T A B L E I1

P

A M I N O A C I D C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D P R E P A R E D B Y DIFFERElvT M E T H O D S

(Results expressed as Amino Acids

Leucine Isoleucine Valine Glycine Threonine Serine Proline Aspartic Acid Glutamic Acid Histidine Arginine Lysine Tyrosine Phenyl Alanine Alanine Methionine Half cystine

Washed total Lipid extract

11.2 7.6 9.5 8.3 11.5 (1KO)* 2.2 2.9 2.4 2.7 2.2 7.4 3.0 8.0 9.0 (6.0)**

Crude Proteolipid

o’,

of total a-amino acid N recovered from acid hydrolysates) Concentrated Proteolipid

I

Chrontatographic Fractions II

III

Water Soluble Proteolipid Protein

11.1 5.9 7.3 9.7 8.4 (7.3)* 2.7 3.7 4.5 2.1 1.7 6.9 4.8 8.3 11.0

11.4 4.9 6.7 10.7 8.3 (7.4)* 3.1 4.0 5.8 1.9 2.0 3.8 4.9 8.3 12.0

11.8 5.3 6.7 9.6 9.0 (5.5)* 3.0 4.3 4.3 1.7 2.7 3.9 4.4 8.6 13.3

11.1 4.9 6.5 10.5 9.0 (5.2)* 2.3 3.9 3.8 1.8 3.1 4.3 4.9 8.2 13.5

11.4 4.8 6.4 10.8 8.8 (5.0)* 2.4 3.9 4.2 2.0 3.2 4.4 4.9 8.2 13.2

11.1 4.9 6.9 10.3 8.5 (8.5)* 2.8 4.2 6.0 1.8 2.6 4.3 4.6 7.9 12.5

(6.0)**

(6.0)**

(6.0)**

(6.0)**

(6.0)* *

(6.0)**

* Serine computed uniformly at 6 % of total Amino Acids. ** Methionine and half cystine computed at 6 % of total Amino Acids.

c)

0

=! 0

2: 0

-I

Z

m

w < 0

10

J. F O L C H - P I

half-cystine have also been computed jointly at 6 %, a concentration consistently found when these amino acids are estimated independently. Tryptophan, which is destroyed by acid hydrolysis, is not included. From optical density values at 280 mp, it can be computed to amount to 3 % or higher. It can be seen that the amino acid composition is indistinguishable from preparation to preparation and very similar to the composition of total chloroform-methanol soluble protein. The same holds true for proteolipid A, B and C, and proteolipids separated by dialysis. However, in the latter case, 10 to 15 % of the protein is found in the diffusate. This dialyzable fraction is protein and not a simple mixture of amino acids. The amino acid composition of the dialyzable material is different from that of the undialyzable proteolipid in that the former contains relatively more glycine, alanine and perhaps threonine. This may represent a real difference or a differential loss of amino acids in the course of hydrolysis because of the presence of a large concentration of lipid in the diffusate. The amino acid pattern of proteolipids has the following features: (a) A relative scarcity of acidic and of basic amino acids; aspartic and glutamic amount jointly to less than 10 % and arginine, lysine and histidine amount jointly also to less than 10 %; (b) A wealth of methionine and half-cystine, as is to be expected from the high concentration of sulfur in proteolipid protein (1.75 %); (c) A relative abundance of the so-called non-polar amino acids, i.e., amino acids that when combined in a peptide chain, offer only non-polar groups to the medium; leucine, isoleucine, valine, glycine, proline, phenylalanine and alanine amount to 57-58 % of total amino acids. If tryptophan is added, over 60 % of amino acids are non-polar ; (d) The relatively high concentration of tryptophan as indicated by the high optical density at 280 mp. On the basis of the least abundant residue, proteolipid protein is computed to comprise 125 amino acid residues of an average size of 100, which gives a minimal molecular weight of 12,500 for the protein moiety of proteolipids (Folch-Pi, 1959). Water-solubleproteolipidprotein - If proteolipids are dialyzed in chloroform : methanol containing HCI to 0.04 N concentration, and then the composition of the outer phase is slowly changed to pure water by gradually decreasing the organic solvent content of the successive outer phases, the retentate is found to consist of protein essentially free of lipids (Tenenbaum and Folch, in press). This preparation has an amino acid pattern indistinguishable from that of the starting proteolipids. It is soluble in acidified aqueous solutions and in chloroform. Apparently, it is the result of a conformational change of the original proteolipid protein (Zand, 1966). The dramatic change in solubility properties is concomitant with the removal of triphosphoinositide. Organization of the proteolipid molecule - Although no complete model of the proteolipid molecule can yet be formulated, it is clear that the lipids in proteolipids exist in different types of binding. Triphosphoinositide is almost certainly bound by an

COMPOSITION O F NERVOUS MEMBRANES

11

electrostatic bond. Other lipids, mainly phosphatidylserine, are bound by ionic linkages which can be dissociated by ionic competition. Finally, other lipids must be bound by more labile types of association. Of these three types of bonds, the first two most likely occur in vivo, while the third type most likely represents in vitro associations. The peculiar solubility properties of proteolipids, which remain unchanged even when the lipid content is reduced to 5 % or less, must be explained in terms of the protein moiety. Since the proteolipid molecule must present a non-polar surface, a tertiary structure must be postulated which would bring to the surface the non-polar groups of the amino acids, while retaining their polar groups in the core of the molecule. A possible structure would involve the stabilization of a particular conformation by triphosphoinositide which, being a polyanion, could combine with the cationic charges of the protein, thus orienting them towards the core of the helical structure and leaving an outer surface occupied mainly by non-polar groups. The release of triphosphoinositide concomitantly with the transformation of proteolipid protein to a water-soluble form would be in favor of this explanation. Neurokeratin - The name of neurokeratin was given by Ewald and Kuhne (1874-77) almost a century ago, to the gastric juice-resistant, pancreatic juice-resistant, fraction of brain proteins. The material was obtained by defatting brain tissue by exhaustive extraction with ethanol and ethyl ether, and submitting the defatted residue to the action of gastric juice and of pancreatic juice, in succession. The final product was an insoluble protein material, rich in S (1.7 %) and free of P. On the basis of its distribution in the nervous system, of the increase in its concentration in temporal relationship to myelination, and of some histochemical evidence, it was concluded that neurokeratin was a myelin constituent, and the name was adopted by histologists to designate the protein framework of the myelin sheath. LeBaron and Folch (1956) were able to prepare neurokeratin by a procedure milder than that used by earlier workers. The trypsin and pepsin resistant material obtained from white matter and designated Trypsin resistant protein residue (TRPR) was resistant to the action of proteolytic enzyme, was characterized by general insolubility, and contained 1.7 % S. In brief, it was very similar to classic neurokeratin except for the important difference that it contained about I .7 % P, almost all of which corresponded to polyphosphoinositide, presumably combined in it by an electrostatic linkage. They also showed that the classical procedure for preparation of neurokeratin resulted in the complete destruction of the constituent polyphosphoinositide, thus yielding a P-free product. The amount of polyphosphoinositide (PPI) present in TRPR accounts for the bulk of the PPI of brain tissue. In the original description of proteolipids (Klenk, 1941), it became obvious that there were many similarities between neurokeratin and proteolipid protein: general insolubility, high sulfur content, relationship to the myelin sheath, similar amino acid composition. The suggestion was made that neurokeratin might, in fact, be a product of breakdown of proteolipids. This suggestion was given further credence by References p.

13-14

J. F O L C H - P I

12

the finding that neurokeratin in its “native” state contained polyphosphoinositide in electrostatic combination just as is the case with proteolipid. This suggestion has been both reinforced and complicated by the recent work on isolated myelin. As already mentioned, it has been found that isolated myelin is completely or almost completely soluble in chloroform : methanol. Operationally this means that neurokeratin and TRPR, which are prepared from the chloroform : methanol insoluble fraction of white matter cannot be prepared from isolated myelin, since it yields no chloroform : methanol insoluble fraction. This forces the conclusion that if, indeed, neurokeratin is a myelin constituent, it exists in it in a form that is soluble in chloroform : methanol after isolation of myelin. This would place neurokeratin in the same category as proteolipids. On the other hand, these observations raise the question of the mechanism by which neurokeratin would become insoluble in chloroform : methanol, and why the same thing would not apply to the proteolipids. There is as yet no answer to these questions. Polyphosphoinositides (PPI) - Many facts pertaining to the discussion of PPI have already been mentioned and the following will only complement them and attempt a brief synthesis of our present knowledge on these interesting compounds. PPI are found mainly in combination in TRPR, which accounts for 80 to 90 per cent of white matter PPT, the balance being found mainly in proteolipids. They are clearly myelin TABLE 111 C O M P A R I S O N O F LEVELS O F P O L Y P H O S P H O I N O S I T I D E S I N D E V E L O P I N G R A T B R A I N A N D O F T R Y P S I N RESISTANT PROTEIN RESIDUE

(TRPR) IN

Rat brain

Age

TPI

DPI

Mouse brain TRPR Total

l(g P/g brain 2 days 4 days 7 days 10 days 16 days 17 days 19 days 34 days 35 days 40 days Adult

8.5 9.2 12.2

-

2.7 3.3

-

11.9 15.5

19.4 44.4

5.7 8.7

25.1 53.1

-

-

11.7

54.5

-

42.8

-

D E V E L O P I N G MOUSE B R A I N

-

TRPR

fresh wt. 0.063 0.115 0.19 0.38 0.40 0.67 0.67 -

TRPR-phosphorus p g P/g brain 5.0 9.4 15.2 30.4

-

32.0 53.6 53.6 -

Results for rat courteously supplied by Doctors J. Eichberg and G. Hauser. Results from mouse obtained or computed from Folch, 1955. Rats were decapitated, the head dropped in liquid nitrogen, and the brain removed without thawing. Mice were anesthesized with ether, the brain removed surgically and placed in a weighing bottle in dry ice. The time elapsed between removal of brain from the living body and its freezing was the time required for the actual freezing of the tissue once placed in contact with the chilled glass wall. The good fitting of values for PPI-P for rat with the values for mice should be regarded as fortuitous. The important analogy is the slope of the increase, which is essentially the same in both species.

COMPOSITION O F N E R V O U S MEMBRANES

13

constituents; they are found in myelin at much larger concentration than in other subcellular fractions of white matter (Eichberg and Dawson, 1965); they are found only in very small amounts, if at all, in non-neural tissues. They appear at the time of myelination and they increase in concentration with the gradual accumulation of myelin. Table 111 gives results on this point, courteously supplied by Doctors J. Eichberg and G . Hauser. They show that from 7 days, before myelination, to 34 days, the concentration of PPI-P increased 5-fold ; for comparison, the concentration of TRPR in the mouse at similar ages is given, both as amount of TRPR and as P (Folch, 1955). There is a remarkable analogy between the total amount of PPI-P and of TRPR-P, a fact that, although not unexpected, bears out strongly the myelinic nature of PPI. Numerous observations attest that PPI exhibits a high rate of P turnover, a fact in sharp contrast with the generally low level of metabolic activity of other myelin components. As yet, no evidence has been forthcoming relating this high metabolic activity of PPI-P to neural function. The marked neural character of PPI, their high metabolic activity, suggests that they must play some crucial role in nerve tissue. What this role is can only be established by further work. ACKNOWLEDGEMENT

The original work described in this discussion was supported by Grants NB-00130 and NB-02840 of the National Institute of Neurology and Blindness, National Institutes of Health. REFERENCES AMADUCCI, L. (1962) The distribution of proteolipids in the human nervous system. J. Neurochem., 9, 153-160. AUTILIO, L. (1966) Fractionation of myelin proteins. Fed. Proc., 25, 764. BLIX,G . (1936) The carbohydrate groups of the submaxillary rnucin. Z. Physiol. Chem., 240.43-54. BROCKERHOFF, H . A N D BALLOU,C. E. (1961) The structure of the phosphoinositide complex of beef brain. 1.Biol. Cherri., 236, 1907-1911. BURTON,R. M., HOWARD,R . E., BAER,S. AND BALFOUR, Y. M. (1964) Gangliosides and acetylcholine of the central nervous system. Biochirn. Biophys. Acta, 84, 441441. DIEZEL, P. M. (1955) Bestimrnung der Neuraminsaure im histologischen Schnittpraparat. Narurwiss., 42, 487-488. DITTMER, J. AND DAWSON, R. M . C. (1961) The isolation of a new lipid triphosphoinositide, and rnonophosphoinositide from ox brain. Biochem. J., 81, 535-540. EICHBERG, J. AND DAWSON, R. M. c. (1965) Polyphosphoinositides in myelin. Biochent. J., 96, 644650. EICHBERG, J., WHITTAKER, V. P. A N D DAWSON, R. M.C . (1964) Distribution of lipids in subcellular particles of guinea-pig brain. Biocheni. J., 92, 91-100. EWALD,A, AND KUHNE,W. (1874-1877) Verharrdl. Naturhist.-Meif.,1, 457. FOLCH,J., (1949); Brain diphosphoinositide, a new phosphatide having inositol rnetadiphosphate as a constituent. J. Biol. Cheni., 177, 505-519. FOLCH,J., ARSOVE, S. AND MEATH, J. A. (1951) Isolation of brain strandin, a new type of large molecule tissue component. J . Biol. Chert?.,191, 819-831. FOLCH,5. AND LEES,M. (1959) Studies on the brain ganglioside strandin in normal brain and in Tay-Sachs’ disease. Arner. J. Dis. Child., 97, 730-738. FOLCH,J. (1955) Composition of the brain in relation to maturation. Biochernisrry of rhe Developing Nervous System, H. Waelsch, Editor, Academic Press, New York, p. 121.

14

J. F O L C H - P I

A N D WOOLLEY, D. W. (1942) Inositol, a constituent of a brain phosphatide. J . Biol. Chem., 142,963-964. FOLCH,J. AND LEES,M. (1951) Proteolipids, a new type of tissue lipoproteins - their isolation from brain. J. Biol. Chem., 191,807-817.

FOLCH,J.

FOLCH,J., LEES,M. AND CARR,S. (1958) Studies of the chemical composition of the nervous system. Exp. Cell Res., Suppl., 5, 58-71. FOLCH,J., WEBSTER, G . R. AND LEES, M. (1959) The preparation of proteolipids. Fed. Proc., 18, 228. FoLcH-Pi, J., (1959); etudes Rkentes sur la chimie du cerveau et leur rapport avec la structure de la gaine myklinique. Exp. Ann. Biochim. Med., 21,81-95. HOWARD, R. E. AND BURTON,R. M. (1964) Studies on the ganglioside micelle. Biochini. Biophys. Acia, 84,435-440. KLENK, E. (1941) NeuraminsBure, das Spaltprodukt eines neuen Gehirnlipoids. Z. Physiol. Chem., 268,50-58. KLENK,E. (1942) Uber die Ganglioside, eine neue Gruppe von Zuckerhaltigcn Gehirnlipoiden. Z. Physiol. Chem., 273, 76-86. KUHN,R.AND WiEcAND-r, H. (1963) Constitution of ganglio-N-tetraose and the ganglioside GI. Chem. Ber., 96,866-880. LEBARON,F. N. AND FoLcii-PI, J. (1956) The isolation from brain tissue of a trypsin-resistant protein fraction containing combined insolitol, and its relation to neurokeratin. J . Neurocliem., 1, 101-108. LEDEEN, R. (1966) The chemistry of gangliosides: A review. J . Amer. O i l Chemists' SOC.,43,57-66. LOWDEN, J. A. AND WOLFE,L. S. (1964) Studies on Brain Gangliosides 111. Evidence for the location of gangliosides specifically in neurones. Canad. J . Biocheni., 42, 1587-1594. MATSUMOTO, M., MATSUMOTO, R. AND FOLCH-PI,J. (1964) The chromatographic fractionation of brain white matter proteolipids. J . Neurochem., 11, 829-838. MCILWAIN, H. (1962) New factors connecting metabolic and electrical events in cerebral tissue. In Ulirasirticture and Metabolism of the Nervous System, Research Publication Association Research Nervous Mental Disease, XL. Williams and Wilkins Co., Baltimore (page 43). MURAKAMI, M., SEKINE, H. AND F U N A H A S H I , s. (1962) Proteolipid from beef heart muSCk-'. Application of organic dialysis to preparation of proteolipid. J. Biochem., 51, 431435. PRITCHARD, E. G . AND FOLCH-PI, J. (1963) Tightly bound proteolipid phospholipid in bovine brain white matter. Biochim. Biopliys. Acia, 70,481483. QUARLES, R. A N D FoLcH-PI, J. (1965) Some effects of physiological cations on the behaviour of gangliosides in a chloroform-methanol-water biphasic system. J . Neurochem., 12,543-553. ROSENBERG, A. AND CHARGAFF, E. (1956) Nitrogenous constituents of an ox brain mucolipid. Biochim. Biophys. Acia. 21, 588-589. SEMiNARio, L. M., HREN, N. A N D GOMEZ,G. J. (1964) Lipid distribution in subcellular fractions of the rat brain. J. Neurochem., 11, 197-209. SPENCE, M. W. AND WOLFE,L. S. (1964) The isolation of a ganglioside-rich membrane fraction from new-born rat brain. Sixth Intern. Congr. Biochem., New York, Abstr., V-,5118, 418. SVENNERHOLM, L. (1964) The gangliosides. J. Lipid Res., 5, 145-155. TENENBAUM, D., AND FOLCH,J., (1966); The prepraration and characterization of water-soluble proteolipid protein from bovine brain white matter. Biochim. Biophys. Acta, 115,141-147. THOMPSON, E.B., KIES, M. W. AND ALVORD,JR. (1963) Isolation of a n encephalitogenic phospholipid-protein complex by dialysis of myelin in organic solvents. Biochem. Biophys. Res. Comm., 13, 198-204. VAN HEYNINGEN, W. E. (1963) The fixation of tetanus toxin, strychnine, serotonin and other substance by gangliosidc. J. Cen. Microbiol., 31,375-387. WEBSTER, G. R. AND FOLCH,J. (1961) Some studies on the properties of proteolipids. Biocliini. Biophys. Acia, 49, 399-401. WHERREIT, J. R. AND M C ~ L W A IH. N , (1962) Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material. Biochem. J., 84,232-337. WOLFE,L. S . (1961) The distribution of gangliosides in subcellular fractions of guinea-pig cerebral cortex. Biocheni. J., 79, 348-355. WOOLLEY, D. W. A N D GOMMI, B. W. (1964) Serotonin Receptors: V, Selective destruction by neuraminidase plus EDTA and reactivation with tissue lipids. Narure, 202, 1074-1075. ZAND,R. (1966) Physical chemical studies on the solution properties of bovine brain white matter proteolipids. Fed. Proc., 25, 736. Z u , L. P. AND HARMON,E. A. (1961) Chloroplast proteolipid. Biochim. Biophys. Acta., 53, 579-58 I.

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DISCUSSION Monday afternoon KATZMAN: I t has been many years since Dr. Folch-Pi characterized and identified the inositol phosphotides and proteolipids and identified the polymer form of inositol phosphate, and it is very exciting to me to see that all these materials are still in the forefront of research. It is most interesting, as Dr. Folch-Pi reported, that the triphosphoinositides, which are so actively turning over, are major constituents of the proteolipids. I would like to ask Dr. Folch-Pi specifically whether it can be demonstrated that in the isolated purified myelin the triphosphoinositides turn over so rapidly. In other membranes where there is a rapid turnover of phosphoinositides, it seems that only a small fraction of phosphoinositide is turning over, and the bulk is not so active. FOLCH-PI:1 don’t think that the triphosphoinositides are especially prominent. The monophosphoinositides, as I am sure you are aware, have a different function from the other inositides and they have a different distribution. Perhaps they are connected with synaptic membranes. In relation to the polyphosphoinositides in myelin, I am not aware that the precise kinetics of a single component’s turnover has been measured. KATZMAN: Has it been shown that the phosphate that is turning over in the purified myelin is specifically the phosphate of the triphosphoinositides? FOLCH-PI: Dawson and others have actually shown the incorporation of P-32 and they have isolated various cell fractions.

MANDEL:There is a turnover of triphosphoinositol in the myelin sheath, but this is quite low compared to the turnover of phosphoinositides in other parts. What is peculiar is that the highest turnover of phosphoinositides is of cardiolipin in the myelin sheith. FOLCH-PI:There is some indication of turnover of cardiolipins, which are the polyanions in mitochondria, but the bulk of the polyphosphoinositides is definitely in the myelin. In isolated proteolipids, LeBaron and Hauser showed years ago that phosphoinositides had a very high turnover of phosphate there. LAJTHA: Can make you any statement on the composition of the proteolipid fractions of the various particulate fractions, and on differences between gray matter and white matter. What I am really driving at is whether you can make any statement about the differences in the composition of the various membranes, whether membranes of glia versus neuronal membranes or particulate membranes. CSAKY:I note that you have isolated the protein from the proteolipids. Could you describe the properties of this fraction, particularly whether you think that this is a typical structural protein? Does it have a high molecular weight? Does it consist of long, thread-like particles? Does it have very high viscosity? Does it respond in its physicochemical properties very readily to ions, and things like that? FOLCH-PI:Some of those measurements have actually been made, although not all of them. However, they are more indicative than exact. Taking the question of molecular size, for example, by the technique of the least abundant component, we get a molecular size of the order of about 12,00015,000. By other physicochemical measurements, for example, sedimentation, diffusion or light scattering, we get somewhat wider scatter with a minimum value of about 20,000 or 30,000. All of these measurements are, of course, done in organic media, since these compounds are insoluble in water. Therefore I would consider every value that we obtained rather comparative only. By Sephadex L-20, molecular size is more between 30,000 and 100,000. Most likely a low polymer formation occurs. We didn’t measure viscosity, again, for the reason that we would have to do that in chloroform and therefore it would have doubtful meaning. But there are people who have done that, for example, Dr. Zahn in Ann Arbor, and Dr. Onkley. We have some information about the tertiary structure, using nuclear magnetic measurements, and in chloroform we get a highly helical structure of about 85 per cent. When the proteins are passed into the water phase, an unfolding of the helixes occurs and they become random coiled.

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J. F O L C H - P I

CSAKY:But would you be willing to say that it could be considered a s a structural protein of the myelin sheath? FOLCH-PI:I think there is very little doubt about that, although I would not make a statement on all membranes. In heart the bulk of the proteolipids are found in mitochondria, and I would think that they must be the components of the structural membranes. The amino acid turnover is very low in these components so that they show a chemical stability, and although not identical, the amino acid composition is fairly similar to Green’s so-called protein isolated from mitochondria. LAJTHA: You don’t think that the lack of turnover is only apparent and is d u e to lack of permeability, that is, the precursor amino acid that you add from the outside doesn’t penetrate to the inside of the myelin sheath, and therefore it is not incorporated. FOLCH-PI:Now we are talking about the mechanism of the stability or how it is actually obtained. It is possible that it is only an apparent one, as you say, but this is very real as far as the living body is concerned. TOWER:Perhaps we are making a mistake when we are talking about fhe structural protein. Wc certainly are dealing with tissues, and the brain in particular, that have many, many membranes with different functions. Our laboratory has some preliminary results which indicate that in membranerich fractions, subfractionated from cerebral microsomal fractions, there is a protein fraction (not necessarily a single protein, although it comes out on a column in a single peak) which exhibits an amino acid composition of about 30 per cent glutamyl plus aspartyl residues. This, theoretically, at least, provides a set of very high negative charges on these molecules (not provided by phosphates in this case but by carboxyl groups), and since this represents a major portion of the proteins of the endoplasmic reticulum, which, in turn, many of us consider may have an important role in transport, it poses some very interesting possibilities. COXON:Dr. Folch-Pi referred to some interactions between ions and the lipids that he is studying. Can he make any statements about selective affinities as between calcium and magnesium and potassium and sodium? FOLCH-PI:There is certainly a difference between the divalent ions and the monovalent ions. The divalent ions have about 500 times higher affinity; something of that order. This is, of course, just a comparative figure. This may be specific for gangliosides, and the carboxyl group of the sialic acid may have an important role. Calcium there certainly displaces sodium, etc. A very interesting point here is that the calcium salt of gangliosides is mainly non-polar, while the magnesium salt is very polar and the polarity changes very much according to the calcium concentration of the medium with which the ganglioside is in contact, and where such shifts happen is around the physiological levels of thc ions. If calcium is somehow sequestered and the ganglioside is facing a relatively low calcium concentration or there would be high magnesium there, there would be a part of the membrane which would be rather lipophobic, but as soon as more calcium came, and I don’t want to speculate how this would occur, that part of the membrane would become much more lipophilic. Therefore, one could postulate this as part of the mechanism of the actual movement of macromolecules in the membrane. This is attractive because it seems to be reversible and doesn’t particularly require energy, and the concentrations required are within the physiological range. The size, from molecular weight measurements, is about 70 Angstroms of these compounds, which well fits within the usual structural arrangement as we picture the membranes. DOBEING: I wonder if you could make any statement about the turnover of these particles that we are discussing, especially since they are often buried beneath several layers, of, for example, proteolipids which, in themselves, do not turn over very rapidly? Is it possible to account for all the turnover as being the turnover of only the exterior part of the sheaths? FOLCH-PI:Now we are in the field of pure speculation. Amaducci did a very careful study several years ago, trying to correlate proteolipids with myelin structures. He not only confirmed what was already known that peripheral nerve contains very little proteolipids, but from a number of structures, such as the optic nerve and the corpus callosum, the brachial plexus and the sciatic nerve, he could

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correlate very well with the sum of the circumferences of the fibers. If you take into account that myelin is usually about half the thickness of the fiber that it surrounds, the myelin is usually related to the surface of the fiber. The smaller the fiber, the greater the sum total of the circumference is, of course, per unit area of the brain. And then he had a very good correlation all the way from the corpus callosurn, which has about 27 parts of proteolipids per loo0 of wet weight basis, down to the brachial plexus which has less then 1 per cent. That could then mean that in the myelin spiral proteolipids would not be distributed uniformly but the distribution somehow would be correlated with the circumference. This could be done by two ways, by having either more or less in the outer turn or the inner turn of the spiral. If you now then equate proteolipids and triphosphoinositides, then you can say that the triphosphoinositides are not equally distributed, and if you postulate that they are richer in the outer turn of the spiral, then you would have the triphosphoinositides much more available than other forms. DOBEING: I f one follows the rate of development of myelin by following the accumulation of the various lipid components, their deposition occurs at different times. On Dr. Folch's hypothesis, it should follow that there would be a different timing for the deposition of the phosphoinositides and o f other components, such as, for example, cholesterol. Could this not be studied in small well-defined areas? FOLCH-PI:I don't think all data were really good enough to put weight on such correlations. In general fashion we have tried such measurements but the data are not really good enough to make too many statements definite. I t did follow the general increase in niyelination and so it showed general correlation. Dawson has done quite a number of such measurements but didn't go into very great detail.

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The Intracerebral Movement of Proteins Injected into Blood and Cerebrospinal Fluid of Mice M I L T O N W. B R I G H T M A N Laboratory of’ Neuroaiiatoiirical Sciences, National Itistilute of Neurological Diseases and Blindness, National Itistitrites of Health, Public Health Service, U. S. Department of Health, Education, and Welfare, Bethesda, Maryland (U.S.A.)

INTRODUCTION

The entry of radioactively labelled protein from blood into cerebrospinal fluid (CSF) and into cerebral tissue is much slower and less in amount (Fishman, 1953; Bering, 1955), than that of tritiated water and certain ions (Sweet and Locksley, 1953; Bering, 1955; Bakay, 1956). The barrier that impedes the entry of labelled protein into the CSF has been interpreted as being responsible also for the exclusion of intravascularly injected acid dyes which, because of their negative electric charge, bind to serum proteins (Tschirgi, 1952). The anatomical site of this barrier to protein movement has been ascribed by light microscopists to the vessel wall (e.g., Broman, 1949) or its surrounding sheath (Tschirgi, 1952). Morphological details of these structures are best resolved by electronmicroscopy, the technique recently used to follow the intracerebral course taken by intravenously administered peroxidase. This protein does not apparently cross the endothelium of cerebral vessels (Reese and Karnovsky, 1967). It has long been known, however, that there are certain regions of the brain where the vessels do not retain acid dyes (Goldmann, 1913), presumably bound to protein, or colloidal particles (Dempsey and Wislocki, 1955; Pappas and Tennyson, 1962; Klatzo et al., 1962). One of these regions is the choroid plexus. The impression gained from these published observations is that, in general, large colloidal particles including some proteins leave the choroidal vessels and accumulate primarily within the perivascular connective tissue stroma (Wislocki and Leduc, 1952; Dempsey and Wislocki, 1955; Brown, 1961 ; Klatzo, et al., 1962; Pappas and Tennyson, 1962). In contrast, certain dyes such as trypan blue (King, 1938; Wislocki and Leduc, 1952), despite its protein-binding tendency, and proflavine hydrochloride (Rodriguez, 1955) are able to go beyond the stroma into the surrounding epithelial cells. The first portion of the present account is an electron microscopic description of the passage of a foreign protein (horseradish peroxidase) from the blood into the stroma and the epithelium of the choroid plexus. When labelled proteins are in.jected into the cerebral ventricles (Bowsher, 1957) Rrfircnir, p. 36-37

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M. W. B R I G H T M A N

or cisterna magna (Lee and Olszewski, 1960) rather than into the blood, there does not appear to be a barrier to their entry into the surrounding brain parenchyma. The second portion of this paper is concerned with this direction of transfer and offers a brief, preliminary description of the movement of peroxidase from ventricular CSF not only toward the vessels of the choroid plexus but also toward the vessels of the cerebral parenchyma. Some observations on the passage of the protein ferritin in this direction are also included. Karnovsky's cytocliemical method ( I 965) of visualizing peroxidase activity is a sensitive one because activity of a few molecules of this enzyme yields a very electron dense reaction product. Six mg of horseradish peroxidase were injected, under Avertin anesthesia, into the femoral vein of mice weighing about 20 g. Fifteen to thirty minutes later the entire periventricular neuropil and choroid plexus were fixed by perfusion through the cerebral ventricles of 3 % glutaraldehyde in phosphate buffer at pH 7.4 (Brightman, 1965a). After immersion for 4 to 16 h in this fixative at about lo" C, frozen sections (about 120 p thick) werecut and incubated at about 25" C for 15 min in a substrate containing 3-3' diaminobenzidineand hydrogen peroxide(Karnovsky, 1965). The sections were then washed in buffer, immersed in phosphate-buffered I 7;osmium tetroxide for 1 to 2 h and prepared for electronmicroscopy. A few blocks, about I p thick, were not frozen, but were otherwise processed in the same way. Henceforth, the terms enzyme (or protein) and reaction product will be used interchangeably. I . M O V E M E N T O F P E R O X I D A S E F R O M C H O R O I D A L B L O O D TO E P I T H E L I U M

A . Endothelial passage

The choroidal vessels, unlike the parenchymal vessels of the brain, have a fenestrated endothelium. The fenestrae are not pores. Although the available techniques led earlier investigators to conclude that the fenestrae were perforations (Maxwell and Pease, 1956) or interruptions (Wislocki and Ladman, 1958), they are now known to be spanned by a thin diaphragm (Pappas and Tennyson, 1962) formed by the apposed outer leaflets of the endothelial unit membrane (Luft, 1966; Wolff, 1966). In such vessels occurring in other organs as well (e.g., Farquhar, 1961), the only continuous part of the endothelial cells are these fused outer leaflets. In tissue fixed 15 min after the intravenous administration of peroxidase, the very dense, amorphous reaction product usually appeared as a coating on the luminal surface of the thicker, non-fenestrated portions as well as the diaphragms of the endothelium (Figs. 2, 3 and 4). Occasionally, and presumably as the result of improper fixation, the dense substance seemed to take the place of the diaphragm and extended from the lumen into the basement membrane of the vessel. In undamaged capillaries, however, the enzyme was restricted to the lumen and appeared to be adsorbed to the entire luminal surface, including its tiny invaginations or pinocytotic pits. In two instances similar pits on the contraluminal surface were coated and it is presumed that these had traversed the endothelium as cytoplasmic vesicles that had originated from the luminal plasmalemma (Fig. 3). Many of these pinocytotic pits and vesicles within the endothelial cytoplasm were completely filled with reaction product.

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Fig. I . Part of a villus from the choroid plexus consists of three epithelial cells with a capillary lumen at the lower right and ventricle at the upper left. The dense, black material is rp (reaction product) of peroxidase that had been injected intravascularly. x 13 000.

Vesicles containing some reaction product were more numerous in the endothelium of choroidal vessels than in that of parenchymal ones. The role of the junctions between these endothelial cells in the passage of protein across the vessels is as yet equivocal. In most cases, peroxidase penetrated the junction for only a short distance (Fig. 2). In several instances, however, reaction product Rcfcwnres p. 36-37

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M. W. B R I G H T M A N

Fig. 2. A fenestrated, choroidal capillary contains pinocytotic vesicles that are eithec rimmed or filled with rp. The fenestrae are indicated by arrows. One junction (JI), between endothelial cells, is cut transversely and contains no rp. Two junctions (Jz and J3), are only partially filled with rp whereas a fourth (54) is more completely filled. The stromal space (S), lined by basement membrane (BM) lies between the vessel and base of an epithelial cell (right margin). x 39 000.

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Fig. 3. In Figs. 3 and 4, the vessel lumen is at the bottom of the figure. A fenestrated, choroidal capillary contains vesicles rimmed by rp at both luminal and contraluminal surfaces. A portion of an epithelial cell is at the top of the figure. x 54 000.

Fig. 4. The junction betwecn these endothelial cells of a choroid plexus capillary is filled with rp. x 73000.

appeared to occupy most or all of the junction (Fig. 4). It is possible that some of the peroxidase within the junction did not enter exclusively from the lumen but was deposited also by vesicular transport. Thus, pinocytotic vesicles arising at the luminal surface may have transported their content to the walls of the junction (Fig. 4) or to the contraluminal surface (Fig. 3). In the latter case, peroxidase may have subsequently diffused from the basement membrane back into the junction.

B. Epithelial entry After crossing the endothelium of the choroid plexus the protein entered the perivascular connective tissue space. This stromal space is bounded by two basement membranes, one apposed to endothelium, the other to epithelium (Fig. 2). The enzyme R r f i r i v w s p. 36-37

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M. W. B R I G H T M A N

Fig. 5. The stromal space, running through the middle of the micrograph, is full of rp that lines the tortuous, communicating spaces between epithelial cell invaginations. Peroxidase has been pinocytosed by numerous vesicles, one of which (arrow, center - left) is continuous with the infolded plasmalemma of an epithelial cell. x 33 000.

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25

penetrated the endothelial basement membrane, some became affixed to collagen fibrils within the space, and appreciable amounts moved across the stromal space to penetrate the epithelial basement membrane. From this site, the enzyme was incorporated within numerous pits and vesicles arising from the highly folded basal and lateral plasmalemmas of the epithelial cells (Figs. I and 5). Many such vesicles migrated to the interior of the cells (Figs. 1 and 6), some moving very close to the free, ventricular surface (Figs. I , 6 and 7). This surface is much more plicated than the lateral or basal surfaces (Figs. 1, 6, 7 and 8), yet the folds are too pleomorphic to constitute a striated or brush border (Maxwell and Pease, 1956). Though the peroxidaseladen vesicles had moved very close to the bases of these free evaginations, none seemed to make contact with their plasmalemma which, moreover, did not appear to have become coated with peroxidase. Occasionally, a flat, broad, inverted, V-shaped cistern intervened between the ventricular surface and the vesicles. Neither these cisterns (Figs. 8, 9 and 10) nor those of the granular endoplasmic reticulum received protein from the vesicles, but vacuoles and multivesicular bodies did. So dense and complete was the filling of the matrix within the multivesicular bodies that the enclosed vesicles appeared to be negatively stained (Figs. 1 and 6). As in the case of fenitin (Brightman, 1965a), these vesicles remained free of peroxidase. C. In tercellular movement

Protein moved between adjacent epithelial cells concurrently with its entry into these cells. The infoldings of the basal and lateral portions oftheircell membranes form many deep, narrow channels (Maxwell and Pease, 1956) that communicate with the large stromal space (Figs. I and 5). Peroxidase left this large space to enter the intercellular channels. These were usually lined with reaction product or sometimes completely filled by it as far as a conical stricture of the interspace (Figs. I and 8). At the stricture, occurring near the ventricular surface, the outer leaflets of adjacent cell membranes presumably approximated each other closely enough to exclude peroxidase. Apical to these appositions the interspace often became distended for a short distance, then it narrowed and was less indented than the space basal to the stricture. Thus a luminal, narrow neck opened into the ventricle but usually contained no protein (Fig. 8). It was evident that peroxidase had moved between adjacent epithelial cells only as far as the strictures and not beyond them into the ventricular CSF. 11. M O V E M E N T O F P E R O X I D A S E A N D F E R R l T l N F R O M V E N T R I C L E I N T O

CHOROIDAL EPITHELIUM A N D CEREBRAL PARENCHYMA

A . Clioroidal entry

Preliminary experiments have demonstrated that 30 min after the injection into the cerebral ventricles of 0.5 mg of peroxidase, a small amount was pinocytosed by the epithelial cells of the choroid plexus. Pinocytotic activity was carried out by that part of the ventricular plasmalemma between the bases of the surface evaginations and Rcfcrmws p . 36-37

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M. W. B R I G H T M A N

Fig. 6. The apical region of an epithelial cell contains many vesicles and at least two multivesicular bodies (B) laden with rp. The ventricle appears at the top. x 34 000.

not by the portion covering them (Fig. 10). The number of vesicles that had engulfed peroxidase was small and they were confined to the apical regions of the cells. Similarly, in one experiment where 35 mg of ferritin had been administered intraventricularly at the same time that 5.0 mg of peroxidase was injected intravenously, the number of ferritin-containing vesicles in the apical cytoplasm was small in contrast to the numerous peroxidase-laden vesicles that had arisen from the folded basal and

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27

Fig. 7. The apical cytoplasm of an epithelial cell contains three peroxidase-laden vesicles, one of which lies very close to this surface. Pleomorphic evaginations of the cell surface o x u p y the ventricle into which ferritin (appesring as micelles about 60 A wide) had been injected. x I17 000.

lateral surfaces (Fig. 7). Neither intraventricular peroxidase nor ferritin passed between adjacent epithelial cells of the choroid plexus in discernible amounts. B. Epetdymal passage

Both proteins, however, passed readily between neighbouring ependymal cells to enter the interstices of the underlying cerebral neuropil. Either protein flowed along the I50 to 200 A-wide clefts between glial and neuronal processes to enter synaptic clefts, as did ferritin in the rat brain (Brightman, 1965b). This continuity ofthe synRc.lcnvico p. 36-37

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M. W. B R I G H T M A N

Fig. 8. The interspace between two epithelial cells is lined with rp as far as the conical stricture near thc ventricular lumen appearing at the top. x 73 000.

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29

Fig. 9. A flattened, agranular cistern occurs commonly near the luminal surface of an epithelial cell. 137 000. Fig. 10. Three pinocytotic invaginations ( * ) are formed by the luminal plasmalemma between the bases of evaginations. A somewhat flattened vesicle contains peroxidase that had been injected into the ventricle (at top). x 128 000. \A

aptic cleft with the rest of the interspaces has recently been confirmed with the use of saccharated iron oxide (Pappas and Purpura, 1966). Peroxidase passed between perivascular glial end-feet ((,f Brightman, 1965b) to spread into the basement membrane surrounding the endothelium of parenchymal capillaries (Fig. 1 1). Either protein was then able t o enter the endothelial pits that communicate with this membrane (Fig. 12). These flask-like invaginations in the contraluminal plasmalemma of endothelial cells thus received either peroxidase or ferritin that had moved extraReferences p. 36-37

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M.

W.

BRIGHTMAN

cellularly from the ventricular CSF. Ferritin also occurred in vesicles within the endothelial cytoplasm but, lacking serial sections, it is unkown whether the vesicle interiors retained continuity with the basement membrane in another plane. None of these vesicles containing protein has yet been observed in contact with the luminal surface of the endothelium; evidence of vesicular transport from outer to luminal surface has, therefore, not yet been obtained. Peroxidase which left the perivascular basement membrane to enter the junctions between endothelial cells could move only a short distancerbefore being stopped. Presumably, the tight junction that blocks the intercellular movement of peroxidase from the blood side of the endothelium (Reese and Karnovsky, 1967) also excludes protein that spread from the parenchymal side. It appears at the present time, that the junctions between endothelial cells of parenchymal capillaries are closed to peroxidase whereas those of choroid plexus vessels are open to it. C. Pinocytosis by presynaptic terminals

Throughout the subependymal neuropil, peroxidase or ferritin spread between glial and neuronal processes and were pinocytosed by these processes. For example, large coated pits in the membranes of dendrites were filled with reaction product. Of special interest, however, was the incorporation of either protein by presynaptic terminals (Figs. 13A and B). Peroxidase or ferritin that had entered the synaptic cleft and the remainder of the periterminal interspace was pinocytosed by the bouton plasmalemma. Small invaginations of the plasmalemma contained peroxidase or one or more molecules of ferritin. Though such pits have not yet been observed i n the membrane fronting the synaptic cleft, it is likely that they form here too (Westrum, 1965). Indeed, in one instance, a peroxidase-laden-vesicle was found very close to a synaptic cleft. Within boutons, protein-containing vesicles had separated from the plasmalemma and had become interspersed among the synaptic vesicles. These pinocytotic vesicles were of the same size as synaptic vesicles and were thus considerably smaller than those occurring, for example, in ependymal and choroidal epithelium. These small pinocytotic vesicles further resembled synaptic vesicles in being non-coated. DISCUSSION

The anatomical barriers to the movement ofperoxidase from blood to ventricular CSF consist of: (a) the structures (which are probably tight junctions) between the apices of the choroidal epithelial cells and, perhaps, (b) their ventricular surface. This surface has been regarded as the barrier to the movement of fluorescent proflavine dyes out of the choroid plexus, though the only part of the epithelial cells enclosing demonstrable dye was the nuclear membrane rather than any portion of the cell membrane itself (Rodriguez, 1955). The conclusion would have been more acceptable if, for example, the basal and lateral plasmalemma had been stained, whereas the apical portion had not. Although no peroxidase-laden vesicles were observed in contact with the ventricular plasmalemma, it is possible that some of the

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Fig. I I . Peroxidase, injected intraventricularly, has entered the perivascular basement membrane ( B M ) from the interspace (arrow) between glial end-feet. x 93 000. Fig. 12. Peroxidase has spread from the perivascular membrane into several pits within the contraluminal plasmalemma of a parenchymal capillary. x 1 I 1 000.

vesicles fused with this membrane and released peroxidase that was immediately washed away by CSF. Such a vesicular transport of a small fraction of intravascularly injected protein could account for the slow entry o f a small amount of labelled albumin into the ventricles observed by others (Fishman, 1953 and Bering, 1955). The ventricular plasmalemma of the choroid epithelial cells is capable, nonetheR&rciu

L’S

p . 36-37

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Fig. 13A. Peroxidase has permeated the interspaces, including synaptic clefts (SC) and has been pinocytosed by vesicles (V) interspersed among synaptic vesicles within presynaptic terminals. x 54000. Fig. 13B. Fertitin has left the cerebral ventricle to enter the neuropil interspaces from which it is pinocytosed by neural processes such as this bouton. The plasmalemmal invaginations (arrows) each contain a molecule of ferritin as does the vesicle (V) that has pinched off from the membrane. x 91 000.

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less, of some pinocytotic uptake of peroxidase. However, a substance that is incorporated into a cell by pinocytosis does not necessarily move across the cell by vesicular transport. Vesicles that had imbibed peroxidase from the ventricle remained in the apical cytoplasm. In contrast, the vesicles that had engulfed protein from the perivascular space had also moved across the cell toward the ventricle. Thus, if vesicular transport does occur, it appears to proceed from the blood-side toward the ventricle and not in the opposite direction. It appears unlikely, therefore, that vesicular transport could account for the nondiffusional movement of nonelectrolytes from a ventricular bath to choroidal blood as suggested by Welch and Sadler (1966). Yet, this possibility cannot be discounted at present because of the imprudence, as emphasized by Bering (l955), of extrapolating between results obtained with different classes of substances. The endothelium of the choroid plexus does not act as a barrier to the movement of intravascularly administered peroxidase but, instead, may allow peroxidase to cross by means of two mechanisms. Perhaps the more important of these may be an intercellular migration along the junctions between endothelial cells. Thorium dioxide, injected into the blood, also enters these junctions in the choroid plexus of the rabbit (Pappas and Tennyson, 1962). The second mechanism is that of vesicular transport, though this route has not here been demonstrated unequivocally. For example, figure 3 may be interpreted in two ways. It may represent the transcellular movement of peroxidase-containing vesicles from the luminal to the contraluminal surface or it may as likely illustrate the uptake by the contraluminal plasmalemma of peroxidase that had permeated the basement membrane after intercellular passage, rather than after vesicular transport. If vesicular transport does occur, it probably takes place across portions of the endothelium that are at least 3001( thick (Brightman, 1965b). More attenuated parts of the endothelium do not contain pinocytotic vesicles (Pappas and Tennyson, 1962). Glial sheets thinner than about 300A do not give rise to focal invaginations and it has been suggested that cell processes in general must attain this minimal thickness before their cell membranes are capable of pinocytotic activity (Brightman, I965b). Throughout the brain, some neurons such as those in the periglomerular regions of the olfactory bulb (Fig. 14) are nearly surrounded by uniformly thin glial wrappings (Reese and Brightman, 1965). Similar thin glial wrappings form a multi-layered, cup-like enclosure around certain synapses in the thalamus of the cat, as we have seen in Dr. Pappas’s presentation (Pappas et a/., Fig. 6, 1966) and in the dorsal motor nucleus of the vagus nerve (Fig. 15) in the rat (Richardson, 1967). This glial-neural configuration implies that the only way in which a large molecule can penetrate the enclosed pericellular and synaptic clefts from the glial channels would be by travelling between the attenuated glial sheets, not across them. Therefore, in the immediate vicinity of such perisomatic and synaptic clefts, the only cell processes that could contribute large molecules, by means of transcytoplasmic vesicular transport, would be neural, not glial. Although the intact fenestral diaphragms are impervious to peroxidase, they may be sites where smaller molecules readily cross the endothelium. Regardless of the R(:lcrcn(xv p . 36-37

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Fig. 14. Two small neurones are partly surrounded by several wrappings of very thin glial sheets (identified by their content of glycogen granules). Periglomerular region of rat olfactory bulb. Os01fixation. (Reese and Brightman, 1965). ,” 15 000. Fig. IS. A stack of thin glial sheets cups that portion of a presynaptic terminal opposite its synaptic cleft (SC). Dorsal motor nucleus of a rat. KMnOA fixation. (Courtesy of K. C. Richardson). i 30 000

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mechanism, the choroidal endothelium allows the passage of peroxidase whereas the parenchymal capillaries do not. Disregarding the risk of extrapolating between results obtained with peroxidase and those with radio-iodinated albumin, one may infer that what little albumin does leave the blood to enter the ventricular CSF (Bering, 1959, does not move across parenchymal, subependymal vessels (Reese and Karnovsky, 1967) but rather across the vessels of the choroid plexus. This inference, however, is at variance with Bering's findings (1955) that such entry into the ventricular CSF occurs after removal of the choroid plexus from an isolated ventricle. The distribution within the cerebral parenchyma of ferritin or peroxidase that had been injected into the cerebral ventricles confirms the observation that the ependyma does not stand as a barrier to many substances including proteins (Lee and Olszewski, 1960; Klatzo et a/., 1962). The results suggest further that the extracellular clefts, about 150-200 A wide, are hydrated channels permitting the movement of hydrophilic substances such as protein. The channels, nevertheless, are variable in content and width (Brightman, 1965b). Those between perivascular end-feet are too narrow to allow passage of ferritin and were, consequently, interpreted as being closed or fused junctions (Brightman, 1965b). The present results, however, support the observation that these particular appositions are narrow but patent (Reese and Karnovsky, 1967) as they are in more primitive species (Kuffler and Nicholls, 1966)and permit the extracellular movement of peroxidase into the perivascular basement membrane. At the ventricular surface, the contents of the intercellular channels are in ultimate communication with the CSF. But our observations tell us nothing about whether the compositions of the fluids are equivalent. The pinocytosis of protein by glial and neuronal processes from their extracellular clefts may signify, conversely, that substances are added to the cleft fluid. The composition of the ambient parenchymal fluid may be thus modulated from cell to cell and could ultimately be quite unlike that of the ventricular fluid. The ability of cerebral nerve terminals to pinocytose ferritin and peroxidase is of particular interest. These proteins are not only incorporated by large (about 80 to 100 p wide), coated vesicles (Brightman, 1965b), characteristic of many different cell types in various species, but are also taken up by presynaptic terminals in vesicles indistinguishable from synaptic vesicles. These pinocytotic vesicles are derived from the bouton plasmalemma; they are smaller than the usual pinocytotic ones, are noncoated, and become interspersed among synaptic vesicles inside the bouton. However, the identification of these pinocytotic vesicles as synaptic has yet to be established. SUMMARY

Following its intravascular injection in mice, the protein, horseradish peroxidase, crosses the fenestrated endothelium of the choroid plexus to enter the stromal space. From there, the protein is pinocytosed by choroid plexus epithelial cells within which peroxidase-containing vesicles migrate as far as the ventricular surface. Concurrently, the enzyme moves between these epithelial cells until stopped by a conical Rrtc~rcwi('s p. 36 37

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M. W. B R I G H T M A N

stricture of the interspace near the ventricular lumen. Unlike parenchymal vessels, the capillaries of the choroid plexus allow the passage of protein that is then taken up by the choroidal epithelium. When peroxidase or ferritin is injected into the cerebral ventricles, relatively little is pinocytosed by the epithelial cells of the choroid plexus and none passes between them. However, either protein readily moves between ependymal cells to penetrate the extracellular clefts of the neuropil. From these channels the proteins are pinocytosed by glial and neuronal processes. In presynaptic endings the plasmalemma forms pits and vesicles incorporating the proteins. These vesicles are morphologically identical with synaptic vesicles among which they become interspersed.

R E F E R E NCES BAKAY, L. (1956) The Blood Brain Barrier. G . C . Thomas, Springfield, Ill. (p. 40-76). BERING,E. A. JR. (1955) Studies on the role of the choroid plexus in tracer exchange between blood and cerebrospinal fluid. J . Nertrosurg., 12, 385-392. BOWSHER, D. (1957) Pathways of absorption of protein from the cerebrospinal fluid: an autoradiographic study in the cat. Anat. Rec., 128, 23-40. BRIGHTMAN, M. W. (1965a) The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. 1. Ependymal distribution. J . Cell Biol., 26, 99-123. -(1965b)The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. II. Parenchymal distribution. Anrer. J. Anat., 117, 193-220. BROMAN, T. ( 1949) The permeability of the cerebrospinal vessels in normal and pathological cortrlitiotrs. Einar Munksgaard, Copenhagen (p. 7-19). BROWN,P. (1961) Albumin, connective tissue and the blood-brain barrier. Bull. Johrrs H o p k i m Hosp., 108, 200-207. DEMPSEY, E. W. AND WISLOCKI, G. B. (1955) An electron microscopic study of the blood-brain barrier in the rat, employing silver nitrate as a vital stain. J . Biophys. Biochcm. Cyrol., 1, 245-256. FARQUHAR, M. G . (1961) Fine structure and function in capillaries of the anterior pituitary gland. Altgiology, 12. 270- 292. FISHMAN, R. A. (1953) Exchange of albumin between plasma and cerebrospinal fluid. Amer. J. Physiol., 175, 96-98. GOLDMANN, E. E. (1913) Experimentelle Untersuchungen iiber die Function der Plex. chorioid. und der Hirnhaute. Verh. cleutsch. Ges. Chirurgie, 42, 107-1 13. KARNOVSKY, M. (1965) Vesicular transport of exogenous peroxidase across capillary endothelium into the T-system of muscle. J . Cell. Biol., 27, 49A. KING,L. (1938) The hematoencephalic barrier. Arch. Neurol. Psychiat., 41, 51-72. KLATZO, I., MIQUEL, J. AND OTENASEK, R. (1962) The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta Neuropathol., 2, 144-160. KUFFLER, S . W. AND NIcHoLLS, J. G . (1966) The physiology of neuroglial cells. Ergeb. Physiol. Biol. Chem. E x p ~ lPharnrakol., . 57, 1-90. LEE,J. c. AND OLszEwsKI, J. (1960) Penetration of radioactive bovine albumin from cerebrospinal fluid into brain tissue. J . Neurol.. 10, 814-822. LUFT.J. (1966) The ultrastructural basis of capillary permeability. In: The /irflamt~ra/oryProcess, B. W . Zweifach, (Ed.:, 1, Ch. 3, 121-159. MAXWELL, D. S. AND PEASE,D. C. (1956) The electron microscopy of the choroid plexus. J . Biophys. Biochem. Cytol., 2, 467-474. PAPPAS,G. D. AND TENNYSON, V. M. (1962) An electron microscopic study of the passage of colloidal particles from the blood vessels of the ciliary process and choroid plexus of the rabbit. J . Cell Biol., 15, 227-240. PAPPAS,G . D. AND PURPURA, D. P. (1966) Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex. Nature, 210, 1391-2.

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PAPPAS, G. B.. COHEN,E. B. A N D PURPURA, D. P. (1966) Fine structure of synaptic and non-synaptic neuronal relations in the thalamus of thecat. The Thalamus. D. P. Purpura and M. D . Yahr (Eds.). Columbia Univ. Press, New York (p. 47-75). R ~ E S FT.. S. A N D B R I G H T M A N , M. w. (1965) Electron microscopic studies on the rat olfactory bulb. Anat. Rec., 151, 492. REESE,T. S. AND KARNOVSKY, M. J. (1967) Fine structural localization of a blood-brain barrier for exogenous peroxidasc. J. CeN Biol. (In Press). RICHARDSON, K. C. (1967) Personal communication. RODRIGUEZ, L. A. (1955) Experiments on the histologic locus of the hemato-encephalic barrier. J . Conrp. Neitrol., 102, 27-39. SWEET, W. H.A N D LOCKSLEY, H. B. (1953) Formation, flow, and reabsorption of cerebrospinal fluid in man. Prac. SOC.Exptl. Biol. Men., 84, 397405. TSCHIRGI, R. D. (1952)Blood-brain barrier, The Biology of Metrtal Health andDisease, Paul B. Hoeber Inc. (p. 34-46). WLLCH,K. A N D SADLER, K. (1966) Permeability of the choroid plexus of the rabbit to several solutes. Arne,.. J. P ~ J J s ~210, o ~ . ,652-660. WESTRUM, L. E. (1965) On the origin of synaptic vesicles in cerebral cortex. J. Physiol., 179 (Proceedings) 4-6. WISLOCKI, G. B. A N D LADMAN, A. J. (1958) The fine structure of the mammalian choroid plexus. Ciba Fobrrnrlatiotr S.vniposiunr on the Cerebrospinal fliiid, Wolstenholme and O'Connor (Eds.). London: Churchill (p. 55-79). WISLOCKI, G. B. A N D LEDUE,E. H. (1952) Vital staining of the hematoencephalic barrier by silver nitrate and trypan blue, and cytologicat comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. Comp. Neurol., 96, 371414. WOLFF,J . (1966) Elektronenmikroskopische Untersuchungen iiber die Vesikulation in dem Kapillarendothel. Z. Zel&wsch., 73, 143-164. DISCUSSION

R. KATZMAN: I would like to ask Dr. Brightman if he can estimate the rate at which the materials move through these pinocytotic vesicles. Dr. Rall's evidence that there is no pinocytosis for inulin would be true only if the time of movement in a pinocytotic vesicle was slower than diffusion. And I wonder if there is some estimate in terms of how many seconds or minutes it takes your marker to move through, e.g., 30 / I . M. BRIGHTMAN: I cannot answer this with certainty. All I can say is that within the shortest time interval (15 minutes), peroxidase had left the choroidal blood, crossed the stromal space and was pinocytosed by the choroidal epithelium. The pinocytotic vesicles, within that interval, moved across these cells (about 15 1) long) to the opposite, ventricular surface. As for the intercellirlar movement from the ventricle across the ependyma, even so large a molecule as ferritin can penetrate the interspaces for at least 30 / t in about 15 minutes, probably less. T. 2. CSAKY: Could you give any indication as to what makes the vesicles move, what is the driving force ? From your pictures it appears that there is a constant ratio gradient. But d o you have any better evidence? Is it a coniplex diffusion? Does it always move away from the place where you put your marker? M. BRIGHTMAN: That is correct. T. 2. CSAKY:But what is the driving force that makes it move?

M . BRIGHTMAN: I can only guess. Cytoplasmic streaming? A pumping activity on the part of the epithelial cells? Although the vesicular traffic appears to be from the blood toward the CSF side of the epithelium. the vesicles ending up near the CSF surface have mixed origins. The lateral (as well as the basal) plasmalemma gives rise to vesicles which need only move a very short distance before reaching the ventricular surface. Moreover, we do not know which proportion of laterally vs basally derived vesicles coalesce, en route, with multivesicular bodies, and so never reach the free surface. But what the forces are that favor this basal to luniinal movement remains unknown.

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D. M. WOODBURY: Did you try the movement of particles across capillaries in other tissue, e.g., muscle capillaries as compared with those in the brain? M. BRIGHTMAN: This is a very good question, anticipating the work of Karnovsky, who found that peroxidase molecules can pass between adjacent endothelial cells in heart muscle capillaries. When Reese and Karnovsky then examined cerebral capillaries, they made the important observation that in this type of blood vessel, the spaces between the endothelial cells were closed to peroxidase. The cerebral endothelium stands as a barrier to the movement of this protein. In the choroid plexus, peroxidase is able to cross the endothelium, in as yet an undetermined way, but the epithelium acts as the barrier to passage into the ventricular fluid.

D. M.WOODBURY: So there is a distinct difference then? M. B R I G H T M A N : Very much so; so that pinocytosis, I daresay, could be rather unimportant, or simply auxillary. D. M. WOODBURY: But what about the smaller molecules? M. BRIGHTMAN: 1 would very much like to find them, but as far as visualizing them, we are limited. D. M . WOODBURY: So you cannot really make any conclusions about these smaller molecules? M. BRIGHTMAN: NO.

K. A. C. ELLIOTT: 1 wondered if Dr. Brightman could straighten me out a little bit: Sometimes you spoke about these particles as though they were just large, inert particles; sometimes you indicated that they were proteins. Now if I am right, they were both proteins and I wonder how this would affect reactions within the tissue. Also, what was the effective size? They could be hydrated and then they would have an effective size that would not be reflected by the molecular weight. And if great big proteins can get in, what about substances like the globulins of the tissue? M . BRIGHTMAN: Both tracers were proteins, apoferritin having a molecular weight of about 480,000 and peroxidase about 40,000.Some sort of binding between protein and cell membrane is a necessary prelude to pinocytosis, as Brandt has so convincingly shown. But the visualization of a protein coat (i.e. peroxidase reaction product) on the cell surface is difficult to interpret. A recent study has shown that many cross-linkages are formed between the dialdehyde, glutaraldehyde, and two different proteins. Thus, the “binding” of one protein (tracer) to another protein (cell membrane) may be purely an event of fixation rather than a linkage that had occurred during life. However, this objection is overcome by the facts that pinocytosis does take place and can be performed only by the responsive membrane of a living cell. Pinocytosis cannot be carried out after fixation. K. A. C. ELLIOTT:What about globulin? Wouldn’t that go in in the same way? M. BRIGHTMAN: 1 think it would.

D. M . WOODBURY: I am glad to hear that the electronmicroscopists d o admit occasionally that there (ire some artefacts! R. V. COXON:Could I first say by way of an addition to what Dr. Brightman has said, that Dr. Simpson-Morgan in my laboratory did study the passage of ferritin out of the cardiac capillaries about a year ago. He found that the process seemed to be determined by the nature of the perfusion fluid. He found that if he kept it perfused with blood, there was practically no escape of ferritin from the capillaries at all. If on the other hand, blood was replaced with saline, then there was a very free penetration of the ferritin into tissue spaces. I would like to, if 1 may, put a perhaps rather wild question to Dr. Brightman: Does he think that his work, showing the uptake of these rather large particles by cells of the nervous system, may give any substance to the claim that has been made that you can transfer learning from one animal to another?

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M. BRIGHTMAN: I have no comment on that question. J. DOBEING:May 1 comment on Dr. Brightman's remarks. First, if he accepts that the BloodBrain Barrier is in the endothelial wall (or rather in the space between it and the parenchyma, such as exists in the heart muscle), we can all go home. But there are other differences than this intracellular gap betwcen cardiac capillaries and brain capillaries. After all, the cardiac capillary is not predominantly ensheathed by glial endfeet. In his other remark towards the end of his paper where he said that the Blood-Brain Barrier was damaged (by which he meant the endothelium) and this material came rushing in; he presuniably damaged more than the endothelium here too.

M. BRIGHTMAN: I would like to say that even though there is a marked difference in the so-called nonBlood-Brain-Barrier areas, within the area postrema and the choroid plexus, there are perivascular connective tissue spaces in both. Olsjewski, for example, found that radioactive proteins got out of the capillaries into the large connective tissue spaces of the area postrema but usually went no further; very little entered the cerebral parenchyma. Some barrier, presumably glial appeared to exclude the protein from the surrounding brain. Now, I emphasize again that I an1 describing barriers to the movement of colloids. When injected into the blood, peroxidase cannot cross the cerebral endothelium to even reach the capillary basement membrane let alone the glia as Reese and Karnovsky have shown. However, as I've illustrated, peroxidase injected into the cerebrospinal fluid compartments has no trouble in reaching the cerebral endothelium. The protein readily moves between the perivascular sheath of glial cells to reach the capillary wall. The glial cells, therefore, do not act as a barrier to the extracellular passage of proteins froni cerebral ventricle to capillary and I see no reason why they would in the opposite direction. The role of the glia and endothelium in the niovenient of amino acids and electrolytes may be, on the other hand, a very different matter. B. D. WYKE:I would like to suggest, contrary to Dr. Dobbing's remark, that we may be even further froni going home than before we arrived. Following the very stimulating dialogue this morning between Dr. Tower and Dr. Csaky on the interesting subject of glucose transport, I would like to suggest for the record that a distinction should be drawn between the mechanisms, whatever they are, of blood and CSF-exchange, and blood and brain exchange. I think it was very noticeable that Drs. Ford and Lajtha, in titling this symposium, p l t the word "systems" in the plural and not in the singular. I feel very strongly that there are at least two systems for glucose exchange (and perhaps more), and that their behaviour is probably very different (Wyke, 1965, in Generd Airuesthesiu, Evans and Gray (Eds.), p. 157, Butterworth, London). If I may, for the benefit of the non-clinical members of the group, illustrate this difference from a human study, 1 would like to remind you of the syndrome of relative cerebral hypoglyceniia that we first described nearly 10 years ago (Wyke, 1959, E/i,c/r.oeticep/i.din. N~wop/i.v.sio/.,/ I : 602). This is a rare condition in which young adults develop the symptoms and the E.E.G. changes of hypoglycemia, although with blood and CSF glucose concentrations that are entirely within normal ranges statistically, both in the fasting and non-fasting states. The only way you can keep these patients symptom free (and the EEG normal) is by maintaining their blood glucose concentrations at figures between 130 and 200 mgm per 100 mL, so that they are in fact in a hyperglycemic state, and some of them are secreting glucose in the urine continuously. However, this does not elevate their CSF-glucose concentrations by more than 10% at the very most, and very often less than that. So here in these patients, you can drive glucose through the Blood-Brain Barrier, whatever that may be, and restore the cerebral metabolic activity to normal by raising the arterial blood glucose concentration, but you cuii not drive it into the CSF to any significant degree. Thus, it seems to me that in this abnormal condition one has a difference in the behavior of the glucose exchange mechanisms between the blood and the brain on the one hand, and between the blood and CSF on the other. Perhaps somebody will have a comment on that? G. PAPPAS: I think this is a rather important aspect. If I might summarize in general: ifyou introduced marker particles into the CSF fluid, the uptake by the choroideal plexus epithelium is via pinocytotic vesicles or vacuoles at the base of the villi. If you introduce marker particles, on the other hand, into the basal portion of these cells, the uptake is much different. They progress via the extracellular space for a while and then later may be pinched up in the vacuoles and vesicles, and we have not clearly shown their cntcring into the cerebrospinal h i d . So that even on a small scale, what you say does seem to hold up rather well, that therc is a difference between the two barriers.

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T. Z. CSAKY: May I point out again that indeed we have to distinguish between the transport of sugar from the capillary into the brain and the sugar exchange between the blood and CSF. When I talked about the choroid plexus I referred to the latter, while the blood-to-brain transport, I believe, is mediated through the glia cells. The two systems are different morphologically and most likely also functionally. One is an epithelial transport mechanism, the other is the glial transport. While we have some limited idea about the epithelium we are in the dark about the transport in the glia.

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Electron Microscopic Cytochemistry and Microgasometric Analysis of Cholinesterase in the Nervous System V I R G I N I A M. T E N N Y S O N , M l R O BRZIN*

AND

PHILIP DUFFY

Departnierit of Pathology, Division of Neuropatliology, College of Physicians and Surgeoris of Columbia University, New York ( US.A.) , arid *Iristitrite of Pathopliy.siology, University of Ljubljana, Ljubljana ( Yugoslavia).

INTRODUCTION

Techniques from different disciplines have been combined in the present investigation in order to provide more meaningful information than would be obtained otherwise. Electron microscopic-cytochemistry and microgasometric analysis have been used to study acetylcholinesterase in the sympathetic and dorsal root ganglia of the frog, using tissue blocks or isolated cells. In addition, some observations on the development of cholinesterase in the nervous system of the embryonic rabbit and human are presented. Cholinesterase activity of isolated sympathetic and dorsal root neurons has been studied by use of the Cartesian diver (Giacobini, 1957, 1959), but the magnetic diver (Brzin et al., 1964; Brzin and Zeuthen, 1964; Brzin et a/., 1965, and Pavlin, 1965) used in the present studies, permits a more sensitive method for quantitative determination of the activity of the enzyme. Isolated neurons have previously been examined with the electron microscope (Roots and Johnston, 1964, 1965; Johnston and Roots, 1965), but no microchemical studies were done on those cells. These investigators (Roots and Johnston, 1965) suggested that in isolated neurons the absence of a physical boundary would undoubtedly alter rates of penetration of substrates into the cell, and recommended that metabolic studies of these cells be monitored by the electron microscope. In previous studies from this laboratory (Brzin et a/., 1966a, 1966b, Tennyson e t a / . , 1966a), isolated neurons, which had been examined with the electron microscope following microgasometric analysis, showed a relationship between chemically measured activity and the morphological condition of the sample. Neurons having intact plasmalemmas and sheath cells showed lower activity values than those in which these structures were ruptured or absent. Ultracytochemistry was then applied to individual neurons, which had been analyzed microgasometrically (Brzin et al., in press). I t was shown that the neural plasmalemma is the ultimate permeability barrier to the substrates acetylcholine and acetylthiocholine. Satellite sheath cytoplasm Rcf<,nviccsp . 59-61

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v.

M. T E N N Y S O N et

al.

often provides an additional cellular barrier, where it is closely applied to the neural plasmalemma over a wide area. The intercellular spaces between folds of sheath cell cytoplasm do, however, permit a limited passage of the substrates. Evidence to support these conclusions will be presented. MATERIALS A N D METHODS

Cervical sympathetic ganglia and lumbar dorsal root ganglia of the frog (Rana pipiens) were used for cytochemical studies. For developmental studies embryonic tissue of rabbits from 9 to 18 days gestation and of human fetuses from 2 to 3 months gestation were employed. The tissue was cut into small blocks and used either unfixed or fixed in 2 % phosphate-buffered glutaraldehyde. For microgasometric studies, individual neurons from frog ganglia were isolated by free hand technique with fine glass needles under a dissecting microscope, and analyzed in the unfixed condition. The cytochemical method used in this study is similar to that described in detail elsewhere (Brzin et al., 1966a). The tissue was treated with a modification (Tennyson et al., 1965) of the copper thiocholine technique for the demonstration of acetylcholinesterase (Koelle and Friedenwald, 1949). The tissue was incubated for 5 to 30 min at pH 6 in the copper-glycinate maleate buffer medium containing 3.5 x 10-3 M acetylthiocholine iodide. Butyrylthiocholine iodide I x 10-2 M, and the inhibitors, iso-OMPA (tetraisopropyl pyrophosphortetramide, Koch-Light, Ltd., England) 2 x 10-4 M or 2 x 10-5 M, and BW 284 C51 (1,5-bis 4 allyldimethylamnioniumphenyl pentan-3-one dibromide, Burroughs-Wellcome, New York) 2 x 10-4 M, and eserine (physostigmine sulfate, Merck, New Jersey) 1 x 10-4 M, were used in control preparations. The tissue was then rinsed in water, fixed for 5 to 10 minutes in 1 % KMnO4, dehydrated, and embedded in Durcupan (Fluka, A. G . , Buchs, S. G . , Switzerland). The cytochemical end production in some specimens was converted to copper sufide (Brzin et al., 1966a) and then post-osmicated. The specimens were sectioned on a Porter-Rlum microtome, placed on formvar-coated copper mesh grids, and stained with routine electron opaque stains. Electron micrographs were taken with a Siemens Elmiskop IA. For the microgasometric procedure unfixed isolated cells were transferred to the usual bicarbonate reaction medium containing 3 x 10-3 M acetylcholine iodide (Zajicek and Zeuthen, 1961). The same procedure was then followed as described previously (Brzin et al., 1964, 1965, 1966a; Brzin and Zeuthen, 1964). Following analysis some of the cells were incubated in the cytochemical medium, fixed, dehydrated and embedded for electron microscopic examination. 1. C Y T O C H E M I S T R Y O F F R O G G A N G L I A

A number of methods have been employed with varying degrees of success for the study of cholinesterase localization in the nervous system with the electron microscope (de Lorenzo, 1961 ; Barrnett, 1962; Torack and Barrnett, 1962; Mori et al., 1964; Lewis and Shute, 1964, 1966; Koelle and Foroglou-Kerameos, 1965; Tennyson et al.,

CHOLINESTERASE IN THE NERVOUS SYSTEM

43

1965b; Lewis el al., 1965; Smith and Treherne, 1965; Schlaepfer and Torack, 1966; Novikoff et a/., 1966; Brzin et al., 1966a; Duffy et al., 1967 a, b). Although there are differences from one paper to another, enzymatic activity has been reported along membranes of the endoplasmic reticulum, the axon and sheath cell membranes, and at some synapses. In previous studies (Shen et a/., 1955; Brzin et al., 1966a) using specific substrates and selective inhibitors in both cytochemical and gasometric studies, it was shown that the enzyme present in the frog nervous system is predominantly acetylcholinesterase. Different cytochemical localizations of the end product of acetylcholinesterase activity are obtained depending on whether the tissue is fixed or unfixed prior to incubation (Brzin et a/., 1966a). End product is located intracellularly in fixed tissue in elements of the endoplasmic reticulum (Fig. I , ER) including the nuclear envelope ( N E ) , subsurface cisternae (arrow), and the agranular reticulum of axons (Fig. 2, arrows). It should be noted that ribonucleoprotein particles usually associated with the endoplasmic reticulum are not well demonstrated in permanganate treated tissue. The presence of end product within subsurface cisternae is of considerable interest since Rosenbluth (1962) has suggested that subsurface cisternae may be a functional component of the neuronal plasma membrane. Most of the components of the Golgi complex are negative when acetylthiocholine is used as substrate, although occasionally Golgi tubules, which may be in continuity with the agranular reticulum, contain end product. Enzyme activity is located predominantly on surface membrane structures if the tissue is unfixed during incubation. The end product is found at the neural or axolemmal surface (Fig. 3, AX), the surface of sheath (S) and Schwann cell (arrow), and at the surface of synaptic endings (Fig. 4, SY). It should be pointed out that the surface of the synaptic endings covered by a satellite sheath (S) exhibits end product, as well as the surface apposed to the neuron (arrow). The endoplasmic reticulum (Fig. 3, ER) and nuclear envelope (NE) do not contain end product in unfixed tissue, unless the cell has been damaged. The presence of end product on surface structures, but its absence intracellularly in unfixed tissue suggests that a permeability barrier is present around the intact unfixed neuron. Fixation diminishes the effectiveness of the barrier, as evidenced by the presence of end product intracellularly in the endoplasmic reticulum. At the same time, however, fixation has an inhibitory effect on cholinesterase, particularly on the enzyme located at surface structures. Microgasometric measurements of ganglion cells before and after glutaraldehyde fixation have shown a decrease of cholinesterase activity from 20 to 53 per cent (Brzin et al., 1966a). It is necessary, therefore, to study tissue which has been incubated in the unfixed condition and after fixation, in order to localize the enzyme on surface structures, as well as intracellularly. The presence of enzymatic activity at the synapse, and at the neural and axonal surface is in keeping with the physiological and biochemical data, which suggest that acetylcholinesterase plays a role in the permeability cycle of excitable membranes during transmission and conduction (Nachmansohn, 1959, 1966). The role which this enzyme plays i n the endoplasmic reticulum, however, is unknown. It may be conR ~ . / r ~ c n ~p c. S9 ~ s 41

44

v. N. T E N N Y S O N et al.

45

CHOLINESTERASE I N T H E N E R V O U S SYSTEM

cerned with processes other than conduction or transmission, particularly since it is found in sympathetic ganglionic neurons, which are not usually considered to be cholinergic. I t is possible that the enzyme in the endoplasmic reticulum and the agranular reticulum may be involved in ion exchanges associated with metabolic processes within the cell.

11. M I C R O G A S O M E T R I C A N A L Y S I S A N D C Y T O C H E M I S T R Y

OF ISOLATED N E U R O N S

Table I presents the results of microgasometric analysis with the magnetic diver of cells isolated from dorsal root and sympathetic ganglia using acetylcholine as the substrate. In a previous study (Brzin et a/., 1966a), it was shown chemically that the cholinesterase present i n these cells is predominantly acetylcholinesterase, since in the presence of an inhibitor of this enzyme, BW 284 C51, there was no appreciable hydrolysis of acetylcholine. On the other hand, the use of iso-OMPA, an inhibitor of butyrylcholinesterase caused only a slight diminution of activity. There was very little hydrolysis when butyrylcholine was used as substrate with or without inhibitors. Eserine ( 1 i: 10-5 M) caused a considerable decrease in activity, but was somewhat less effective than BW 284 C 51. It can be seen from Table I that there is a considerable difference in activity from pl one cell to another. Dorsal root cells ranged in activity from 1.8 to 59.4 x COr/h/sample and sympathetic cells ranged in activity from 2.1 to 51.8 pl COz/h/ sample. Certain questions arise concerning the interpretation of these findings: ( I ) Do the results indicate a true quantitative difference from one cell to another? (2) Are the isolated samples single neurons devoid of sheath cytoplasm? (3) How well preserved is the specimen and will its condition affect the chemical measurements? Histochemical studies using light microscopy (Koelle, 1955 ; Holmstedt and Sjoqvist, 1959) and electron microscopy (Brzin et a/., 1966a) have shown that there are differences i n the amount of end product present from one cell to another. There is the possibility, therefore, that the measured activity could represent a real difference from one cell to another, but the other factors mentioned might also have some influence on the results. I n an attempt to obtain information concerning these questions, electron microscopic examination was carried out on isolated cells following microgasometric analysis (Brzin e t a / . , 1966a).Table I I shows that the amount of measured activity can be correlated with the degree of preservation of cell structure. Damaged

Fig. I . Electron micrograph of a glutaraldehyde-fixed sympathetic neuron reacted for AChE activity. The end product is present in the endoplasmic reticulum (ER), nuclear envelope (NE), and in subsurface cisternae (arrow) located near the postsynaptic surface of an axosomatic synapse (SY). The mitochondria ( M )are negative. Ribonucleoprotein particles are not visualized in this preparation, because it was treated with KMnO4. AThCh t iso-OMPA, 30 min. x 29 000. Fig. 2. Electron micrograph of an unmyelinated sympathetic axon fixed in glutaraldehyde and reacted for AChE activity. End product is present in the agranular reticulum (arrows). AThCh iso-OMPA, 5 min. post-tixed in KMnOJ. x 49 000. (Reprinted from Brzin ef a / . , 1966a).

+

R ~ ~ f i w n r cp ~ 59 \ 6/

v.

46

M. T E N N Y S O N et

a/.

Fig. 3. Electron micrograph showing the localization of AChE activity in tissue which was unfixed prior to incubation. End product is present along the folds of satellite cell (S) and sheath cell (arrow) cytoplasm, and also along the surface of the axon (AX). The endoplasmic reticulum (ER) and nuclear envelope (NE) do not contain end product. AThCh iso-OMPA, 30 min, post-fixed in KMn04 x 56 000. (Reprinted from Brzin et a/., 1966b). Fig. 4. Electron micrograph of a sympathetic neuron, which was unfixed prior to incubation for AChE activity. End product is present around the nerve endings (SY)at the neural (arrow) and sheath (S) surfaces. AThCh iso-OMPA, 30 min, post-fixed in KMn04 x 25 200. (Reprinted from Brzin et al., 1966a).

+

+

CHOLINESTERASE I N T H E N E R V O U S SYSTEM

47

TABLE I ChE

A C T I V I T Y O F ISOLATED C E L L S

ACTIVITY EXPRESSED I N

1'1

coz

X Io-'/h/SAMPLE

Dorsal rooi cells Syrnpaiheiic cells

I .8 3.3 4.8 7.5 12.3 38.4 49.5 59.4

2.1 8.8 9.6 11.2 14.4 16.0 16.0 17.3 20.8 22.4 23.5 25.6 26.6 31.6 51.8

TABLE I1 R E L A T I O N S H I P H E T W E E NChE A C T I V I T Y A N D S T R U C T U R A L C O N D I T I O N O F C E L L S I S O L A T E D

P R O M T H E D O R S A L ROOT G A N G L I O N . THE A C T I V I T Y IS E X P R E S S E D

IN,^ COZ x 10-6/h/sample

(Reprinted from Brzin ef al., 1966a) Cells with high aciivify

Cells with low aciiviiy

63.3 Partially ruptured sheath, neural plasmalemma only partially intact, short segment of axon 57.0 Sheath and neural plasmalemma absent

6.9 Two neurons. Sheaths surround both neurons, neural plasmalemma intact

54.6 Sheath and neural plasmalemma absent 49.5 Sheath and neural plasmalemma absent 49.5 Ruptured sheath, neural plasmalemma only partially intact, short segment of axon 47.1 Sheath and neural plasmalemma absent

5.4 Two neurons. Sheaths surround both neurons, neural plasmalemma intact 4.5 Sheath surrounds neuron, neural plasmalemma intact 3.6 Sheath surrounds neuron, neural plasmalemma intact, short segment of axon 3 .O Sheath surrounds neuron, neural plasmalemma intact, short segment of axon 2.4 Sheath surrounds neuron, neural plasmalemma intact, short segment of axon

neurons having ruptured or absent neural plasmalemmas and satellite sheaths (Figs. 5 , 6) exhibited considerably higher cholinesterase activity than those having intact plasma membranes and sheaths (Fig. 7, S). This suggested that the intact neural plasmalemma, and possibly, the satellite sheath formed a permeability barrier to the substrate. The condition o f t he isolated cell, therefore, appears to have a considerable influence on the results of microgasometric analysis. In order to test this supposition further, and to determine what structures in the Riferencrs p. 5 9 4 1

48

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M. T E N N Y S O N et a / .

49

C H O L I N E S T E R A S E IN T H E N E R V O U S S Y S T E M

T A B L E III RELATIONSHIP

OF

MORPHOLOGY

TO

CHEMICAL

AND

CYTOCHEMICAL

DETERMINED ON T H E S A M E U N F I X E D S A M P L E . A C T I V I T Y IS EXPRESSED I N

ChE }I1

coz

ACTIVITY X

m5/h/

SAMPLE.

(Reprinted from Brzin et al., 1967) Activity

Group I

Morphologic condition of the sample

2.8

Surfaces of axon, synapse, perikaryon and sheath

Most of cell intact, but one side damaged. Endoplasmic reticulum reasonably well preserved.

3.0

Surfaces of axon, synapse, perikaryon and sheath

Sheath and neural plasmalemma intact. Subsurface cisternae badly swollen.

6.2

Surfaces of axon, synapse, perikaryon and sheath

Sheath damaged, but neural plasmalemma intact. Endoplasmic reticulum reasonably well preserved.

8.8

Surfaces of axon, synapse, perikaryon and sheath

Sheath and neural plasmalemma intact. Endoplasmic reticulum reasonably well preserved.

10.1

Surfaces of axon, synapse, perikaryon and sheath

Sheath and neural plasmalemma intact. Endoplasmic reticulum somewhat swollen.

11.7

Surfaces of axon, synapse, perikaryon and sheath

Sheath and neural plasmalemma intact. Endoplasmic reticulum reasonably well preserved.

36.0

Surfaces of axon, synapse, perikaryon and sheath

Sheaths and plasmalemmas of both neurons intact. Endoplasmic reticulum reasonably well preserved. Several axon processes present.

32.3

Endoplasmic reticulum, surfaces of axon, synapse, perikaryon and sheath

Cell intact on one side, but damaged on other. Endoplasmic reticulum swollen.

33.2

Endoplasmic reticulum, surfaces of axon, synapse, perikaryon and sheath

Cell intact on one side, but damaged on other. Endoplasmic reticulum swollen.

43.5

Endoplasmic reticulum, surfaces of axon, synapse, perikaryon and sheath

Sheath and neural plasmalemma ruptured. Endoplasmic reticulum swollen.

(2 neurons)

Group I1

D i d product localization

Fig. 5 . Phase micrograph of an isolated dorsal root neuron in Table 11 having a high AChE activity of 54.6 .iIC51'1 COz/h. This granular neuron retained its contour, but the surface is slightly roughened and damage is evident at the base. x 1 250. (Reprinted from Brzin el al., 1966a). Fig. 6 . Electron micrograph of the roughened surface of the cell in Fig. 5. The sheath and neuroplasmaiemma are absent. x 23 200. (Reprinted from Brzin et al., 1966a). Fig. 7 . Electron micrograph of a portion of the two neurons in Table 11 which showed a low activity of 5.4 x I G F 1'1 COz/h. A thin satellite sheath 6 ) and a neuroplasmalemma surrounded each neuron. The cisternae of the endoplasmic reticulum (ER) are somewhat swollen, but the nucleus and mitochondria (M)do not appear t o be altered. x 9000. Rc.Jerrni ('s p. 59-61

50

v.

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C H O L I N E S T E R A S E IN THE N E R V O U S S Y S T E M

51

cell are responsible for the chemically measured activity, ultracytochemical studies were done on isolated cells following microgasometric analysis (Brzin et al., 1967). As expected damaged cells had higher measured cholinesterase activity than intact cells (Table 111). The cytochemical end product of cholinesterase activity in low activity intact cells was localized at the neural plasmalemma (Figs. 8, 9, NP), sheath cell surfaces ( S ) , and at the surfaces of the axons (AX), and synaptic endings (Fig. 10, SY). No end product was present within the endoplasmic reticulum (Figs. 8, 9, 10 ER) of these cells. In the higher activity cells which were damaged, end product was found in remaining surface structures, as well as in the cisternae of the endoplasmic reticulum (Fig. 11, ER and arrows). In these experiments, therefore, morphological, cytochemical and quantitative information obtained on the same cell has shown that its morphologic condition significantly influences chemical data. An intact unfixed neural plasmalemma is an effective permeability barrier to thesubstrates used. Acontinuous sheet ofsatellite sheath cytoplasm probably provides an additional cellular barrier where it is closely applied to the surface of a neuron. In those portions of the sheath consisting of multiple interrupted layers of cytoplasm and numerous intercellular spaces, the barrier is less effective. A limited passage of substrate occurs through these anastomosing channels as far as the neural surface. Passage of acetylcholine through intercellular clefts in the insect nervous system has previously been discussed by Smith and Treherne (1965). Diffusion of substances through intercellular channels may be an important means of exchange between the extracellular space and the neuron under normal conditions. The patency of at least some of the intercellular spaces in the nervous system has previously been shown by the use of marker particles (Lazansky and Wald, 1962; Rosenbluth and Wissig, 1964; Brightman, 1965; Pappas and Purpura, 1966). Ion exchange studies have indicated that the main pathways for the rapid movements of sodium and potassium ions and sucrose is through intercellular clefts in the nervous system rather than through glial cytoplasm (Nicholls and Kuffler, 1964; Kuffler etal., 1966). It should be emphasized, however, that the demonstration of penetration barriers and of permeability channels depends on the substances tested. A barrier to a water soluble charged molecule, such as acetylthiocholine, may nevertheless permit penetration of a lipid soluble substance. Although certain substances may diffuse through intercellular spaces, the same substances, as well as different ones, may be actively transported into cells. Fig. 8. An electron micrograph of a n isolated sympathetic neuron which had a low activity of 8.8 pl COz x lO-S/h. Thin layers of satellite cell cytoplasm (S) can be seen surrounding the neuron. The nucleus (N) and endoplasmic reticulum (ER) are reasonably well preserved, but the mitochondria (M) are swollen. Nuclear pores (arrow) are evident in the nuclear envelope. Histochemical end product is present between folds of satellite cell cytoplasm (S), a t the neural plasmalemma (NP) and a t the surface of the axon (AX). Incubated unfixed, AThCh, 15 min. X 19 200. Fig. 9. A higher magnification of another surface area of the cell in Fig. 8. End product of AChE activity is present at the perikaryal-sheath interface (NP), the axon (AX)-sheath interface, and between layers of sheath cell cytoplasm (S). N o end product is present in the endoplasmic reticulum (ER) or in nuclear envelope (NE). The neural plasmalemma (NP) is intact. X 29 OOO. (Reprinted from Brzin ct al., 1967). Rc.jerences p . 5 9 4 1

52

v. M. T E N N Y S O N et a/.

C H O L I N E S T E R A S E I N T H E N E R V O U S SYSTEM

53

111. C Y T O C H E M I S T R Y O F D E V E L O P I N G N E R V O U S S Y S T E M

Cholinesterase activity has been studied intensively by light microscopy in the chick embryonic nervous system (Bonichon, 1958; Bonichon and Gerebtzoff, 1958; Gerebtzoff, 1959; Strumia and Baima-Bollone, 1964). The following is a summary ofthe findings of these investigators. Cholinesterase activity was identified i n the ventral horn of the spinal cord of the chick embryo during day 4 of gestation. The mantle layer and a thin marginal layer had formed by this period. The cells containing enzyme activity had not differentiated into neuroblasts, when judged by light microscopic criteria. It was concluded, therefore, that biochemical differentiation of neuroblasts takes place before morphological differentiation. Activity appears in the dorsal root neurons a day later, after a well defined grouping of cells have formed into a ganglion with central and peripheral roots. As differentiation proceeds, cholinesterase activity becomes localized in the periphery of the perikarya, then appears in the processes. The first fiber bundle to show enzyme activity is the tract of Lissauer. This is followed by the dorsal root fibers, and later by the ventral root fibers. In previous electron microscopic-cytochemical reports from this laboratory (Tennyson et a/., 1966b, 1967; Duffy er a/., 1967b) it was shown that cholinesterase activity actually appears at an earlier stage of development than can be recognized by light microscopy. Enzymatic activity could be identified by electron microscopy in the neural tube of rabbit embryos during the beginning of day 10 of gestation before the marginal and mantle layers have formed. A small amount of end product is present in the nuclear envelope (Fig. 12, NE) and later in an isolated cisterna of the endoplasmic reticulum of an occasional cell located in the ventrolateral region of the neural tube. Near the end of day 10, evidence of the onset of enzyme activity can be found sporadically in bipolar cells, which are migrating from the neural crest to form the dorsal root ganglion. The presumptive neurons from both regions are still undifferentiated morphologically, but have begun to move to the site where development may take place. The cytoplasm of these cells is filled with ribonucleoprotein particles, but relatively few cisternae of the endoplasmic reticulum are present (Tennyson, 1962; Tennyson, 1965). As the cells differentiate, the number of cisternae increases greatly. The source of this nearly formed membrane is not known, but it has been suggested

Fig. 10. An electron micrograph of the isolated sympathetic neuron in Table 111 which had a moderately low activity of 11.7 / r l COZ x I e 5 / h . Although the neural plasmalemma (NP) has become detached from the synaptic boutons (SY), it appears intact. An intact layer of satellite sheath cytoplasm surrounds the neuron and is reflected in loose folds over the boutons. Most of the endoplasmic reticulum (ER) is reisonably well preserved, but the mitochondria ( M ) are swollen. End product of AChE activity is present at the surfaces of the boutons (arrow) and along sheath surfaces (S). (Incubated unfixed, AThCh, 15 min KMn04 fixation). x 23 000. (Reprinted from Brzin et a / . , 1967). Fig. 1 I . An electron micrograph of a n isolated sympathetic neuron in Table 111 which had a relatively high activity of 32.3 pI C02 x I W i h . The neural plasmalemma (NP) and satellite sheath (S) are ruptured in many places along the surface. The endoplasmic reticulum (ER) and subsurface cisternae (SSC) are badly swollen, and contain end product of cholinesterase activity (arrows). The nuclear envelope ( N E ) also contains end product. The mitochondria (M) are swollen. (Incubated unfixed, AThCh 15 rnin K M n 0 4 fixation). x 27 000. Rtfermces p. 5 9 6 1

54

v. M. T E N N Y S O N et al.

C H O L I N E S T E R A S E I N T H E N E R V O U S SYSTEM

55

that contributions may come from the nuclear envelope or Golgi complex (Tennyson, 1965). Since the enzyme appears in the cell in the nuclear envelope first, the question arises concerning its subsequent appearance in the endoplasmic reticulum. The enzyme could develop in situ in the endoplasmic reticulum as it did in the nuclear envelope. It is also possible that portions of the nuclear envelope having enzyme forming potential could remain in the cytoplasm, after a previous mitosis, and become the site of enzyme synthesis in the endoplasmic reticulum. On the other hand, portions of the outer nuclear envelope containing enzyme could project into the cytoplasm and pinch off to become cisternae of the endoplasmic reticulum. Out-pocketings of the outer nuclear envelope are frequently seen in early neuroepithelial cells. Although the basic mechanisms underlying the appearance of the enzyme are unknown, its presence in a morphologically undifferentiated cell is one of the first signs of further development. The onset of neuroblast differentiation, therefore, follows just after the appearance of cholinesterase in the nuclear envelope and endoplasmic reticulum of the cell. These findings are in agreement with the conclusion of Bonichon (1958) and Gerebtzoff ( I 959) that biochemical differentiation precedes morphological differentiation. Because of the higher resolving power of the electron microscope, however, both cytochemical and morphological differentiation can be identified at an earlier stage, i.e., before the mantle and marginal layers have formed. Enzyme activity gradually increases in the ventral horn and dorsal root ganglion during the next few days. More cells contain end product and itsdistribution throughout the cisternae of the individual neuroblasts becomes more widespread. The axolemmal surface of fibers in the tract of Lissauer begins to show enzyme activity by day 1 I , but is more pronounced during day 12 (Fig. 13, arrows). End product appears in the agranular reticulum of some axons during the latter part of this period. Many of the neuroblasts during day 17 have developed into immature neurons, although most are still in late neuroblast stages. End product of cholinesterase activity can be found in the nuclear envelope (Fig. 14, NE) and endoplasmic reticulum (ER) of most of the neuroblasts and immature neurons examined. In addition, satellite cells, which show some differentiation by this time, also show activity in the nuclear envelope and endoplasmic reticulum. Examination of the spinal cord of a three month gestation human fetus shows a high amount of cholinesterase activity in similar locations as seen in the rabbit -~

Fig. 12. An electron micrograph illustrating the presumptive ventral horn region of the neural tube of a 10 day gestation rabbit embryo. The first sign of ChE activity appears in the nuclear envelope (NE) in an occasional undifferentiated cell having scant perinuclear cytoplasm. The endoplasmic reticulum (ER) is sparse at this stage and the mitochondria (M)have few cristae. Increased membrane density (arrow) which may indicate a junctional contact, appears between the thin cellular process and the adjacent cells. The cells rest on the external limiting membrane (EX). Since this specimen was treated with KMnO.1, the ribonucleoprotein particles, which normally fill the cytoplasm of these immature cells, are not well visualized. AThCh, 30 min. x 15 400. Fig. 13. An electron micrograph of a cross section through the tract of Lissauer from a rabbit embryo during day 12 of gestation. The tissue was unfixed prior to incubation for ChE activity. End product (arrows) is present at the axolemmal surface of many of the nerve fibers (AX). Incubated unfixed, AThCh I iso-OMPA, 30 min KMn04 fixation. x 11 200. R i f i r i w r s p . 59-61

56

v.

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CHOLINESTERASE I N T H E N E R V O U S SYSTEM

57

fetus. End product is present in the nuclear envelope (Fig. 15, NE) and endoplasmic reticulum (ER) of the immature neurons. I t is also present in the agranular reticulum (arrow) of axon processes in the neuropil. A possible relationship between the enzyme in the endoplasmic reticulum and that at the neural surfaces has been suggested by Fukuda and Koelle (1959). These investigators suggested that cholinesterase might be synthesized in the endoplasmic reticulum of the normal adult perikaryon, and then be transported via its canaliculi to the surface of the neuron and its processes. If this were the case, one would expect the enzyme to be found in embryonic development first in the neural perikaryon, then in the agranular reticulum of the proximal portion of the axons, and finally at the surface of the distal portion of the axons. Evidence so far from the embryological studies of this laboratory has shown a different developmental sequence. In the case of neurons of the dorsal root ganglia, the enzyme does appear first in the perikaryon, but then it is found at the axolemmal surface of the distal portion of the nerve fibers, i.e., i n the tract of Lissauer. This occurs before there is any appreciable amount of enzyme in the agranular reticulum of the proximal portion of the axon processes, i.e., in the dorsal root. This suggests that the enzyme at the axolemmal surface develops independently of that in the agranular reticulum. Moreover, one would question whether cholinesterase is free to flow through the cisternae of the endoplasmic reticulum as implied by the suggestion of Fukuda and KoelIe (1959), since the available chemical evidence suggests that the enzyme is firmly bound to membranes (Toschi, 1959). It is possible that the enzyme in the agranular reticulum of the axon may originate in embryonic life by cytoplasmic streaming of this organelle from the perikaryon into the axon, but there is no data so far to show that the enzyme is then transferred from this site to the axolemmal surface. There is evidence that cholinesterase can be formed in the axon itself (Koenig, 1965) even though ribonucleic acid concentration is low. The functional role of the intracellular enzyme is unknown. The absence of a sequential proximal-distal relationship in the development of cholinesterase, however, supports the concept that the enzyme in the elements of the endoplasmic reticulum may have a different function from the enzyme at the axolemmal and synaptic surfaces. SUMMARY

Acetylcholinesterase has been studied in frog sympathetic and dorsal root ganglia by

Fig. 14. An electron micrograph of a well developed transitional bipolar neuroblast from the dorsal root ganglion of a 17 day gestation fetal rabbit. The nuclear envelope (NE) of the eccentrically located nucleus contains end product of ChE activity, as well as most of the cisternae of the endoplasmic reticulum (ER)in the cytoplasm. Numerous mitochondria ( M ) and several distinct groupings of the Golgi complex ( G )are present. (AThCh i iso-OMPA 15 min post-fixed in KMn04). x 15 600. Fig. 15. An eleztron micrograph of a glutaraldehyde-fixed neuron from the spinal cord of a three month gestation human fetus. End product is present in the nuclear envelope (NE) and in the endoplasmic reticulum (ER)of the neuron and also in the agranular-reticulum (arrow) of axon (AX) processes. (AThCh I iso-OMPA, 40 min, converted to CuS. oso4 fixation). x 27 000. RrJiwtrws p. 59-61

58

v.

M. TENNYSON

et al.

a combination of electron microscopic-cytochemistry and microgasometric analysis with the magnetic diver. The enzyme was demonstrated in two principle areas depending on whether the tissue was fixed or unfixed prior to incubation. End product was present in fixed tissue on the inner surface of the membranes of the endoplasmic reticulum, including the nuclear envelope and subsurface cisternae, as well as the agranular reticulum of axons. The enzyme is most readily demonstrated at the surface of the synapse, the axolemma, the neural plasmalemma, and the surface membranes of the satellite and Schwann cells in unfixed or very briefly fixed tissue. Ultracytochemistry was carried out on isolated neurons following microgdsometric analysis. By this means, morphological, cytochemical and quantitative information was available on the same cell. The low activity cells exhibited an intact neural plasmalemma and usually an intact satellite sheath. The cytochemical end product was located at the surfaces of the axon, synapse, perikaryon, and sheath cells, but not within the cytoplasm of the cell. This supports the concept that the neural plasmalemma, and to a lesser extent, the satellite sheath, forms a permeability barrier to the substrates used. The neural plasmalemma of the high activity cells was ruptured and the cytochemical end product was present in the endoplasmic reticulum, as well as on the remaining surface structures. These investigations show the value of electron microscopic-cytochemical observation in the interpretation of biochemical data. The development of cholinesterase activity has been studied in the nervous system of the rabbit embryo and human fetus. The enzyme appears very early in development, just before the onset of morphological differentiation of the neuroblast. Activity appears first in the nuclear envelope of a few scattered cells in the ventrolateral region of the neural tube. It is then found in the endoplasmic reticulum, particularly as differentiation proceeds. Shortly thereafter the enzyme can be found in cells migrating from the neural crest to form the dorsal root ganglion. The presence of cholinesterase in these relatively undifferentiated cells permits their identification as presumptive neuroblasts, since studies of these areas in the next stage of development have shown that all of the cells containing cholinesterase activity are neuroblasts. Enzyme activity does not appear in satellite cells until later in development, when the neuroblasts are differentiating into immature neurons. Activity appears at the axolemmal surface of fibers in the tract of Lissauer soon after the posterior funiculus becomes established. End product is then found in the agranular reticulum of the axons. Neurons and axons of three month gestation human fetuses contain a high amount of cholinesterase activity in similar locations as described in the rabbit fetus. ACKNOWLEDGEMENTS

This work has been supported by the Clinical Research Center for Parkinson’s and Allied Diseases NB 05184 and the Parkinson Information Center, a part of the National Information Network of NINDB under contract no. PH 43 64 54, and NB03304, 5-TI-NB-5062, 2-ROI-HD-00964, HE-5906, National Science Foundation Grant No. NSF-GB-4844, and the Boris KidriE Foundation, Ljubljana, Yugoslavia. The authors wish to express their gratitude to Dr. David Nachmansohn for his

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critical discussions and constructive advice, and to Dr. Melvin Yahr for providing the opportunity to do these studies. The invaluable technical assistance of Miss Mary Budininkas, and Mr. Gamil Debbas, the electronic management of the instrument by Mr. Moshe Rosen, and the phase contrast microscopy by Mr. Lewis Koster is gratefully acknowledged. Figs. 2 and 4 and Table II have been reprinted by permission of the Rockefeller University Press from the Journal of Cel/ Biology, November, 1966, Volume 3 / , No. 2, p p . 215-242. Figure 10 and Table I I I were reprinted by permission of Pergamon Press from the International Journal of Neuropharmacology, and Figure 3 was reprinted by permission of Raven Press. REFERENCES BARRNETT, R. J. (1 962) The fine structural localization of acetylcholinesterase a t the myoneural junction. J. Cell Biol., 12, 247-262. BONICHON, A. (1958) L’acetylcholinesttrase dans la cellule et la fibre nerveuse a u cows du dtveloppement. I. Ann. Histochini., 3, 85-93. M. A. (1958) L’acetylcholinesttrase dans la cellule et la fibre BONICHON,A. AND GEREBTZOFF, nerveuse au cours du developpement. 11. Ann. Histochim., 3, 171-178. BRIGHTMAN, M. W. (1965) The distribution within the brain of ferritin injected into the cerebrospinal fluid compartments. 11. Parenchymal distribution. Amer. J. Anut., 117, 193-220. BRZIN,M., DETTBARN, W. D., ROSENBERG, P. A N D NACHMANSOHN, D. (1965) Cholinesterase activity per unit surface area of conducting membranes. J. Cell Biol., 26, 353-364. BRZIN, M., KOVIC,M. AND OMAN,S . (1964) The magnetic diver balance. c . R. Trav. Lab. Carhberg, 34,407426. BRZIN,M.,TENNYSON, V. M. AND DUFFY, P. E. (1966a) Acetylcholinesterase in frog sympathetic and dorsal root ganglia: a study by electron microscope cytochemistry and microgasometric analysis with the magnetic diver. J . Cell Biol., 31, 215-242. -, ( I 966b); Combined cytochemical-electron microscopic localization of cholinesterases in the nervous system. Syrnp. Biochenr. Pharmacol. Basal Ganglia. E. Costa, L. J. Cote, M. D. Yahr, Editors, Hewlett, New York, Raven Press (p. 65). - (1967) Ultrastructural, cytochemical, and microgasometric studies of isolated neurons of the frog. Intern. J. Neriropharmacol., 6, 265-272. BRZIN,M. AND ZEUTHEN, E. (1964) Notes on the possible use of the magnetic diver for respiration measurements (error plihour). C.R. Trav. Lab. Carlsberg, 34, 427431. DE LORENZO, A. J. D. (1961) Electron microscopy of the cerebral cortex. 1. The ultrastructure and histochemistry of synaptic junctions. Brill. Joltns Hopkins Hosp., 108, 258-267. DUFFY,P. E., TENNYSON, V. M.AND BRZIN,M. (1967a) Acetylcholinesterase in mammalian nervous system; A study of human cerebral cortex, and rabbit hypothalamus using a combined cytochemical and electron microscopic technique. Truns. Atner. Neurolog. Ass., (in press). - (1967b) Cholinesterase in adult and embryonic hypothalamus. A combined cytochemical electron microscopic study. Arch. Neurol., 16, 385-403. T. AND KOELLE, G. B. (1959) The cytological localization of intracellular neuronal acetylFUKUDA, cholinesterase. J. Bi0ph.v~.Biocheni. Cyrol., 5 , 433440. GEREBTZOFF, M. A. (1959) Choliiresteruses. New York, Pergamon Press. E. (1957) Quantitative determination of cholinesterase in individual sympathetic cells. GIACOBINI, J . Neurochem., 1, 234244. - (1959) The distribution and localization of cholinesterases in nerve cells. Acta fhysiol. Scand., 45, Suppl. 156, 1 4 5 . HOLMSTEDT, B. AND SJOQVIST, F. (1959) Distribution of acetylcholinesterase in the ganglion cells of various sympathetic ganglia. Acra fhysiol. S c a d , 47, 284-296. JOHNSTON, P. V. AND ROOTS,B. 1. (1965) The neurone surface. Nature, 205, 778-780. KOELLE,G. B., (1955); The histochemical identification of acetylcholinesterase in cholinergic, adrenergic and sensory neurons. J . fhurmaiol. Expfl. T h e r p . , 114, 167-184. KOELLE,G. B. AND FORWLOU-KERAMEOS, C. (1965) Electron microscopic localization of cholinesterases in a sympathetic ganglion by a gold thiolacetic acid method. LVe Sci., 4, 417424.

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KOELLE,G. B. AND FRIEDENWALD, J. S., (1949); A histochemical method for localizing cholinesterase activity. Proc. SOC.Exptl. Biol. Med., 70, 617-622. KOENIG,E. (1965) Synthetic mechanisms in the axon. I. Local axonal synthesis of acetylcholincsterase. J. Neurochem., 12, 343-355. KUFFLER, S. W., NICHOLLS, J. G . AND ORKLAND, R.K.(1966) Physiological properties of glial celk in the central nervous system of amphibia. J. Neuropliysiol., 29, 768-787. LAZANSKY, A. A N D WALD,F. (1962) The extracellular space in the toad retina as defined by the distribution of ferrocyanide. A light and electron microscopic study. J. Cell Biol., 15, 463479. LEWIS,P. R. AND SHUTE,C. C. D. (1964) Demonstration of cholinesterasc activity with the electron microscope. J. Physiol., London, 175, 5P (abstr.). - (1966) The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell Sci., 1, 381-390. LEWIS,P. R., PINE, A. B. AND SHUTE,C. C. D. (1965) The electron microscopic distribution of cholinesterase in rat brain. J. Physiol., London, 131, 15P (abstr.). MORI,S., MAEDA, T. AND SHIMIZU, N. (1964) Electron-microscopic histochemistry of cholinesterases in the rat brain. Histochem., 4, 65-72. NACHMANSOHN, D. (1959) Chemical and Molecular Basis of Nerve Activity. New York, Academic Press, Inc. (pp. 1-235). -(1966) Chemical control of the permeability cycle in excitable membranes during electrical activity. Ann. New York Acad. Sci., 137, 877-900. NICHOLLS, J. G. AND KUFFLER. S. W. (1964) Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neurophysiol., 27, 645-67 I . NOVIKOFF. A. B., QUINTANA. N., VILLAVERDE, H. AND FORSCHIRM, R. (1966) Nucleoside phosphatase and cholinesterase activities in dorsal root ganglia and peripheral nerve. J. Cell Biol.,29, 525-545. PAPPAS,G. D. AND PURPURA, D. P. (1966) Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex. Nature, 210, 1391-1 392. PAVLIN, R. (1965) Cholinesterases in reticular nerve cells. J. Neurocheni., 12, 515-518. ROOTS,B. 1. AND JOHNSTON, P. v. (1964) Neurons of ox brain nuclei: their isolation and appearance by light and electron microscopy. J. Ultrastruct. Rex, 10, 35C361. - (1965) Isolated rabbit neurons: electron microscopical observations. Nature, 207, 3 15-316. ROSENBLUTH, J. (1962) Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol., 13, 405421. ROSENBLUTH, J. AND WISSIG,S. L. (1964) The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons. J. Cell Biol., 23, 307-325. SCHLAEPFER, W. W. AND TORACK,R. M. (1966) The ultrastructural localization of cholinesterase activity in the sciatic nerve of the rat. J. Histochem. Cytocliem., 14, 369-378. SHEN,S. C., GREENFIELD, P. AND BOELL,E. I. (1955) The distribution of cholinesterase in the frog brain. J. Comp. Neurol., 102, 717-743. SMITH,D. S. AND TREHERNE, J. E. (1965) The electron microscopic localization of cholinesterase activity in the central nervous system of an insect, Periplaneta aniericana L. J . Cell Biol., 26, 445465. STRUMIA, E. AND BAIMA-BOLLONE, P. L. (1964) AChE activity in the spinal ganglia of the chick embryo during development. Acta Anatomica, 57, 281-293. TENNYSON, V. M. (1962) Electron microscopic observations of the development of the neuroblast in the rabbit embryo. Fiflh International Congress for Electron Microscopy, Philadelphia. S . S . Brecsc, Editor. New York, Academic Press, Vol. 11, N-8. -, (1965); Electron microscopic study of the developing neuroblast of the dorsal root ganglion of the rabbit embryo. J. Comp. Neurol., 124, 267-318. TENNYSON, V., BRZIN,M. AND DUFFY, P. (1965) Electron microscopic localization of acetylcholinesterase in the neur0r.s of frog sympathetic ganglia. J. Cell Biol.,27, 105 (abstr.). - (1966a) Ultracytochemical and microgasometric analysis of acctylcholinesterase in frog dorsal root and sympathetic neurons. Sixth International Congress for Electron Microscopy, Kyoto. R. Uyeda, Editor. Tokyo, Maruzen Co., Vol. I1 (pp. 101-102). - (1966b) Cholinesterase localization in the developing nervous system of the rabbit embryo and human fetus by electron microscopic histochemistry. Anat. Rec., 154, 432, (abstr.). - (1967) Cholinesterase localization in the dorsal root ganglion of the rabbit embryo by electron microscopic histocheniistry. J. Neuropathol., 26, 136-1 37.

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TORACK,R. M. A N D BARRNETT, R. J. (1962) Fine structural localization of cholinesterase activity in the rat brain stem. Expll. Neiirol.. 6, 224-244. TOSCHI, G . (1959) A biochemical study of brain microscomes. Expll. Cell Res., 16, 232-255. ZAJICEK,J. AND ZEUTHEN, E. (1961) Quantitative determination by a special “ampulla-diver” of cholinesterase activity in individual cells, with notes on other uses of the method, in General Cy/ocheriiical Mrrhods. J. F. Danielli, Editor. New York, Academic Press, Vol. 2 (pp. 131-152)

DISCUSSION Monday afternoon MANDEL: I t seems to me that one of the explanations for your findings is that the synthesis of the compound occurs in the endoplasmic reticulum and then it moves to other places from there. This is the same with cytochrome oxidase. which is synthesized in the endoplasmic reticulum and then goes into the mitochondria. TENNYSON: Fukuda and Koelle (J.B.B.C. 5:433, 1959) postulated that the enzyme in the endoplasmic reticulum moves down the axon and is transferred to the surface. We think that it is likely that the acetylcholinesterase in the agranular reticulum does originate in the cell body and moves into the axon. However, we have not been able to demonstrate histochemically that the reticulum-bound enzyme is transferred directly to the axolemmal surface. For instance, the agranular reticulum containing active enzyme is not found closely approximated to the surface of the axon. Some mechanism other than direct transfer of active enzyme must be involved if the reticulum-bound enzyme is the source of the surface AChE. We think, instead, that the acetylcholinesterase in each site is part of a different intracellular system. Indeed there are some differences in its properties between the two sites. The surface enzyme is more sensitive to inhibition by eserine and fixatives. The reticulum-bound enzyme may be concerned with the functioning of the endoplasmic reticulum in general, whereas the surface enzyme may play a more specific role in nerve impulse conduction. ELLIOTT:Your enzyme could be attached first in onc place, then be in transit when you would not see it and then, again, be attached at another place where you can find it again. At this other place it may have very diffcrent properties, depending on what lipoprotein complexes it happens to be stuck on.You would just not observe theenzyme while it is in transit. TOWER:There is a little support for the point of view of Koelle since Edward Koenig in Buffalo has shown the presence of RNA in the axon itself.

TENNYSON: If there is RNA in the axon, why can’t it synthesize the enzyme at the periphery? Why does the enzyme in the active form have to come from the cell body? TOWER:You can also assume that you have an extension of endoplasmic reticulum right down the axon, which is what Waelsch and Koelle originally had in mind. In their experiments the results cannot be explained that way, but the possibility exists.

TENNYSON: You also have to consider that in sympathetic neurons catecholamines are thought to be liberated at the endings. There is, nevertheless, a larg: amount of AChE in the perikarya of these cells. CSAKY:It looks, from rccent cxperiments, as if acetylcholine may be the primary liberator of everything, including catecholamines. At least, Burns has published a number of papers on this subject. TENNYSON: I still think there is a considerable amount of acetylcholinesterase in the cell body which needs to be accounted for. I t could be there for intracellular ion exchange and other metabolic processes of the perikaryon itself. All this enzyme is not necessarily present in the cell body solely for transport down the axon.

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KOENIG:I would like to make a comment on the biosynthetic renewal of acetylcholinesterase. Actinomycin-D, an effective inhibitor of RNA synthesis, and secondarily protein synthesis, administered intraspinally in microgram quantities, produces a chronic neuron disease. A gradual depletion of intraneuronal and glial RNA results, together with a remarkable inhibition of protein synthesis. Acetylcholinesterase activity, demonstrated histochemically by the Koelle method, is rapidly lost from neuronal cytoplasm and from axoplasm, indicating that this enzyme turns over rapidly (Koenig, Lu, and Jacobson, Transactions Amer. Nerrrol. Ass'rt, 1967, in press).

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The Fine Structure of the Choroid Plexus: Adult and Developmental Stages V I R G I N I A M. T E N N Y S O N

AND

G E O R G E D. P A P P A S *

Departrneiit of Pathology, Divisiori of Neuropathology, College of Physiciuns and Surgeons, Columbia University New York, New York ( U . S . A . ) .

INTRODUCTION

The mechanism of formation and absorption of cerebrospinal fluid is probably the net result of many processes involving active transport, diffusion, and reabsorption. It is generally agreed that the choroid plexuses play a primary role in the production of cerebrospinal fluid. The entire lining of the ventricular system and subarachnoid space, however, including the ependyma, the pia-glial membranes, and the blood vessels in the subarachnoid space may also modify the composition of the fluid. Studies of the fine structure of the choroid plexus (Dempsey and Wislocki, 1955; van Breemen and Clemente, 1955; Millen and Rogers, 1956; Maxwell and Pease, 1956; Wislocki and Ladman, 1958; Case, 1959; Tennyson, 1960; and Tennyson and Pappas, 1961, 1964) reveal details which support the concept of this structure as a secretory organ. These studies have shown that the choroidal epithelial cells possess certain characteristics found in other cells concerned with fluid transport. The choroidal epithelium, the ciliary epithelium of the eye, the epithelium lining the striated duct of the salivary gland, and the epithelium of the proximal convoluted tubule of the kidney have extensive invaginations and foldings of their surfaces. Pease (1956) proposed that plication of cell membranes provides a greater surface area where transport phenomena could occur. The choroid plexuses from the lateral and fourth ventricle of adult, newborn and embryonic rabbits, from day 14 to day 20 of gestation, were fixed by injection into the ventricle of 2 % buffered osmium tetroxide (Palade, 1952) or 6.25 % buffered glutaraldehyde (Sabatini et al., 1963) followed by osmium post-fixation. In addition specimens from six human embryos, ranging from 8 to 12 weeks were studied. The specimens were then treated according to routine electron microscopic procedures. I. A D U L T RABBIT CHOROID PLEXUS

The adult choroid plexus (Fig. 1) is formed of a single-layered cuboidal epithelium

* Present address: Department K c f k v m s p. 83-85

of Anatomy, Albert Einstein College of Medicine, New York.

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which surrounds a highly vascular connective tissue core. The vesicular nuclei (N) of the choroidal epithelial cells are centrally located in the cytoplasm. A brush border consisting of multiple thin polypoid projections of the cell membrane, the irregular microvilli (MV), is present on the luminal surface. Cilia may or may not be present. Small vesicles, about 300 to 700 A in diameter (Figs. 1 and 2 V) are found close to the surface and throughout the cytoplasm of the cells. Some vesicles, which exhibit a radially striated coating (Fig. 3, V), are formed by the process of pinocytosis in which inpocketings of the cell membrane pinch off to form free vesicles in the cytoplasm. This can be demonstrated by injecting a marker particle, such as thorium dioxide (Tennyson and Pappas, 1961) or ferritin (Brightman, 1965, 1968) into the cerebrospinal fluid. These particles adhere to the microvilli and are then found within vesicles beneath the cell surface. Current theories concerning cerebrospinal fluid dynamics favor a bidirectional movement of substances across the linings of the ventricular system. The normal occurrence of these vesicles in choroidal epithelial cells suggests that pinocytosis by microvesicles (Palade, 1953) may be one of the mechanisms whereby some substances move through the choroid plexus. Adjacent choroidal epithelial cells are attached apically by a specialized junction (Fig. 1, J), which is characterized by a closer approximation and increased density of the juxtaluminal lateral membranes (Brightman, 1968). These junctions closely resemble the zonula occludens or tight junction in other cells, which is considered to be a seal or diffusion barrier (Farquhar and Palade, 1963). Brightman (1968) showed that the apical junction in the choroid plexus of the mouse was impervious to the passage of peroxidase injected into the cerebrospinal fluid. Beneath the apical junction (Fig. 2) the lateral cell membranes are closely apposed and relatively straight except near their bases. Frequently flattened cisternae of the endoplasmic reticulum (arrows) are directly applied to the inner surface of opposing membranes. This formation closely resembles the subsurface cisternae of neurons and other excitable cells, which Rosenbluth (1962) has suggested may modify the properties of the surface membrane. Subsurface cisternae have also been reported to occur in register with corresponding cisternae in adjacent epiphyseal cells (Wolfe, 1965). In the basal region a complex infolding of portions of contiguous cells occurs (Figs. I and 3, I). These membrane elaborations greatly increase the area of the cell surface which is available for the exchange of substances. Adenosine triphosphatase activity has been localized in these infoldings (Torack and Barrnett, 1964; Yasuzumi and Tsubo, 1966) in combined electron microscopic-histochemical studies. The localization] of this enzyme is pertinent to the discussion of secretion by the choroid plexus.

Fig. I . The cubical choroidal epithelial cells of an adult rabbit, have rounded centrally located nuclei (N). The luminal surface is elaborated into multiple polypoid microvilli (MV). Adjacent cell membranes are closely apposed at the apex (J). The lateral surfaces of the cells are relatively straight except near the base where extensive folds and interdigitations ( I ) occur. The basal surface is relatively straight or shows slight undulations. The cytoplasm contains a Golgi complex (G),short cisternal or tubular elements of the endoplasmic reticulum (ER), numerous mitochondria (M), vesicles (V) and dense bodies (B) with a heterogeneous content. A basement membrane (BM) separates the epithelium from the connective tissue (CT). x 9000. ReJiwvrws p. 83-85

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A significant quantity of a sodium-potassium activated adenosinetriphosphatase,

which is presumed to play a role in active cation transport, has been found by biochemical assay in the choroid plexus (Bonting et ul., 1961). The basal surface of the choroidal epithelial cell is relatively straight or slightly undulated. It may be interrupted occasionally by inpocketings of the cell membrane (Fig. 3, V, and arrow). The cells rest on a basement membrane (BM), which has the usual electron lucent zone and moderately dense filamentous components. It continues beneath the junction of the cells without entering into the lateral intercellular space. The basement membrane of the adult choroidal epithelium has been considered at least a partial barrier to certain substances, such as silver particles (Dempsey and Wislocki, 1955) and thorium dioxide particles (Tennyson and Pappas, 1960), but not to peroxidase (Brightman, 1968). The choroidal epithelial cells typically contain large numbers of mitochondria (Figs. I and 3 M), which are probably the source of the high respiratory metabolism determined biochemically in the choroid plexus (Krebs and Rosenhagen, I93 1 ; Fisher and Copenhaver, 1959). These organelles could readily s~ipplythe energy utilized in active transport processes. The Golgi complex (Fig. I , G ) ,a system of vacuoles, vesicles and flattened sacs, generally occupies a para- or supra-nuclear position in the cell. Although this organelle is prominent in choroidal epithelium, its function in this cell is not known. The endoplasmic reticulum in adult choroidal epithelial cells is usually scattered randomly throughout the cytoplasm as rough surfaced tubuli and canaliculi, although cisternal elements (Fig. 2, ER) may be found. The choroid plexus contains a varying number of single membrane-bounded dense bodies (Fig. I , B), which have a moderately dense granular matrix, and often a heterogeneous content. They are usually considered to be lysosomes, because similar bodies in other cells exhibit acid phosphatase activity (Novikoff, 1961). These bodies incorporate foreign particles which have gained access to the cell following experimental procedures. Silver particles (Wislocki and Ladman, 1958), hemosiderin granules (Case, 1959) and thorium dioxide particles (Tennyson and Pappas, 1961) have been reported in these bodies. Lipid droplets and pigment bodies are common. Large vacuoles, often reported in light microscopic studies as evidence for secretory activity, probably represent artificial swelling of organelles or dissolved lipid inclusions. The capillaries of the connective tissue core of the choroid plexus often have an unusually large diameter lumen compared to thinness of the endothelial wall. Fig. 2. The apical portion of adjacent adult choroidal epithelial cells shows microvilli (MV), vesicles (V). and flattened cisternae (arrows) closely apposed to the lateral membranes of adjacent cells. x 33000. Fig. 3. An elaborate basal interdigitation (1) between adjncent adult choroidal epithelial cells. Coated pinocytotic vesicles enter the cells from the space between the interdigitations (arrows) as well as from the base of the cell (V). Numerous mitochondria (M) are present in thc cytoplasm. Pores are present in the nuclear envelope (NE). A moderately thick filamentous basement membrane (BM) separates the epithelium from the connective tissue area, which contains collagen fibers (C). . 21 500. Fig. 4. The attenuated capillary endothelium frequently contains pores (arroHs). A moderately dense basement membrane (BM) surrounds the capillaries. Cross sections of collagen fibers (C) are present. x 82 000.

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Organelles, such as mitochondria, vesicles, and a small amount of endoplasmic reticulum are present predominantly in the thicker perinuclear areas oftheendothelial cell. The Golgi complex is usually located between the nucleus and the capillary lumen. Vesicles are present throughout the endothelial cytoplasm except in the most attenuated areas, which may be narrower in diameter than the vesicles. Frequently "pores'' or fenestrae covered by a thin diaphragm (Fig. 4,arrows) are present in these areas. A basement membrane (BM) surrounds the capillaries even i n its most attenuated portions. 11. D E V E L O P M E N T O F T H E R A B B I T C'HOROID P L E X U S

The choroid plexus in most areas forms as invaginations into theventricle of a thin vascularized roof plate, the choroid epithelial lamina. The anterior portion of the telencephalic choroid plexus, however, arises from a thicker, more stratified roof plate in the angle between the paraphyseal arch and the medial wall of the hemisphere (Bailey, 1916). This area, therefore, undergoes an additional stage of development i n which a pseudostratified epithelium is transformed intoa thinner columnar type, similar to the choroid epithelial lamina present in other areas. Recent light microscopic studies have appeared on the histogenesis of the choroid plexus (Kappers, 1958; Birge, 1962; Strong, 1964; Shuangshoti and Netsky, 1966). Kappers (1958) has divided the histogenetic development of the human telencephalic choroid plexus into three stages. In the first phase at approximately 6 weeks, the choroid plexus is represented by a simple fold into the lateral ventricle of the neural tube consisting of pseudostratified epithelium. The mesenchymatous stroma is filled with developing blood cells, and strands of angioblasts formingcapillaries. The second phase appears at about 8 weeks when the plexiis becomes lobular and increases i n size until it occupies almost the entire ventricle. The epithelium now consists of a single layer of low columnar cells containing glycogen. The stroma appears spongy and contains gelatinous connective tissue with few cells. During the third period of development when the fetus is in its fourth month, the choroid plexus decreases i n volume with respect to the ventricle. The amorphous ground substance of the stroma decreases, and fibrillar connective tissue appears. Most of the epithelial cells still contain glycogen, but some cuboidal cells closely resembling the mature epithelium have appeared.

Fig. 5. Pseudostratified and tall columnar epithelium of the telencephalic choroid plexus of a rabbit during day 15 of gestation. The nuclei (N) are usually elongated and often indented. Short irregular microvilli (MV) are present on the luminal surface. The lateral surface between the apical junction ( J ) and the base of the cells (arrows) is relatively straight. Mitochondria (M) are present throughout the cytoplasm. The endoplasmic reticulum (ER) and Golgi complex ( G )are located predominantly in the apical cytoplasm. Clusters of ribonucleoprotein particles (R)fill the basal cytoplasm. A thin basement membrane (BM) separates the epithelium from the connective tissue space, which contains capillaries, but very little collagen. x 8000. (Inset) A high magnification illustrating an apical junction ofchoroidal epithelial cells from a rabbit during day 16 of gestation. Just beneath the lumen the adjacent cell membranes are denser and more closely apposed to one another than elsewhcre. A material of moderate density is present on the cytoplasmic surface of the membranes. The intercellular space widens beneath the junction. x 33 500.

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I n an electron microscopic study of the development of the choroid plexus in the rabbit similar developmental stages were found (Tennyson and Pappas, 1964). These stages have been classified on the basis ofthe morphology ofthechoroidal epithelial cell. Stage I - Pseudostratijied epithelium

In the initial stage of the development of the telencephalic choroid plexus the epithelium consists of a pseudostratified layer of two or three cells in thickness (Fig. 5). The nuclei (N) are large and elongated and contain multiple polymorphous nucleoli. The apical surface is formed of irregular microvilli (MV), bulbous protrusions, and occasional cilia. The apical junction (J, and inset) has closely apposed dense membranes even at this early stage of development. Below the junction the cell membranes are relatively straight, even at the base (arrows), exhibiting very little of the complexity or folding characteristic of adult choroidal epithelium. The base of the cell is straight and rests on a delicate basement membrane (BM), which is thinner and less dense than that of the adult. The Golgi complex (G),which consists of flattened sacs and vesicles lies in a plane perpendicular to the lumen in the supranuclear cytoplasm. The larger vacuolar component of the complex found in the adult is usually not present in the early embryo. Rough surfaced endoplasmic reticulum (ER) is found in the immature cells, particularly in the apical region. The particulate component of the endoplasmic reticulum, the ribosome (R), is abundant throughout the cytoplasm and occupies most of the finely fibrillar ground substance of the basal cytoplasm. Mitochondria (M) are present throughout the cytoplasm. It is unlikely that these pseudostratified cells are functioning in the same manner as in later embryonic periods or in the adult because of the simplified arrangement of their surfaces and the different distribution oforganelles. Woodbury (1968) has shown quantitative differences in the ionic composition of embryonic cerebrospinal fluid, as compared to the adult. Stage JJ - Granular columnar epithelium As the telencephalic choroid plexus undergoes further development the pseudo-

stratified layer becomes a single layer of granular columnar epithelial cells, which is typical of the choroidal epithelial lamina in other areas. The myelencephalic and telencephalic choroid plexuses develop multiple villus folds, and then both undergo the same changes in fine structure from this period until the mature plexuses are formed. Fig. 6. The apical portion of the choroidal epithelial cells from the fourth ventricle of a rabbit during day 16 of gestation. The irregular microvilli (MV) may have been formed by partitioning of bulbous protrusions by coalescence of rows of vesicles (arrow). Finely fibrillar material is present in the cytuplasm near the narrow apical junctions (J). Rows of cisternae of the endoplasmic reticulum (ER) are prominent. Small aggregates of dense glycogen particles (GL), which begin to appear at this time in the ground substance are readily distinguishable from thesmaller less dense ribonucleoprotein particles attached to the cisternae. x 31 000. Fig. 7. Large accumulations of glycogen particles (GL) are present in the basal portion of many of the choroidal cells from the same specimen as in Fig. 6. The lateral surfaces of these cells show incipient infolding (I). A thin basement membrane (BM) is evident beneath the epithelium, but is not distinct around the blood vessel (BV). x 30 000.

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The surface elaborations become more complex during this period, but they are not as fully developed as in the adult. Increased numbers of microvilli (Fig. 6 , MV) appear on the apical surface, but they are not as long and do not have the regular polypoid formation seen in the adult. In some areas bulbous protrusions (arrow) containing chains of vesicles are present on the surface. The irregular microvilli may form by coalescence ofthesevesicles, thereby partitioning the protrusion into multiple processes. lnfoldings and interdigitations (Fig. 7, 1) appear between adjacent cells at their bases. The base of the cell is often highly irregular. The basement membrane (BM) is somewhat more distinct than in the earlier period but is not as prominent as in the adult. The number of organelles increases during this period, particularly the apical cisternae of the endoplasmic reticulum (Fig. 6, ER), although these may now be found anywhere in the cytoplasm. Rosettes of ribonucleoprotein particles are numerous as before, but now small aggregates of glycogen (GL) appear in the finely granular ground substance among the ribonucleoprotein particles. Glycogen continues to increase in the cell during this stage filling large areas of cytoplasm, particularly in the basal portion of the cell (Fig. 7, GL). The formation of microvilli and infolded basal surfaces are pertinent to the question of the onset of secretion of cerebrospinal fluid, since these surface elaborations are thought to be important for secretion in the adult. Of interest in this respect is the work of Weed (1917), who studied the formation of cerebrospinal fluid with the dye, Prussian blue. He noted that the first escape of fluid into the subarachnoid space corresponded to the formation of villus tufts of the choroid plexus in the fourth ventricle. Weed interpreted this to indicate the onset of secretion of cerebrospinal fluid in the embryo. Stage 111 - Glycogen-laden epithelium

The choroid plexus has increased in size and fills most of the lateral ventricle of the fetus. The cytoplasm of the epithelium becomes engorged with glycogen (Figs. 8 and 10, GL) distending its surface complexities and displacing organelles toward the periphery of the cell. Only a few short microvilli (MB) remain on the apex of the cell. The basal infoldings have become less complex. The nucleus (Fig. 8, N) is usually indented and eccentrically located in the cell. The glycogen remains in the choroidal epithelium of the rabbit until birth. It is depleted within the first two weeks after birth and then the cells assume mature characteristics (Tennyson and Pappas, 1961).

Fig. 8. Telencephalic choroidal epithelial cells during day 20 of gestation. The indented nucleus ( N ) and most of the cytoplasmic organelles (M) have been displaced to the apical region or periphery by the large accumulation of glycogen (GL). The apical membrane is relatively simple, having only a few short microvilli (MV). The lateral membrane is straight from the apical junction (J) to the base. Thorium dioxide particles, which had been injected intravascularly are present in the intercellular space (arrow) and in vesicles and vacuoles (V) within the cytoplasm. x 7500. (Inset) A higher magnification illustrating the marker particles within the cytoplasmic vacuole. x 24 500. Fig. 9. A higher magnification of the basal area in Fig. 8, illustrating the presence of marker particles in the intercellular space (arrow) and in vesicles (V) within the cytoplasm. x 20500.

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FINE STRUCTURE OF CHOROID PLEXUS

Glycogen has previously been described in immature choroidal epithelium (Loeper, 1904; Goldmann, 1913; Askanazy, 1914; Weed, 1917; Sundberg, 1924; Kappers, 1958). The functional significance of the glycogen content of fetal choroidal epithelium is not known, but several possibilities were suggested by Kappers (1958): ( I ) Glycogen might be related to the synthesis of mucoproteins of the basement membranes and ground substance. (2) Glycogen might be utilized in the transformation of thegelatinous stroma into fibrous connective tissue. (3) Glycogen, a source of energy under anaerobic conditions, might have a nutritive function. Glycogen could be converted to glucose and then diffuse through the cerebrospinal fluid to be taken up by ependymal cells. This theory is supported by studies of species which hibernate during the winter (Oksche, 1958). Glycogen disappears from the choroid plexus during hibernation, and can be found within ependymal cells and their processes in the neuropil. Additional evidence in support of the nutritive theory is obtained from the finding of greater concentrations of glucose in the cerebrospinal fluid of premature infants than in older children or adults (Otila, 1948). The reaction of embryonic choroidal epithelial cells to experimentally introduced thorium dioxide particles was tested. Marker particles introduced into the cerebrospinal fluid attach to the microvilli (Fig. 10, MV), then are incorporated intothecytoplasm in pinocytotic vesicles (V). The particles are then segregated into multivesicular bodies (B). In agreement with the findings of Brightman (1965, 1968), marker particles are not found within the apical tight junction (J), but may gain access to the intercellular space by the discharge of particles from pinocytotic vesicles (arrow). Thorium dioxide injected intravascularly caused considerable damage and rupture of the delicate embryonic choroidal capillaries, with subsequent release of marker particles into the interstitial space (Figs. 8, 9). These particles were found in vesicles (Fig. 9, V) and vacuoles (Fig. 8, V and inset) in the cytoplasm, and also in the intercellular space (Figs. 8, 9, arrow). Interstitial area

The fine structure of the capillaries and of the interstitial tissue differs very little from embryonic to fetal stages. The increase in the gelatinous ground substance (Kappers, 1958) is reflected in the increased width of the connective tissue area, but the ground substance itself is not apparent in these preparations. The capillaries usually present an irregular sinusoidal appearance and are often filled with immature blood cells. The endothelial walls are extremely thin, and a few “pores” similar to those in the adult may be seen during the latter part of Stage 11. The basement membrane surrounding the capillaries is thinner and less dense than that of the adult, and may even be absent (Fig. 7) during early embryonic periods. Although some finely fibrillar material may be ~~~

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Fig. 10. The apical portion of a telencephalic choroid plexus from an embryo during day 20 of gestation. Thorium dioxide particles, which had been injected into the cerebrospinal fluid, adhere to the microvilli (‘MV) and are incorporated into vesicles (V) within the cytoplasm. The particles are then segregated into multi-vesicular bodies (B) and dense bodies. Marker particles were not found within the narrow apical junction (J). A vesicle (arrow) appears to be discharging a particle into the intercellular space beneath the junction. Glycogen‘particles (GL) have accumulated in the cytoplasm. x 3 1 000. HrJi~rrnrrsp . 83 3 5

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found in the connective tissue area, very little mature collagen is present. Scattered fibroblasts and phagocytes may be found in the Connective tissue area. 111. H U M A N E M B R Y O N I C C H O R O I D P L E X U S

The specimens studied so far in the human embryo correspond very closely to Stage I I or Stage 111 of the rabbit, with some variations. Apically the cells have irregular microvilli (Figs. I I and 12, MV) and tight junctions (J) between adjacent cells. Organelles such B S mitochondria (M), endoplasmic reticulum (ER), and ribosomes are numerous, particularly i n the apical cytoplasm. Depending on the stage of differentiation, small or large accumulations of glycogen (Figs. I 1 and 13, GL) are present. Some cells within the human fetal choroidal epithelium differ from the others by having a considerable number of filaments and microtubules (Figs. I I , 13 and 14, T) longitudinally oriented in their cytoplasm. Areas along their lateral surfaces and those of the adjacent cell are characterized by increased density of the cell membranes and subjacent cytoplasm (Figs. I I , boxed area and inset; 13, arrows). Since the intercellular space is not narrowed, these areas more closely resemble fascia adhaerens (Farquhar and Palade, 1963; Richardson, 1964), rather than fascia occludens, as is found i n the apical junction. The basal cytoplasm of these cells (Fig. 13, Cell 11) may form irregular extensions partially surrounding other cytoplasmic processes (P). Collections of mitochondria (Figs. 1 I , 13 and 14 M), and an assortment of bodies (DB) and vesicles (Fig. 14, V) of varying density are found in the basal cytoplasmic extensions. Agranular tubular profiles (Fig. 14, arrow) are often present. The dense bodies are usually spheroidal and range in diameter from 0.1 to 0.3 p, however, elongate and dumbbell shapes also occur. The bodies are membrane-bounded, and are separated from the dense granular matrix by a light zone. In addition to the dense bodies, there is a heterogeneous assortment of clear vesicles, coated vesicles, and vesicles with a central core (V). The latter vesicles are approximately I300 A to 1500 A in diameter with pale or moderately dense central material. The nature of these bodies and vesicles is not known, but the large dense bodies resemble a heterogeneous group of structures classified as lysosomes commonly seen in all cells. Usually, however, lysosomes are randomly dispersed in the cytoplasm, or else are located in the perinuclear Golgi zone, rather than grouped together in the basal cytoplasm as seen here. The vesicles with a core of variable density are similar to but larger than the vesicles found among the Fig. 11. Tall columnar choroidal epithelial cells from the fourth ventricle of a 12 week gestation human fetus clos-ly resemble the cells in Stsge I I of the rabbit. The nuclei (N)are indented, and cilia ( C ) and irrcgulir pdypoid microvilli ( M V ) are present on the apical surface. A tight junction (J) joins the Juxtalurnin~lsurface of adjacent cells. Mitochondria, endoplasmic reticulum (ER)and aggregates of glycogen psrticles (GL) are present throughout the cytoplasm. A pxtion of one cell differsfromthe others in t h n t it has large numbers of filaments and microtubules (T) throuehout the cytoplasm. Collections of mitochondria (M), dense bodies (DB) and vesicles are present in the b x d region. These cells often form intermediate type junctions (boxed area) with the adjacent cell. Thc process (P),which has finely filamentous cytoplasm and a paucity of organelles, diffcrs from the adjacent epithelial cells. i9000. (Inset) A serial section near the boxed area s h o w several region5 o f increised density of thc cell merllbrine and subjocent cytoplasm. Unlike the apical tight junction, the intercellular cleft is the same or slightly larger than in other areas. x 49 000.

FINE STRUCTURE OF CHOROID P L E X U S

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typical dense core vesicles present in adrenergic nerve endings (Grillo and Palay, 1962; Richardson, 1964). It is interesting to note that adjacent to the basal extension containing the dense bodies and pale core vesicles there is sometimes a large intercellular process (Figs. 1 I and 13, p). These processes show some evidence of membrane specialization with the adjacent epithelial cells (Fig. 13, boxed area and inset). Serial sections of these profiles have shown that they are part of an elongated process, and not just an oblique section of choroidal epithelium. The finely filamentous character of their cytoplasm and the absence of ribonucleoprotein particles from section to section differs from the cytoplasm of the surrounding epithelial cells. These profiles may represent sections through a nerve fiber, since there is evidence from light microscopic studies using silver impregnations that intra-epithelial nerve endings are present in the choroid plexus of the human fetus (Clark, 1934), as well as in other species (Junet, 1926; Clark, 1928, 1934; Tusker, 1947). Clark (1928) reported that 4 or 5 large nerve fibers pass from the region of the cranial extension of the nucleus cuneatus through the tela choroidea to the medial tufts of the choroid plexus of the fourth ventricle. These fibers ended within the choroidal epithelium and were quite distinct from those found in the connective tissue i n relation to blood vessels. Junet (1926) hypothesized that intraepithelial nerve endings could be sensitive to the variations in the constituents of the cerebrospinal fluid or of the pressure of the fluid. Interstitial area

The interstitial area of the human choroid plexus is similar in many respects to that of the rabbit. The capillaries have a large lumen with moderately thin to very attenuated walls. Pores are seen from time to time in the embryo. There is usually a perivascular cuff of connective tissue fibers (Fig. 15, C) around the blood vessels (BV), and also a larger number of fibers just beneath the epithelium than those found in the embryonic rabbit. As in the rabbit, however, large spaces (*) lacking fibers are present, which in I ~ I W probably contain gelatinous ground substance (Kappers, 1958). A large number of interstitial cells extend long interlacing processes into this space. These cells have large vacuoles (V), in which is contained a flocculant material similar to that seen in some extracellular areas. These cells may be forming some of t b materials i;. the ground substance. Kappers (1958) noted in a light microscopic study the large number of angioblasts and hemocytoblasts in the stroma of the human embryonic choroid plexus. He suggested that the interstitial area of the embryonic choroid plexus might be concerned with the production of blood cells during early development. In the present electron microscopic study the immature blood cells were found predominantly in the Fig. 12. The apical region of choroidal epithelial cells from the fourth ventricle of a 12 week gestation human fetus. The nuclei ( N ) are indented. Occasional cilia ( C ) are interspersed between irregular microvilli (MV). Vesicles and cytoplasmic particles are present in many bulbous processes (arrow). At the apical region one o r more areas ( J ) appear to from tight junctions. Mitochondria (M),vesicles (V) and cisterndl elements of thc endoplasmic reticulum (ER) are common in the apical cytoplasm. x 17 500. R t f e ~V I I ( vs p. 8.Z-8.5

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capillary lumen rather than in the connective tissue area. There wasadistinct tendency, however, for the human to exhibit more extravascular immature cells than the rabbit. Whether these cells are developing in the interstitial area, or have merely migrated there from the blood vessels cannot be determined in this study. Since this tissue was not perfused, cells might have entered the interstitial tissue artifactually during the preparatory procedures. It is likely, however, that many of the cells that appear extravascular in light microscopic studies are actually intravascular. The sheath surrounding the blood cells may not be apparent in light microscopy, because of the thinness of the endothelium and the lack of a well defined basement membrane. The presence of large numbers of immature blood cells may merely reflect the circulation through the vessels, rather than be evidence of a blood forming organ. SUMMARY

The fine structure of the choroid plexus of the adult rabbit, and of embryos from the rabbit and human has been described. The mature choroidal epithelium is characterized by having numerous mitochondria and extensive elaborations of cell surface membranes, such as apical polypoid microvilli and highly infolded lateral surfaces near the base of adjacent cells. Elaborated membranes are commonly found in cells involved in fluid transport. The cells of the first anlage of the telencephalic choroid plexus of the rabbit embryo are pseudostratified and have relatively simple surfaces, although a few bulbous apical processes and irregular microvilli are present. Most of the membraneous cell organelles are present in the apical cytoplasm, whereas the base contains predominantly ribonucleoprotein particles. With further development the microvilli elongate and the basal surfaces of adjacent cells show incipient folding. The organelles have increased in number and have become more randomly dispersed in the cytoplasm. Glycogen particles appear randomly, and later accumulate in such quantity that they fill much of the cytoplasm, distending the cell and flattening out the membrane specializations. Experimentally administered thorium dioxide particles enter into the apical portion of adult and embryonic choroidal epithelial cells by pinocytosis from the cerebrospinal fluid. lntravascularly injected thorium dioxide particles enter into the interstitial area, but usually encounter a barrier at the epithelial basement membrane in the adult. In the embryo, however, marker particles which have gained access to the interstitial area, are present in vesicles in the cytoplasm, and may also be found in the intercellular space between choroidal epithelial cells. Fig. 13. A large process (P), which may be a cross section of a nerve fiber is present in the intercellular space between choroidal epithelial cells from the fourth ventricle of a human fetus. The process contains mitochondria ( M ) and some aggregates of glycogen particles (GL) in its finely filamentous ground substance. A cytoplasmic extension of Cell 11, which partially surrounds the process, contains mitochondria ( M ) , large and small dense bodies (DB), and microtubules (T) in addition to the usual ribonucleoprotein and glycogen particles (GL). Areas of increased density of the membrane and subjacent cytoplasm occur between Cell 1 and I 1 (arrows), and between the process (P)and Cell I1 (boxed area). A 16 500. (Inset) A serial section of the boxed area illustrates filamentous material oriented obliquely to the dense membrane attachment. x 46 500. Rvfirencru p. 83-85

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The human embryonic choroid plexus shows similar immature membrane specializations, organelles, and glycogen accumulations, as in the rabbit. In addition some epithelial cells have numerous filaments and microtubules, as well as small and large dense bodies and vesicles containing a pale core in the basal cytoplasm. These cells form specialized junctions along the lateral surface with adjacent choroidal epithelial cells and with processes which may represent profiles of inter-epithelial nerve fibers. ACKNOWLEDGEMENTS

This work has been supported by the Clinical Research Center for Parkinson’s and Allied Diseases NB05184 and the Parkinson Information Center, a part of the National Information Network of NINDB under contract no. PH 43 64 54, and 5-TI-NB5062, HE-5906, N B 03448, and United Cerebral Palsy Research and Educational Foundation, R-216-67 and R-190-65. The authors wish to express their gratitude to Miss Mary Budininkas for skilful technical assistance and to Mr. Moshe Rosen for the electronic management of the microscope. REFERENCES ASKANAZY, M. (1914) Zur Physiologie und Pathologie des Plexus chorioidei. Zentr. allgem. Pathol. palhol. Anat., 25, 390-391. BAILEY, P. (1016) Morphology of the roof plate of the forebrain and the lateral choroid plexus in the human embryo. J . Comp. Neurol., 26, 79-120. BIRGE,W. J. (1962) Induced choroid plexus development in the chick mesencephalon. J . Comp. Neurol., 118, 89-96. BOWING,S. L., SIMON,K . A. A N D HAWKINS, N. M. (1961) Studies on sodium-potassium activated adenosine triphosphatase. Arch. Eiochern. Biophys., 95, 416423. BRIGHTMAN, M. W. (1965) The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. I. Ependymal distribution. J . Cell Eiol., 26, 99-123. - (1968) Intra-cerebral movement of proteins injected into the blood and cerebrospinal fluid. (this volume). CASE,N. M. i1959) Hemosiderin granules in the choroid plexus. J. Biophys. Biochem. Cytol., 6, 527-530.

CLARK, S. L. (1928) Nerve endings in the choroid plexus of the fourth ventricle. J. Comp. Neurol., 47, 1-21. -- (1934) Innervation of the choroid plexuses and the blood vessels within the central nervous system. J . Comp. Neurol., 60,21-35. DEMPSEY, E. W. A N D WISLOCKI, G. B. (1955) An electron microscopic study ofthe blood-brain barrier in the tat, employing silver nitrate as a vital stain. J . Biophys. Eiochem. Cyrol., 1,245-256. FARQUHAR, M. G. AND PALAVE,G. E. (1963) Junctional complexes in various epithelia. J . CeN Eiol.. 17. 375412.

Fig. 14. A higher magnification of the basal portion of a cell in Fig. 11 showing details of the mitochondria (M), dense bodies (DB), clear and coated vesicles, and vesicles with a core of moderate density (V). Agranular tubular profiles (arrow), and microtubules (T) are present. A basement membrane (BM) is present between the cell and the interstitial area. x 33 000. Fig. 15. The gelatinous interstitial area of the myelencephalic choroid plexus of a 12 week gestation human fetus. Connective tissue fibers (C) are concentrated around the walls of the capillaries (BV). Few fibers are found in the wide intercellular spaces (*I. The interstitial cells have long thin interlacing processes giving the interstitial area a “honey-comb” appearance. Large vacuoles (V) containing a flocculant material are present in these cells. X 5000.

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FISHER,R. G. AND COPENHAVER, J. H. (1959) The metabolic activity of the choroid plexus. J. Neurosurg., 16, 167-176. GOLDMANN, E. (1913) Vitalfarbungam Zentralnervensystem (Beitrag zur Physio-pathologie des Plexus choroideus und der Hirnhaute. Abhandl. preuss. Akad. Wissensch., Phys.-Matheni. KI., 1, 1-60. S. L. (1962) Granule-containing vesicles in the autonomic nervous system. GRILLO,M. A. AND PALAY, In: Electron Microscopy, 5th International Congress for Electron Microscopy, S. S . Breese (Ed.) Vol. 2, U-1, Academic Press, New York. JUNET,W. (1926) Terminaisons nerveuses intrakpitheliales dans les plexus chorioides de la souris. C . R . SOC.Bid., 95, 1397-1398. J. ARIENS(1958) Structural and functional changes in the telencephalic choroid plexus KAPPERS, during human ontogenesis. Ciba Found. Symp. Cerebrospinal Fluid. G. E. W. Wolstenholme and C. M. O'Connor (Ed.). Boston, Little Brown & Co. (pp. 3-31). KREBS,H. A. AND ROSENHAGEN, H. (1931) Uber den Stoffwechsel des Plexus chorioideus. Z . ges. Neurol. Psychiat., 134, 64348. LOEPER, M. M. (1904) Sur quelques points de I'histologie normaleet pathologiquedesplexuschoroides de I'homme. C . R. SOC.Biol., 56, 1010-1012. MAXWELL, D. S. AND PEASE,D. c. (1956) The electron microscopy of the choroid plexus. J. Biophys. Biochem. Cytol., 2,467476. MILLEN,5. W. AND ROGERS,G . E. (1956) An electron microscopic study of the choroid plexus in the rabbit. J. Biophys. Biochem. Cytol., 2, 207-215. NOVIKOFF, A. B. (1961) Lysosomes and related particles, in: The Cell. II. Cells and their Coniponetrt Parts, J. Brachet and A. E. Mirsky (Eds.). Academic Press, New York. OKSCHE,A. (1958) Histologische Untersuchungen iiber die Bedeutung des Ependyms, der Glia und der Plexus chorioidei fur den Kohlenhydratstoffwechsel des ZNS. Z. Zellforsch. mikrosc. Anat., 48,74129. OTILA,E. (1948) Studies on the cerebrospinal fluid in premature infants. Acta paediat. (Uppsala), 35, 1-100, suppl. PALADE, G. E. (1952) A study of fixation for electron microscopy. J. Exptl. Med., 95, 285-298. -, (1953) Fine structure of blood capillaries. J . Appl. Phys., 24, 1424, abs. PAPPAS,G. D. AND TENNYSON, V. M. (1962) An electron microscopic study of the passage of colloidal particles from the blood vessels of the ciliary processes and choroid plexus of the rabbit, J. Cell Biol., 15, 227-239. PEASE,D. C. (1956) Infolded basal plasma membranes found in epithelia noted for their water transport. J. Biophys. Biochem. Cytol., 2,203-208, suppl. RICHARDSON, K. C. (1964) The fine structure of the albino rabbit iris with special reference to the identification of adrenergic and cholinergic nerves and nerve endings in the intrinsic muscles. Amer. J. Anat., 114, 173-205. ROSENBLUTH, J. (1962) Subsurface cisterns and their relationships to the neuronal plasma membrane. J. Cell Biol., 13, 405-421. SABATINI,D. D., BENCH,K. AND BARRNETT, R. J. (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17, 19-58. SHUANGSHOTI, S. AND NETSKY,M. G. (1966) Histogenesis of choroid plexus in man. Amer. J . Anat., 118,283-316. STRONG,L. H. (1956) Early development of the ependyma and vascular pattern of the fourth ventricular choroid plexus in the rabbit. Amer. J. Anat., 99,249-290. - (1964) The vascular and ependymal development of the early stages of the tela choroidea of the lateral ventricle of the mammal. J. Morphol., 114, 59-82. SUNDBERG, C. (1924) Das Glykogen in menschlichen Embryonen von 15, 27 und 40 m.m. Z. ges. Anat., 73, 168-246. TENNYSON, V. M. (I960) Electron microscope studies in the developing telencephalic choroid plexus in normal and experimentally induced hydrocephalus. Thesis, Columbia University, New York. TENNYSON, V. M. AND PAPPAS,G. D. (1961) Electron microscope studies of the developing telencephalic choroid plexus in normal and hydrocephalic rabbits. Disorders of the Developing Nervous System. W. Fields and M. Desmond (Eds.). Springfield, Charles C. Thomas. - (1964) Fine structure of the developing telencephalic and myelencephalic choroid plexus in the rabbit. J. Comp. Neurol., 123, 379412. 'IORACK, R . L .AND BARRNETT, R. J. (1964) The fine structural localization of nucleoside phosphatase activity in the blood-brain barrier. J. Neuropathol. Exptl. Neurol., 2 3 , 4 6 5 9 .

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TSUKER, M. (1947) Innervation of the choroid plexus. Arch. Neurol. Psychiar., (Chicago), 58,474483. V A N BREEMEN, V. L. A N D CLEMENTE, C . D. (1955) Silver deposition in the central nervous system and the hematoencephalic barrier studied with the electron microscope. J. Biophys. Biocheni. Cytol., 1, 161-166. WEED.L. H. (1917) The development of the cerebrospinal spaces in the pig and man. Conrrib. Embryo/., No. 14, Carnegie Itist. Wash., No. 225. WIsLocKi, G. 9. AND LADMAN, A. J. (1958) The fine structure of the mammalian choroid plexus. Ciba Found. Syuip. Cerebrospinal Fluid, G . E. W. Wolstenholme and C. M. O’Connor (Eds.). Boston, Little Brown & Co. WOLFE,D. E. (1965) The epiphyseal cell: an electron-microscopic study of its intercellular relationships and intracellular morphology in the pineal body of the albino rat. In: Progr. Brain Res., 10, J. Ariens Kappers and J. P. Schade (Eds.). Elsevier, Amsterdam. D. M. (1968) (this volume) WOODBURY, YASUZUMI, G. AND TSUBO,I. (1966) The fine structure of nuclei as revealed by electron microscopy. I l l . Adenosine triphosphatase activity in the pores of nuclear envelope of mouse choroid plexus epithelial cells. Exptl. Cell Res., 43, 281-292.

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Transport and Effects of Cationic Dyes and Tetrazolium Salts in the Central Nervous System* H A R O L D KOENIG Neurology Service, VA Research Hospital, arid Departnient of Neurology arid Psychiatry, Northwestern University Medical School, Chicago, Illiiiois ( U.S.A.)

INTRODUCTION

This communication describes some recent investigations of the distribution and effects of two classes of organic cations, basic dyes and tetrazolium salts in the central nervous system. These agents exhibit interesting but divergent neurobiological behavior which depends in part upon their selective affinity for different cytoplasmic organelles. Basic dyes accumulate mainly within lysosomes and cause remarkable but reversible changes in the structure of these particles without altering their function. Tetrazoles, on the other hand, concentrate within mitochondria, undergo reduction to the insoluble formazan derivatives and cause serious disruption of mitochondria1 structure and function. A. C A T I O N I C DYES

Anionic dyes such as trypan blue do not normally enter the central nervous system in mammals, perhaps partly because they bind to serum protein. Cationic dyes, on the other hand, readily penetrate the blood-brain barrier, probably in the unionized form (Davson and Danielli, 1943), and impart a distinct color to the brain. Cationic dyes are concentrated within pre-existing cytoplasmic inclusions of nerve cells that have been purported to be the Golgi apparatus (Cove11 and Scott, 1928), ribonucleoprotein (Nissl) masses (Bradley and Wolf, 1960; Zeiger and Harders, 1951), and lipochondria (Shafiq, 1953; Thomas, 1948). More recently these structures, which occur also in glia and other neural cells, have been identified on morphological (Koenig, 1962a, b, 1964a) and biochemical (Koenig et a/., 1964) grounds as lysosomes, a class of cytoplasmic particles which contain numerous, potentially destructive acid hydrolases in an inactive form (reviewed in De Duve, 1963). A wide variety of plant and animal cells also possess cytoplasmic inclusions that are stained preferentially in vivo by basic dyes (see Baker, 1958). These particles also resemble lysosomes, as demonstrated by histochemical staining for acid phosphatase activity, in their morphology and intracellular location (Allison and Mallucci, 1964; Brachet, 1957; R r f i r m c w p. l l X - I 0

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Byrne, 1964a; Koenig, 1962b, 1963a, b; Mulnard, 1961 ; Ogawa et a/., 1961 ; Robbins etal., 1964; Schmidt, 1962; Tanaka, 1961). Over the past few years my colleagues and 1 have investigated the uptake of basic dyes in the central nervous system of rats with particular reference to their sequestration within and effect upon lysosomes. I n these studies we used mainly neutral red because it is less toxic for rat and can be permanently retained in microscopic sections after appropriate processing. Several other cationic dyes have also been studied. Physiological eflects Neutral red ( I solution in water), given intraperitoneally to adult rats in a dose of 0.4 g/kg, exerts potent central actions that closely resemble the effects of the phenothiazines, a class of organic cations chemically related to thiazine dyes like methylene blue. Spontaneous motor activity diminishes and rats become lethargic soon after dye injection. Signs of autonomic activation appear coincident with this change in behavior; these include piloerection and increased formation of lacrimal and nasal secretions which are stained by dye. Generalized convulsive seizures may occur and a few animals, approximately 15 %, die. Concurrently the neuraxis becomes bright red, gray matter more than white, reaching a maximum I to 3 h after dye injection (Fig. I). Animals recover from the behavioral and autonomic affects by about 8 hours,

Fig. I . Rat brain 1 h after neutral red. Gray matter is deeply stained while white matter is only slightly colored.

at which time the brain is no longer visibly stained. Acridine orange, methylene blue and toluidine blue, given intraperitoneally in a smaller dose, 0.15-0.25 g/kg, because of their greater toxicity, exert a similar central action accompanied by visible staining of the neuraxis. The pharmacologic effects and the intensity of neural staining of these dyes, in accord with the lesser dose, are less marked than those of neutral red.

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localizat ioit

Fresh squash preparations of vitally stained spinal cord, sensory ganglia and cerebral cortex have been examined by light, phase and fluorescence microscopy. lnitially the cytoplasm is diffusely colored. Within a few minutes some pre-existent cytoplasmic granules of neurons and other neural cells become preferentially stained by these dyes in vivo. When acridine orange is administered by vein, stained cytoplasmic inclusions are visible in the fluorescence microscope within 30 sec. The number and staining intensity of granules increase with time, reaching a maximum at I to 2 h. As reported earlier (Koenig, I962a, b, 1963a, b, I964a) the general morphology of these granules is identical to that of the acid phosphatase-positive lysosomes.

Fig. 2. Squash preparation from spinal cord of rat given 25 mg of acridine orange intraperitoneally 1 h before sscrifice. Spinal motoneuron exhibits numerous metachromatically stained lysosomes. The neuronal nucleus exhibits a weak orthochromatic staining, while glial nuclei are more distinctly stained. Extraneuronal, metachromatically stained lysosomes probably belong to glia. Fluorescence photomicrograph, x 640.

Lysosomes stained by the basic fluorochrome, acridine orange, emit a n orange fluorescence which denotes metachromatic staining. Nuclei may reveal a feeble green fluorescence indicating slight orthochromatic staining (Fig. 2). Methylene blue and toluidine blue confer upon lysosomes a dark purple color also characteristic of metachromatic staining, but do not visibly stain other cytoplasmic or nuclear structures. Neutral red stains lysosomes red without coloring other structures. When viewed in fluorescence optics, however, the unstained cytoplasm and nuclei emit a yellow fluorescence, indicating the presence of neutral red in the un-ionized form (Strugger, 1940). Exposure of neutral red-stained lysosomes to the ultraviolet beam for a few minutes causes a decolorization of these structures. Irradiated lysosomes then exhibit a strong yellow fluorescence presumably characteristic of un-ionized neutral red. However, lysosomes stained in vivo by methylene blue or toluidine blue are not readily decolorized by ultraviolet light. R&-rwccs

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Fresh squash preparations suffer from several disadvantages: ( 1 ) they are impermanent; (2) cell morphology and relationships are distorted by compression of tissue fragments; and (3) secondary staining procedures cannot be applied for histochemical study of stained inclusions. After considerable experimentation, I found that neutral red granules (NRG) are preserved in frozen sections of tissues which have been fixed by vascular perfusion with 4 % neutral formaldehyde. Unfortunately other basic dyes are not retained in situ by perfusion fixation. The identification of NRG as lysosomes has been confirmed by a direct demonstration of acid phosphatase activity within these structures. This was accomplished by comparing photomicrographs of the same vitally stained neurons before and after staining fixed-frozen sections for acid phosphatase activity by Gomori’s lead glycerophosphatase method (Fig. 3).

Fig. 3. Fixed-frozen section of cerebral cortex from rat receiving neutral red 2 hours before sacrifice. (a) Neutral red granules (NRG) are demonstrated in two adjacent neurons. These arc typically

“swollen” or enlarged at this stage. (b) Same neurons stained for acid phosphatase activity by Gomori’s lead glycerophosphate method. Incubation period 4 h (normal incubation 45 niin). Most of the NRG arc acid phosphatase-positive. x 1500.

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Mechanism of vital metachromasia of lysosomes What is the basis for the vital metachromasia of lysosomes? In fixed tissues metachromatic staining is generally ascribed to electrostatic binding of basic dyes in dimeric or polymeric form to acidic macromolecules, designated chromotropes (Baker, 1958). The metachromasia of lysosomes differs in that it is abolished by fixation, suggesting that it does not depend upon recognized chromotropes as these retain their metachromasia after fixation. However, unfixed lysosomes in cell particulates suspended in sucrose media do stain metachromatically at 4" C. Hence energy-yielding or other "vital" cellular activities are not actually required for the staining of lysosomes in vivo. The putative role of electrostatic binding in the vital metachromasia of lysosomes was tested by observing the influence of various cations and pH on lysosomal staining TABLE I F A C T O R S I N F L U E N C I N G A C R I D I N E O R A N G E M E T A C H R O M A S I A O F C E R E B R A L LYSOSOMES

in vitro*

Metachromatic staining is inhibited or prevented by the following cations: a. Ag+, Cu++and Pb++(0.1-5.0 mM) b. Chlorprornazine (0.2-5.0 mM) c. Protamine, histone and polylysine (0.1-1 mg/ml) 2. Cationic inhibition may be reversible if dye concentration is increased 10-100 X 3. Na+, K+ and C a t + (25-100 mM) are non-inhibitory or only weakly inhibitory. 4. Lysosomal staining is pH** sensitive: - no staining a. p H 3 b. p H 4-5 - orthochromatic staining c. pH 5.5-10 - metachromatic staining increasing with pH 1.

-

* These results are based on two or more experiments for each agent tested. * *' The following buffers were used in final concentration of 20 mM: p H 4-6, acetate; pH 7-8, tris-HCI; pH 9-10, glycine-NaOH. Mitochondrial fractions prepared from rat brain (Koenig el a/., 1964) were suspended in 0.25 M sucrose (0.1 g fresh tissue equivalent per ml). After pretreatment with cation or buffer in 0.25 M sucrose at 4" C, suspensions were stained with acridine orange (final concentration 0.0034.001 %). Small drops were placed on slides, dried and examined under oil immersion lens in the fluorescence microscope.

by acridine orange in vitro. Mitochondrial fractions from rat brain were used in these staining experiments. The results are summarized in Table 1. Cerebral lysosomes emit a distinctive orange or yellow-orange fluorescence characteristic for metachromatic staining by acridine orange (Fig. 4). Mitochondria and other subcellular particles are unstained or emit a weak green fluorescence denoting slight orthochromatic staining under these conditions. Pretreatment of particulate suspensions with a variety of inorganic and organic cations caused varying degrees of impairment in dye binding ranging from a slight diminution of metachromasia, manifested by a shift from orange toward yellow fluorescence, to no staining at all. With intermediate degrees of dye binding, lysosomes were stained orthochromatically, i.e., they fluoresced Rekrcnces p. 118-120

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Fig. 4. Fresh particulate suspension of rat brain lysosomes stained in virro with acridine orange (0.0015%) and photographed with fluorescence optics. This is fraction E in which lysosornes are concentrated approximately five-fold over the homogenate. Lysosomes stain metachromatically. Other structures such as mitochondria are unstained or exhibit a weak orthochromatic staining. x 640. (From Koenig ef a/., 1964).

green. The degree of inhibition was more marked with higher concentrations of cations. Moreover, inhibition of staining was reversible, at least at lower concentrations of some cations, since it could be overcome by increasing dye concentration. These findings suggest that cations compete with dye molecules for anionic binding sites in lysosomal particles. The negligible effect of even high concentrations of the physiological cations, K+, Na+, and Ca++,on lysosomal staining indicates that they bind weakly or not at all to the anionic sites of lysosomes. The influence of pH on lysosomal staining is also consistent with the hypothesis that cationic dye molecules bind electrostatically to acidic sites in lysosomal granules. High pH would favor ionization of anionic binding sites with consequent enhancement of dye binding by lysosomes. At pH values near or below the isoelectric point of these acidic groups, binding would be reduced or abolished. The experimental results are in accord with these expectations. The basophilia and metachromasia of lysosomes increased progressively as the pH of the staining medium was elevated. The isoelectric point of cerebral lysosomes, measured by quenching of metachromatic staining, is approximately pH 5.5-6.0. However, as judged by quenching of orthochromatic staining the isoelectric point is in the vicinity of pH 4. The pH dependence of lysosomal staining by basic dyes has been verified by quantitative measurements of toluidine blue and neutral red bound in vitro by purified renal lysosomes (Koenig, unpublished). It may be concluded from these experiments that basic dye binding in vitro, and inferentially in vivo, depends upon electrostatic interaction forces between dye molecules and anionic sites in lysosomal particles. Metachromasia appears when the concentration of dye molecules attains a critical level, and may be due to an aggregation or a “stacking” (Bradley and Wolf, 1960) of bound dye molecules.

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Let us now consider the possible nature of these acidic dye binding sites. The common chromotropes, e.g. acidic mucopolysaccharides and nucleic acids, d o not, as far as is known, occur in lysosomes. However, phospholipoproteins are present, and for the reasons which follow are the most likely candidate for this role. Purified phospholipids, particularly those with unsaturated fatty acids, exhibit a metachromasia in model staining experiments that is attributable to ionized phosphate groups (Byrne, 1962; Sinapius and Thiele, 1965). Polyunsaturated phospholipids occur as lipoprotein complexes in the matrix of lysosomes in neural and other tissues, as judged by histochemical staining reactions (Koenig 1964a, c, 196%). Efforts to investigate the non-enzymatic components, i.e., the structural matrix, of cerebral lysosomes by biochemical methods have been thus far hampered by our inability to isolate these structures as a homogeneous fraction (Koenig el a/., 1964). However, lysosomes have been isolated from rat kidney and found to possess a significant phospholipid content, approximately 110 pg per mg protein, together with lesser amounts of cholesterol, 30 pg, and glycolipid (cerebroside?), 10 pg, per mg protein. Sphingomyelin and lecithin are the predominant phospholipids in renal lysosomes, each comprising about 35% of the total (Koenig et a/., in preparation). Lipids seem to be necessary for basic dye binding, as ethanol and the non-ionic detergent, Triton-X 100 (0.1 %) abolish the staining of cerebral and other lysosomes by acridine orange in vitro. Phospholipid phosphate has been specifically implicated as a potential anionic binding site by experiments employing the enzyme phospholipase C . Preincubation of lysosomal suspensions with phospholipase C , which splits phospholipids into phosphoesters and diglycerides, diminishes or abolishes the metachromasia of cerebral and other lysosomes (Koenig, 1965a; Koenig and Gray, 1964), but does not completely prevent basic dye binding. Hence the carboxyl groups of associated proteins may also serve as anionic binding sites. In any event the integrity of native lipoprotein macromolecules seems to be required for basic dye binding, since lysosomal staining is abolished by 4 % neutral formaldehyde, a treatment which denatures proteins without extracting lysosomal phospholipids during brief fixation periods (Koenig et a/., in preparation). It seems likely that the lipoproteins of the lysosome matrix differ significantly from lipoproteins in other structures, e.g., mitochondria and endoplasmic reticulum, since the latter do not exhibit vital metachromasia. We may conclude from the foregoing experiments that lysosomes in vivo possess free or available anionic binding sites and can therefore be regarded as polyanionic lipoprotein granules (Koenig 1962b, 1964a). It seems reasonable to assume that this electrically charged matrix is related to the functions of lysosomes. One such function is the protection of host cells from injury by exogenous cations, e.g. basic dyes and heavy metallic cations (Koenig, 1963a), by temporary (or permanent) storage of these toxic agents. The negatively charged matrix of lysosomes also may play a role in storage of the acid hydrolases, some of which are known to be cationic proteins (Zeya e t a / . , 1966). De Duve and associates (De Duve, 1959, 1963) consider the lysosome to be an inert sac delimited by a lipoprotein membrane which serves as a physical barrier to separate the contained enzymes from the cell substrates. The vital metachromasia of lysosomes, together with other observations, have led me to propose, as Rcfiwncrs p . 118-l20

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an alternative to the membrane hypothesis of De Duve, that the enzymes are complexed to lysosomal matrix by electrostatic and possibly other chemical forces (1962a, 1964a). This model of the lysosome accounts for the structural latency of the lysosomal enzymes by postulating that the active sites of these enzymes, which may include basic amino groups, are occluded through ionic linkage to acidic sites in lysosomal matrix (Koenig, 1964a). This mechanism is analogous to the non-specific, reversible inhibition of cationic enzymes in vitro by a variety of macromolecular polyanions or “niacroanions” described by Spensley and Rogers (1954) and others (Bernfeld, 1963). Morphological changes in Iysosomes

For these studies rats were injected intraperitoneally with neutral red, 0.4 g per kg, and killed from 5 min to 24 h after dye injection by vascular perfusion with 4 % neutral Figs. 5-8. Photomicrographs of spinal cord from frozen-fixed sections of rats given neutral red intraperitoneally. All are photographed at x 1000.

Fig. 5. Five minutes after neutral red injection. Note diffuse staining of cytoplasm and some NRG in motoneurons.

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Fig. 6. Thirty minutes after neutral red injection. (a) Numerous, relatively small NRG are evident in bright field optics. (b) NRG appear as dense particles in the phase microscope.

Fig. 7. One hour after neutral red injection. (a) Numerous swollen NRG are now evident in rnotoneuronal perikarya and dendrites. (b) NRG are highly refractile and phase-rare as viewed by phase microscopy. Rtfereerenccs p. 118-120

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Fig. 8. Four hours after neutral red injection. NRG are smaller and less brilliantly stained than at I h (Fig. 7).

formaldehyde. Spinal cord and brain were freeze-sectioned and examined with an oil immersion objective by bright field, phase and fluorescence optics. Granular inclusions in the nerve cells which correspond to lysosomes are stained by dye within a few minutes following injection (Fig. 5 ) . The number of NRG and their staining intensity increase with time, reaching a maximum by I to 2 h (Figs. 6a, 7a). Subsequently NRG diminish in numbers and in dye content, disappearing 6 to 8 h after dye injection (Fig. 8). NRG, as viewed in bright field and phase optics, undergo distinctive changes in size and refractility. Initially NRG are phase dense but reveal no other discernible change in structure (Fig. 6b). After about 45 min NRG are perceptibly enlarged, and by 1 to 2 h they measure two to three times their normal diameter. Concomitantly N RG become highly refractile and phase-rare relative to the cytoplasm, suggesting an attenuation of lysosomal matrix (Fig. 7b). After about 3 h, NRG undergo a progressive reduction in size and refractility paru passu with loss of neutral red staining. At 8 h when NRG are no longer evident, lysosomes, identified by acid phosphatase staining, are normal in size and non-refractile. For reasons that are not entirely clear, a high concentration of neutral red in lysosomes interferes with the histochemical demonstration of acid phosphatase activity by Gomori's method. Inasmuch as the enzymatic deposition of lead phosphate in NRG, as seen in the electron microscope, is not impaired, it is likely that neutral red in some manner interferes with the secondary conversion of lead phosphate to opaque lead sulfide. NRG are most conspicuous in spinal motoneurons which possess numerous, relatively large lysosomes. Neurons in cerebral cortex tend to have fewer and smaller NRG (Fig. 9). Glia in white and gray matter also contain NRG, but these are relatively minute and sparse. Adventitial, leptomeningeal, and chorioidal epithelial cells (Fig. 10) reveal coarse NRG, while the NRG of ependymal cells are small (Fig. 10). In all these cell types NRG correspond morphologically to lysosomes

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and also reveal a uniform pattern of structural changes, increasing in size and refractility as dye concentration rises, and reverting toward normal as dye content diminishes.

Fig. 9. Rat cerebral cortex I h after neutral red injection. NRG in cortical neurons are sparser and

smaller than those in spinal motoneurons. x 1000.

l-.ig. 10. Chorioidal plexus and wall of lateral ventricle of rat cerebrum one h after neutral red injection. NRG of chorioidal plexus (CP) are numerous and coarse, while those of ependymal (E) cells are sparse and relatively small. X 2000. RrJcrenci2s p. 119-121

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Electron microscopic observations Remarkable ultrastructural changes have been observed in lysosomes of neurons, glia and ependymal cells upon uptake of neutral red. These changes are fully reversible in 8 to 12 hours (McDonald and Koenig, 1965, 1966; Koenig, McDonald and Jacobson, in preparation). Several different fixatives were used with similar results, including buffered 2 osmium tetroxide, and vascular perfusion with buffered 4 glutaraldehyde followed by immersion in buffered osmium tetroxide. Tissue blocks were dehydrated and imbedded in epon or araldite. Ultrathin sections were stained with uranyl acetate or lead hydroxide for examination in an RCA EMU-3F electron microscope. Neuronal lysosomes normally show considerable variations in fine structure which have to be kept in mind in evaluating abnormalities. In neurons of cerebral cortex and spinal cord of young adult rat lysosomes typically consist of a finely granular, moderately dense matrix which is surrounded by a unit membrane (Fig. 11). A variety of structures is discernible in the matrix. These include small osmiophilic granules (Fig. 12) and/or vesicles. Sometimes the matrix material consists only of vesicles, designated multivesicular bodies (Fig. 13). Occasional dense bodies contain a large, relatively electronlucent zone or vacuole (Fig. 14), and more rarely lamellated areas (Fig. 15). During the first hour after neutral red injection, increasing numbers of lysosomes show progressive changes in matrix ultrastructure which are readily distinguished from the normal variations. However, the electronlucent vacuoles, which are devoid Figs. 11-15. Electron micrograph showing neuronal lysosomes (“dense bodies”) from control rats

Fig. 1 1 . Typical lysosomcs contain a finely granular, moderately electron-dense matrix and a delimiting unit membrane. Caulfield’s fixative, uranyl acetate. x 54000.

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Fig. 12. Lysosoines showing strongly osmiophilic and electron dense granules within matrix. Glutaraldehyde-0s04 fixation, uranyl acetate. x 57 0oO.

Fig. 13. Multivcsicular body. Glutaraldehyde-0s04 fixation, uranyl acetate. x 52 500. Rcfirenrrs p. 119-121

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Fig. 14. Lysosome showing numerous osrniophilic granules and vesicles, and a large electronlucent droplet. OsOr fixation, lead citrate. x 52 500,

Fig. 15. Lysosome with numerous osmiophilic larnellae arranged circumferentially. Caulfield’s fixative, uranyl acetate. x 72000.

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Figs. 16-26. Electron micrographs of neuronal lysosonies from rats given neutral red.

Fig. 16. Fifteen minutes after neutral red. Note presence of a number of parallel osmiophilic laniellae and areas of increased electronlucency in the matrix. OsO.l,'uranyI acetate. x 39 OOO.

Fig. 17. Thirty minutes after neutral red. Lysosome is somewhat swollen and bulk of matrix is finely granular. A small portion of the matrix is condensed, lamellated and osmiophilic. 0 ~ 0 4 ,uranyl acetate. x 36 600. Hcfi*nwces p. 119-I21

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Fig. 18. One hour after neutral red. One lysosome is markedly swollen and consists of a large electronlucent zone (“vacuole”). The matrix occupies a small area and is granular and vesicular. Glutaraldehyde-Caulfield, uranyl acetate. x 36 600.

Fig. 19. One hour after neutral red. Lysosomes are swollen and vacuolated. The matrix is heterogeneous, consisting of granular, vesicular, lamellated and condensed, osmiophilic material. Glutaraldehyde-Caulfield, uranyl acetate. x 36 600.

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Fig. 20.. Survey electron micrograph of cortical neuron 2 h after neutral red. All lysosomes are enlarged, vacuolated, and contain a condensed osmiophilic matrix of variegated configurations. The general morphology of the nucleus, perikaryon and neuropil is normal. Caulfield, uranyl acetate. x 12000.

of acid phosphatase activity (Kreutzberg and Hager, 1966), are unaffected. The earliest changes, already evident at I5 min in many lysosomes, is the formation of a few dense parallel lamellae (Fig. 16). Subsequently lamellation increases and is accompanied by the appearance of vesicles and other membranous structures, rarefaction of granular matrix, and vacuolation (Fig. 17). The most advanced changes in lysosomal fine structure appear at I to 2 h. These changes coincide with maximum staining by neutral red when lysosomes are seen to be enlarged, refractile and phaserare in the optical microscope. At this stage virtually all lysosomes are two or three times their normal diameter, equivalent to a 16 to 27 fold increase in volume. The matrix is a markedly rarefied and empty space, which represents water incorporated ir? viva and accounts for a large fraction of the area of these lysosomes. The matrix material assumes various appearances, including: a diffuse attenuation of granular material ; membranous structures such as multiple parallel lamellae and vesicles R
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Fig. 21. Several lysosomes from Fig. 20 at higher magnification showing the separation of the matrix into large electronlucent regions, "vacuoles", containing sparse granular material, and smaller areas of osmiophilic electron dense material. x 54 000.

Fig. 22. Six hours aftcr neutral red. Lysosomes are much smaller, vacuolated zones arc reduced in area, and matrix is made up of osmiophilic lamellae in circular arrays. G~utaraldehyde-Caiillield, uranyl acetate. x 36 600.

Fig. 23. Seven hours after neutral red. Two lysosomes are nearly normal in size and internal structure. One lysosome still contains a lamellated matrix. Glutaraldehyde-Dalton. x 54 000.

-

. 7 . A

Figs. 24-26. Electron micrographs of rat cerebrum stained for acid phosphatase activity. The enzyme product, lead phosphate, is electron-dense.

Fig. 24a

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b Fig. 24. Control neuron. (a) Lysosomes show rather heavy deposits of enzyme product distributed diffusely throughout matrix of lysosomes. Glutaraldehyde, Caulfield. Y I7 000. (b) Lysosome shows absence of enzyme product in electronlucent vacuole. Inner two lamellae of the Golgi apparatus exhibit acid phosphatase activity. Glutaraldehyde, Caulfield. x 55 000.

(Fig. 18, 19); and condensed, irregular osmiophilic masses (Fig. 19).The limiting unit membrane always appears to be intact. At 2 and 3 h virtually all lysosomes are severely vacuolated (Figs. 20, 21). By 4 h, lysosomes show indications of incipient return towards a normal structure as manifested by an increasing compactness of matrix and a reduction in size and number of vacuoles. Many lysosomes appear normal at 5 to 7 h, but some still reveal a pleomorphic matrix containing lamellae, vesicles and other formations (Figs. 22, 23). By 10 to 12 h neuronal lysosomes are indistinguishable from those in control animals. The subcellular sites of acid phosphatase activity were investigated with the electron microscope in a few experiments. Sections of glutaraldehyde-fixed tissue blocks were cut at a thickness of 50 ,u with the Mcllwain chopper (Smith and Farquhar, 1963),

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Fig. 25. Three hours aftcr neutral red injection. Numerous vacuolated lysosomes in cortical neuron show product of acid phosphatase activity over matrix. A satellite oligodendrocytewith single reactive, non-vacuolatcd lysosome is also evident. Glutaraldehyde, Caulfield. x 24 000.

incubated in Gomori’s lead glycerophosphate medium for acid phosphatase staining and processed for electron microscopy. As reported by others (e.g., Novikoff et a / . , 1963; Osinchak, 1964; Kreutzberg and Hager, 1966), acid phosphatase activity in normal neurons is localized largely to lysosomes and to inner lamellae of the Golgi apparatus (Fig. 24). In experimental animals the reaction product of acid phosphatase activity remained confined to lysosomes despite the extreme alterations in their fine structure (Figs. 25,26). Although the other lysosomal enzymes were not investigated in this study, it seems likely that they also are retained within NRG. The total absence of autolytic changes in the cytoplasm throughout the cycle of lysosomal vacuolation supports this inference. Rcfcrcnws p . 119-121

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Fig. 26. Three hours after neutral red injection. Neuronal lysosomes stained for acid phosphatase activity. The association of enzyme product with lysosomal matrix and its absence from vacuoles is well demonstrated. Glutaraldehyde, Caulfield. x 52 500

In all cells studied the acid phosphatase activity in N R G was restricted to the matrix material. Enzyme product has never been detected within vacuoles. Byrne (1964b) and Brewer and Heath (1964) have made comparable observations in vacuolated lysosomes of pancreas and liver respectively. These findings are of great importance with respect to the functional organization of lysosomes. If the intralysosomal compartmentation and structural latency of the acid hydrolases is due only to the barrier function of the membranous envelope, as postulated by De Duve (1959, 1963), then acid phosphatase should also occur in the vacuolar fluid. The invariable association of acid phosphatase activity with matrix material in these vacuolated lysosomes is at variance with the membrane hypothesis and comprises compelling evidence in support of the hypothesis that lysosomal enzymes are chemically bound to matrix. Moreover the large electronlucent vacuoles that occur in some lysosomes of normal neurons are analogous to the fluid vacuoles of N R G in that they also are devoid of acid phosphatase activity (Kreutzberg and Hager, 1966). Since these vacuoles are enclosed together with normal matrix inside a common limiting membrane, we may assume that they, like the fluid vacuoles of NRG, have no “affinity” for acid phosphatase, i.e., this enzyme is bound to the normal matrix. The normal matrix seems to be rich in unsaturated phospholipids since its osmiophilia is blocked by bromination and by extraction with chloroform-methanol (Koenig, 1964b, 1965c), whereas these vacuoles evidently contain lipase-sensitive neutral triglycerides (Torack, 1967).

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a

v,

[ L I

,

Acid Phosphatase

‘Neutral Red

Fig. 27. Distribution of neutral red and acid phosphatase activity in primary fractions of rat brain 15 min after neutral red injection. The nuclear (N) and microsoma1 (R)fractions contain the highest concentration of neutral red, expressed as relative specific activity (RSA), while the mitochondrial fraction ( M ) contains the highest concentration of acid phosphatase.

4

Control

3

f

Hour

$ Hr 2 Hrs I Hr

NeutralRed

Fig. 28. The effect of neutral red on the distribution of acid phosphatase activity in subfractions prepared from a crude mitochondrial fraction by spinning over a discontinuous sucrose density gradient (Koenig ct a/., 1964). Fraction A (0.3-0.8 M ) contains myelin; B (0.8-1.0 M), nerve endings; C (1.0-1.2 M), nerve endings and mitochondria; D (1.2-1.4 M), mitochondria; E (pellet from 1.4 M sucrose), rich in lysosomes. Note shift of acid phosphatase from E into lighter fractions peaking at 9.Neutral red occurs in highest concentration in B and C, even before a marked shift in lysosomes appears.

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Biochemical studies Additional information regarding the uptake, subcellular distribution and effects of basic dyes on lysosomes in rat brain have been obtained in biochemical investigations (Koenig and Hurlebaus, 1965; Koenig et a/., in preparation). The results of these experiments have confirmed and extended the microscopic observations. ( I ) Subcellular distribution of neutral red - Rat brain homogenates were fractionated by differential and density gradient centrifugation (Koenig et a/., 1964) to study the subcellular distribution of basic dyes and lysosomal acid phosphatase activity. The results of a typical experiment with neutral red (0.4 g/kg intraperitoneally) are presented in Fig. 27. Neutral red was recovered from all primary fractions, but occurred in highest concentration in the nuclear and microsomal fractions. Dye uptake periods in these experiments ranged from 15 min to 6 hours. The distribution of acid phosphatase activity was not well correlated with that of neutral red in the primary fractions or in subfractions of the crude mitochondrial fraction obtained by spinning the latter over a discontinuous sucrose density gradient. Usually subfractions B and C, which are rich in nerve endings (Koenig et a/., 1964), contained the highest concentration of dye (Fig. 28). Moreover fractions rich in nerve ending membranes bound a considerable amount of neutral red in vivo (Koenig et a/., 1965). Thus, although neural lysosomes are the only structures that are visibly stained, neutral red is also associated with other neural components, in accord with the results of fluorescence microscopy. Acridine orange and methylene blue reveal a similar subcellular distribution in rat brain homogenates.

(2) Sedimentation characteristics of vitally stained lysosomes - Brain homogenates from neutral red-injected rats, when subjected to differential centrifugation, revealed a small increase in soluble acid phosphatase activity probably due to an increased fragility of altered lysosomes. Despite the marked changes in the morphology of lysosomes, the subcellular distribution of acid phosphatase, as revealed by differential centrifugation, was essentially unaltered. However, on isopycnic centrifugation of the crude mitochondrial fraction over a discontinuous gradient consisting of 0.3, 0.8, 1.0, 1.2 and 1.4 M sucrose (Koenig et al., 1964), lysosomal acid phosphatase activity underwent a striking shift into lighter subfractions (Fig. 28). This redistribution manifested itself in a reduced relative specific activity (RSA) of acid phosphatase activity in fraction E, and an increased RSA in lighter subfractions which was most pronounced in fraction B. This shift in lysosomal acid phosphatase was seen as early as 15 minutes after dye injection and was most marked between one and two hours. Subsequently the distribution of particulate acid phosphatase gradually returned toward normal, complete restoration occurring between 6 and 12 h . I n a small number of experiments with acridine orange (20-25 mg/ 103 g), a qualitatively similar, but quantitatively less marked, redistribution of lysosomal acid phosphatase activity was observed in this gradient system. From these studies, it is evident that cerebral lysosomes stained in vivo with basic dyes undergo a significant reduction in buoyant density. This density change is directly correlated with the dye concentration and the morpholo-

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gical changes in cerebral lysosomes, and is due to the attenuation of lysosomal matrix by water. Evidently the increased size of these organelles compensates for their diminished density so that their sedimentation characteristics are but little altered on differential centrifugation. NRG were compared with normal lysosomes with regard to accessibility of their contained acid phosphatase to substrate. This was done by measuring free and total acid phosphatase activity in particulate fractions of control and experimental rat brain according to Gianetto and De Duve (1955). The total acid phosphatase activity of rat brain was unaffected by neutral red nor was the accessibility of acid phosphatase to substrate, as measured by the percent free activity, increased in experimental brain. Thus despite the marked changes in structure of NRG, thecontained acid phosphatase, which we have shown to be bound to the matrix material, is still largely inactive, i.e., not free to interact with the added substrate, glycerophosphate. Concluding Retnarks

Lamellar structures have been found in lysosomes in diverse circumstances, e.g., in macrophages after uptake of India ink (Karrer, 1960), in liver cells incorporating bile pigment (Essner and Novikoff, 1960), and kidney tubule cells after uptake of trypan blue and thorium dioxide (Trump, 1961). Vacuolation of lysosomes has been induced in liver by the administration of dextran (Daems and van Rijssel, 1961), the detergent Triton WR-1339 (Wattiaux et a/., 1963) and in liver (Brewer and Heath, 1963) and kidney cells (Trump and Janigan, 1962) by hypertonic sucrose solutions. Vacuolation and lamellation occur in lysosomes of frog sympathetic neurons soon after x-irradiation (Pick, 1965). Similar ultrastructural changes have been reported in lysosomelike structures of non-neural tissues after uptake of basic dyes, e.g., cytosomes (Schmidt, 1962), segresomes (Tanaka, I964), autophagic vacuoles (Byrne, 1964a; Morgan e t a / . , 1966), and multivesicular bodies (Robbins ef a/., 1964). The present investigation demonstrates for the first time that extreme changes in the structure of lysosomes in the nervous system, and inferentially in other tissues, can be completely reversible within a short time interval. The binding of neutral red to lysosomes initiates an influx of water into these particles with consequent attenuation of the matrix material. Of great interest is the observation that the granular matrix of lysosomes is readily interconvertible to parallel arrays of lamellae, myelin figures, vesicles and other membranous structures. These morphological changes evidently represent reversible physical or physicochemical rearrangements among the lipoprotein macromolecules ofthe matrix due to binding ofcharged (cationic) molecules and/or the consequent influx of water. The unit membrane which surrounds these particles is apparently tough and also elastic, as it can undergo extensive stretching as lysosomes enlarge and equivalent contraction during the stage of lysosomal restoration. Lt seems likely that this membranous envelope prevents the disintegration of swollen lysosomes and consequent release of the contained enzymes into the cell. It is truly remarkable that, despite the marked changes in the fine structure of the matrix of lysosomes, lysosomal acid phosphatase, and presumably the other lysosomal Refirmri,s p . 119-161

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enzymes, remain associated with the matrix of these particles and exhibit no notable increase in free activity, i.e., in accessibility to substrates. B. T E T R A Z O L I U M S A L T S

Tetrazolium salts are colorless organic cations which contain four nitrogen atoms in a five-membered heterocyclic ring. These compounds are avid electron acceptors which upon reduction undergo ring cleavage with consequent formation of a colored insoluble formazan. It should be noted that HCI is simultaneously produced (Fig. 29).

cc

Tetrazoliurn Chloride

Forrnozon

N-N-R~

c L-

CL Dilelrazolwrn Chloride

Dilorrnazm

Fig. 29. Diagram showing the reduction of monotetrazoliunis and ditetrazoliums.

Fig. 30. Rat brain 6 days after injection of neotetrazolium (0.1 mg) into the lateral ventricle. The colored formazan derivative is restricted to ependymal lining of ventricular system and leptomeninges.

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Fig. 3 I . Fixed-frozen section of wall of 3rd ventricle 15 min after injection of neotetrazolium (1.0mg) into the lateral ventricle. Colored formazan deposits occur in dilated mitochondria of ependymal cells. x 1500.

Because of this property, tetrazoliums have been extensively used in dehydrogenase histochemistry. Recently 1 undertook an investigation of the uptake of tetrazolium in vivo with the expectation that these cations would localize to lysosomes in a manner analogous to basic dyes. Preliminary results seemed to bear this out. When injected intraperitoneally in rat in a dose of 2-3 mg/day for several days, neotetrazolium and NB-tetrazolium were localized as the colored formazan derivatives within lysosomes of a number of organs, including nerve and satellite cells of spinal and autonomic ganglia (Koenig, 1965d). However, these tetrazoles did not seem to traverse the bloodbrain barrier, as no formazan granules were found in cells of the central nervous system (Wolf et al., 1956; Koenig, 1965d). lntracellular Localization

The present experiments were undertaken in an effort to secure penetration of tetrazoles into the central nervous system. The intrathecal route was used to bypass the blood-brain barrier. When neotetrazolium chloride is injected into the lumbar subarachnoid space in cat, the pial surface rapidly becomes violet colored due to the formation of formazan deposits within cytoplasmic inclusions of leptomeningeal cells. A few formazan granules can be seen in the cytoplasm of adventitial cells and glia near the pia, but neural parenchyma is otherwise unstained. Administration of tetrazolium saltsinto the lateral ventricle of rat in a dose of 0.01-1.0 mg resultsincoloration of the ependymal lining and chorioidal plexuses and leptomeningeal s rface of the brain and cervical spinal cord within a few minutes. However, the eural parenchyma is still essentially unstained (Fig. 30). Examination of fixed-frozen secRcercnccr p . 119-121

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Figs. 32-35. Electron micrographs of ependymal cells of 3rd ventricle or rat cerebrum. Fig. 32. Normal ependyma. x 37 500.

tions in the light microscope revealed the presence of formazan deposits in discrete intracytoplasmic particles mainly within ependymal, chorioidal epithelial and leptomeningeal cells (Fig. 31). With doses of 0.1 mg intraventricularly, rats become somnolent for a period but usually survive indefinitely. Larger doses cause a variety of neurological disturbances often leading to fatal outcome, and some formazan deposits are formed in the subependymal tissues. Morphological Efects

Electron microscopic studies were carried out with Drs. S . Jacobson and M. J . Feldman. The ependymal lining of the third ventricle was fixed by intraventricular injection of buffered OsOe and processed for examination by light and electron microscopy. Tetrazoliums have a molecular weight of about 700 and seem to penetrate the plasma

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Fig. 33. Ten minutes after intraventricular injection of 0.1 mg neotetrazolium. Note dilatation of mitochondria, electronlucency of their matrix and incipient clumping of nuclear chromatin. x 37 500.

membrane of ependymal cells at a molecular level, as no pinocytotic vesicles are formed. Within minutes after the intraventricular injection of 0.1 mg of neotetrazolium, the mitochondria in some ependymal cells swell and their matrix becomes less dense (Figs. 32, 33). At one hour, the limiting double membranes of mitochondria are still intact but few cristae are seen (Fig. 34). By two hours, mitochondria appear as distended bags with scant finely granular matrix (Fig. 35). At six hours, the surrounding cytoplasm in some ependymal cells is markedly condensed, apparently because of a massive shift of water into the intramitochondrial compartment (Fig. 36). Many ependymal cells are in various stages of cytolysis. Other acute changes usually occur. Nuclear chromatin is severely clumped. Concurrently the basement membrane and the subjacent parenchymal tissue usually reveal edematous changes. Damaged or dying ependymal cells become detached, often in sheets, from the ventricular wall. However, lysosomes do not reveal ultraR,,li,r.vntr,.\ p. 119-1.'1

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Fig. 34. One hour after 0.1 mg neotetrazolium. Mitochondria are markedly dilated but the lysosome appears normal. x 52 500.

structural changes (Fig. 34). Considerable regenerative activity is seen in surviving ependymall cells of chronic animals. Some of these contain large pleomorphic dense bodies which may be autophagic vacuoles.

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Fig. 35. Two hours after 0.1 mg neotetrazolium. Mitochondria are even more distended and cristae are absent. Nuclear chromatin is severely clumped. x 34000.

Concluding Remarks

Clearly the formazan-containing inclusions seen in the optical microscope correspond to dilated mitochondria. The selective localization of formazan deposits within mitochondria in viva depends upon the combined presence of electron donors generated during oxidative phosphorylation and the appropriate dehydrogenases. The resultant mitochondria1 swelling indicates an accumulation of osmotically active particles within the mitochondria1 cavity. These may include chloride ions and/or hydrogen ions formed during reduction of tetrazolium, and Krebs cycle intermediates whose interconversions probably are blocked. The clumping of chromatin which is seen early R c f c r c n e s p. 119-1.?1

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Fig. 36. Eight hours after 0.1 mg of neotetrazolium. Mitochondria are swollen and electronlucent. Intervening cytoplasm is electron dense. x 34 000.

may be partly due to interference with oxidative metabolism. An electrostatic interaction of tetrazoliums with the phosphate groups of DNA may contribute to the condensation of chromatin. Ependymal cell injury and death seem to be secondary to this mitochondria1 lesion. The subependymal edema is due to swelling of astrocytes and their processes, and probably reflects a similar injury to the mitochondria of these cells since some formazan granules occur in the subependymal tissues when large doses of tetrazolium are administered. Moreover, the neurological disturbances which occur in the latter indicate a direct action on neural parenchyma. The failure of tetrazoles to penetrate the blood-brain barrier may be due to the strongly basic character of these salts, i.e., they probably are completely ionized at physiological pH. The large molecular weight of these compounds, about 700, may be a contributory factor. The impenetrability of neural parenchyma to tetrazoles when these are

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administered subarachnoidally or intraventricularly is attributable to intramitochondrial trapping of these cations as the insoluble formazans within the lining cells. When these cells are fully saturated, free tetrazoles can penetrate further into neural tissue.

ACKNOWLEDGEMENTS

My collaborators in these studies were Drs. S. Jacobson and T. F. McDonald. Valuable technical assistance was rendered by Mrs. A. Hurlebans, Mr. M. J. Feldman, Miss. B. Sheppard, Miss. T. Ronga, Mrs. D. Thomas, Mr. C. Hughes and Mr. E. Washington. Mrs. T. Howell prepared the typescript. Assisted by grants from N I H (NB04493, NB05509 and NB06838), AEC(AT(I1-I) I180), and National Multiple Sclerosis SOC.(No. 372).

REFERENCES ALLISON, A. C. A N D MALLUCCI,L. (1964) Uptake of hydrocarbon carcinogens by lysosomes. Nature, 203, 1024-1027. BAKER, J. R. (1958) Pritrciples ofBiologica1 Microtechirique. New York, John Wiley and Sons (p. 243). BERNFELD, P. (1963) Polyanionic inhibitors. Metabolic Inhibitors. R. M. Hochster and J. H. Quastel, Editors. New York, Academic Press (p. 437). BRACHET, J. (1957) Bioclreiiiical CjVo1og.v. New York, Academic Press (p. 52). BRADLEY, D. F. A N D WOLF,M. K. (1960) Neurochemistry of polynucleotides. The Neurocheiiiistry of Nucleotirles a i d Aiiriiio Acids. R. 0. Brady and D. B. Tower, Editors. New York, John Wiley and Sons (p. 89). BREWER D. B. AND HEATH,D. (1963) Lysosomes and vacuolation of the liver cell. Nature, 198, 1015-1016. - (1964) Development of sucrose vacuoles from liver cell lysosomes. J. fathol. Bacteriol., 87, 405-408. BYRNE,J. M. (1962) The uptake of dyes by extracted phospholipids and cerebrosides. Quart. J. Microscop. Sci., 103, 47-56. - (1964a) An electron microscopical study of neutral red granules in mouse exocrine pancreas. Quart. J. Microscop. Sci., 105, 219-225. - (1964b) Acid phosphatase activity in neutral red granules of mouse exocrine pancreas. Quart. J. Microscop. Sci.. 105, 343-348. COVELL,W. P. AND SCOTT,G . H. (1928) An experimental study of the relation between granules stainable with neutral red and the Golgi apparatus in nerve cell. Aiiat. Rec., 38, 377400. DAEMS,W. TH. A N D V A N RIJSSEL, TH.G. (1961) The fine structure of the peribiliary dense bodies in mouse liver tissue. J. Ultrastr. Rex., 5, 263-290. DAVSON, H. A N D D A N I E L L I , J. F. (1943) The Permeability of Natural Menrbraiws. Cambridge, Cambridge University Press (p. 295). [IF: DUVE,C. (1959) Lysosomes, a new group of cytoplasmic particles. Subcellular Particles. T. Hayashi, Editor. New York, Ronald Press (p. 128). -(1963) The lysosome concept. Ciba Foundation Symnposiuiii 011 Lysosotires. A. V. S. de Reuck and M . P. Cameron, Editors. Boston, Little, Brown and Company (p. 1). ESSNER,E. AND NOVIKOFF, A. B. (1960) Human hepatocellular pigments and lysosomes. J . Ultrastr. Res., 3, 374. GIANETTO, R. AND DE DUVE,C. (1955) Comparative study of the binding of acid phosphatase, /l-glucuronidase and cathepsin by rat liver particles. Bioclreiii. J., 59, 433438. KAHKER, H . E. (1960) Electron microscopic study of the phagocytosis process in the lung. J. Biophys. Biocheiir. Cytol., 7, 357.

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KOENIG,H. (1962a) Histological distribution of brain gangliosides: lysosomes as glycolipoprotein granules. Nature, 195, 782-784.

- (1962b) Lysosomes as polyatiionic glycolipoproteiti particles. Abstracts of Papers Presented at the

Second Annual Meeting of American Society for Cell Biology a t San Francisco, November 5-7 (P. 96). (1963a) lntravital staining of lysosomes by basic dyes and metallic ions. J. Histochem. Cytochem., 11, 120. (1963b) Staining of lysosomes by acridine orange. J. Cell Biol., 19, 87A. (1964a) Studies of brain lysosomes. Response of the Nervous System fo lotiizitig Radiation. T. Haley and R. Snider, Editors. Boston, Little, Brown and Company (p. 403). (1964b) Histochemical study of lysosomes and lipofuscin granules in the nervous system. Anal. Rec., 148, 303. (1964~)Neuronal lipofuscin in disease. Its relation to lysosomes. Trans. Amer. Neiirol. Ass., 89,212. (1965a) The staining of lysosomes by basic dyes. J. Histochern. Cytochem., 13, 20-21. (1965b) Vital staining of neural lysosomes by basic dyes. J. Neiiropathol. Exptl. Neurol., 24, 171. (1965~) Histochemical studies of lysosomes and lipofuscin granules in the nervous system. Proceedings of the 5th Intertiational Corrgress of Neriropathology, Zurich, 1965. Amsterdam, Excerpta Medica Foundation (p. 476). - (1965d) Intravital staining of lysosomes and mast cell granules by tetrazolium salts. J. Histocheni. Cytochem., 13, 411-413. KOENIG,H. AND GRAY,R. (1964) Action of phospholipase C on lysosomes. J. CeN Biol.,23, 5OA. KOENIG,H. AND HURLEBAUS, A. (1966) Biochemical and cytochemical studies of neutral red uptake into brain. J. Neciropathol. Exptl. Neurol., 25, 112. KOENIG,H., GAINES,D., MCDONALD, T., GRAY,R. AND SCOTT,JR. J. (1964) Studies of brain lysosomes - 1. Subcellular distribution of five acid hydrolases, succinate dehydrogenase, and gangliosides in rat brain. J. Neurochem., 11, 729-743. KOENIG,H., GAINES,D., NELSON,R., HURLEBAUS, A. AND CAVENDER, M. (1965) Some studiesof synaptic vesicles. Trans. Amer. Neurolog. Ass., 90, 189. KREUTZBERG, G. W. AND HAGER,H. (1966) Electron microscopical demonstration of acid phosphatase activity in the central nervous system. Histocherti., 6, 254-259. MCDONALD, T. F. A N D KOENIG,H. (1965) Ultrastructural changes in neural lysosomes induced by in vivo staining with neutral red. Anat. Rec., 151, 385. - (1966) Ultrastructural changes in neuron lysosomes induced by in vivo staining with neutral red. J. Neuropathol. Exptl. Neurol., 25, 130. J. A N D ALOUSI,M. A. (1966) Studies of the biological activity of neutral MORGAN,W. S., FERNANDO, red. Exptl. Molecular Puthol., 5, 491-503. MULNARD, J. (1961) Analyse des inclusions mktachromatiques in vivo dans des cellules de poulet en culture. Arch. Biol.,72, 525-572. N. (1963) Relation of endoplasmic reticulum, Golgi NOVIKOFF, A. B., ESSNER,E. AND QUINTANA, apparatus and lysosomes. J. Microsc. (Paris), 2, 3. OGAWA,K., MIZUNO,N. AND OKAMOTO, M. (1961) Lysosomes in cultured cells. J . Hisfochem. Cytochem., 9, 202. OSINCHAK, J. (1964) Electron microscopic localization of acid phosphatase activity in hypothalamic neurosecretory cells of the rat. J. Cell Biol.,21, 3547. PICK,5. (1965) The fine structure of sympathetic neurons in x-irradiated frogs. J . Cell Biol., 26, 335-351. ROBBINS,E. AND MARCUS,P. I. (1963) Dynamics of acridine orange-cell interaction. I. Interrelationships of acridine orange particles and cytoplasmic reddening. J. Cell Biol., 18, 237-250. ROBBINS, E., MARCUS,P. I. AND GONATAS, N. K. (1964) 11. Dye-induced ultrastructural changes in multivesicular bodies (acridine orange particles). J. Cell Biol.,21, 49-62. SCHMIDT,W. (1962) Licht- und electronenmikroskopische Untersuchungen uber die intracellulare Verarbeitung von Vitalfarbstoffen. Z. Zellforsch., 58, 572-637. SHAFIQ,S. A. (1953) Cytoplasmic studies of the neurones of Locusta migratoria. I - Cytoplasmic inclusions of the motor neurons of the adult. Quarf. J. Microscop. Sci., 94, 319-328. SINAPIUS, D. AND THIELE, 0. W. (1965) Uber die Methylenblaubindung von Lipiden. Untersuchungen an Modellsubstanzen. Histochern., 4, 553-562. M. G. (1963) Preparation of thick sections for cytochemistry and elecSMITH, R. E. AND FARQUHAR, tron microscopy by a nonfreezing technique. Nature, 200, 4907. SPENSLEY, P. C.AND ROGERS, H. J. (1954) Enzyme inhibition. Nature, 173, 1190-1191.

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STRUGGER, S. (1940) Neues uber die Vitalfarbung pflanzlicher Zellen mit Neutralrot. Protoplusrnu, 34, 601-608. TANAKA, H. (1961) Segresome and the cellular uptake of varied substances as revealed in the electron microscope. Ann. Rept. Inst. Virus Res., Kyoto Univ., 4, 118. - (1964) Electron microscope studies on the neutral red vacuoles. Intrucellular Membranous Structures. S. Seno and E. V. Cowdry, Editors. Okayama, Japan, Japan Society for Cell Biology (p. 293). THOMAS, 0. L. (1948) A study of the spheroid system of sympathetic neurons with special reference to the problem of neurosecretion. Quart. J. Microscop. Sci., 89, 333-350. TORACK, R. M. (1967) The nature and significance of compound lipid cytoplasmic bodies in progressive dementia. J. Neuroputhol. Exptl. Neurol., 26, 151-152. TRUMP,B. F. (1961) An electron microscopic study of the uptake, transport and storage of colloidal materials by the cells of the vertebrate nephron. J. Ultrustritc. Res.. 5, 291. TRUMP, B. F. A N D SANIGAN, D. T. (1962) The pathogenesis of cytologic vacuolization in sucrose nephrosis. Lah. Invest., 11, 39541 I . WATTIAUX, R., WIBO,M. AND BAUDHUIN, P. (1963) Influence of the injection of Triton-WR-1339 on the properties of rat liver lysosomes. Cibu Foririrlutiori Sy~~iposiitrn on Lysosonies. A. V. S. de Reuck and M. P. Cameron, Editors. Boston, Little, Brown and Company (p. 176). WOLF,A. A N D COWAN, D. (1956) Reduction of neotetrazolium in the satellite cells ofthe cerebrospinal and sympathetic ganglia. J. Nerrroputhol. Exptl. Neurol., 15, 384. ZEIGER,K. AND HARDERS, H. (1951) Uber Vital-Fluorochromfarbung des Nervengewebes. Z. Zellforscli., 36, 62-78. ZEYA,H. I., SPITZNAGEL, J. K. AND SCHWAB, J. H. (1966) Antibacterial action of P M N lysosomal cationic proteins resolved by density gradient electrophoresis. Proc. SOC.Exptl. Eiol. Med., 121, 250-253.

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QUADBECK: If you use the reduction of tetrazolium salts, you not only produce formazon but also hydrochloric acid, which in turn could produce disruption of the Blood-Brain Barrier. Can y o u exclude the effect of this hydrochloric acid on mitochondria? : The tetrazolium salts were dissolved in buffer which was in considerable excess. Indeed, KOENIG the phosphate buffer at times induced convulsions in the animal because of hypocalcaemic effects. In later experiments, using much lower doses of tetrazolium, we got the same morphological effects. The formation of HCI within mitochondria by reduction of tetrazolium cannot be prevented.

CSAKY:I would like to make a comment, as far as the barrier to dyes is concerned, which fits in with Dr. Steinwall’s interesting remarks. When we talk about selectivity, there is not any magic about it. Substances can penetrate cells, depending on their lipid solubilities, or they may penetrate by a specific transport mechanism. The lipid solubility strictly depends on the pKa of the substance and the pH of the medium. What Dr. Koenig showed was that the pKa of the neutral red is probably low enough that at pH 7 (the blood pH) perhaps 10 per cent was un-ionized and thus was trapped in an acid medium in the lysosome or adsorbed there to something. I t is interesting that this adsorption caused a swelling which would mean that the osmotic properties of the lysosome were suddenly changed. What intrigues me very much is the reversibility. What could have happened is that perhaps the dye was very rapidly metabolized and the metabolite leaked out again, or whatever it was combined with was broken down and was very rapidly resynthesized. KOENIG:It is likely that the unionized form of neutral red penetrates the barrier. Once inside the nervous system, a portion becomes ionized and is bound primarily to lysosomes, which in the living cell seem to be polyanionic particles. The changes then produced in lysosomes, which have a lipoprotein matrix, are interesting, particularly the rapid revesibility of these changes. Within ten hours the whole sequence returns to normal. Lysosomes thus exhibit a structural plasticity which wasn’t recognized hitherto. Upon uptake of neutral red, these organelles go through a remarkable sequence of morphological changes, including the formation of myelinlike structures. The positive charge of the dye molecules may disturb the morphology of the lipoprotein molecules in such a manner that they assume an orderly linear or lamellar arrangement. What this really means on a molecular level, I don’t know. The swelling and evacuolation of lysosomes is due to an influx of water. This may be due to an increase in number of osmotically reactive particles, the limiting membrane functioning as a semipermeable membrane. Alternatively, the influx of water may be due to increased solvation of the colloidal matrix to which dye molecules are bound. As for the disposition of the dye, Morgan, some ten years ago, recovered only about 30 or 40 per cent of administered neutral red in the urine, and suggested that the tissues degraded the rest. I don’t know whether neutral red is degraded in lysosomes or merely leaks out in time. CSAKY: In order to leak out either the pK or the pH has to be changed.

KOENIG:I don’t know what happens to the dye, except that it disappears in five or six hours.

I understand that you have buffered your solution, but have you also buffered your mitochondria?

QUADBECK:

KOENIG:That is a very good point. The mitochondria1 matrix probably has considerable buffering capacity. Whether the formazon causes the changes or the HCI is not clear. It is interesting that the tetrazolium does localize to the mitochondrion in the form of the insoluble formazon. The remarkable swelling of mitochondria suggests that there is an increase in the number of osmotically active particles within this organelle. MCILWAIN:Relationships can be seen between the dyestuffs and isolated cerebral tissues. We incubated isolated cerebral tissues with neutral red and a number of other related compounds and found uptake by the neutral red granules; but this was accompanied by quite large metabolic changes in the tissue as a whole. There was a great increase in glycolysis, and these changes could fit in very well with the disorganization of the lysosomes, described by Dr. Koenig, with the liberation of some active phosphatases. Do you postulate any relationship between the cytological alterations and the other changes that you describe? Is there any similarity in time sequence?

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KOENIG:We were hoping to demonstrate such a relationship between the central effects of neutral red and a druglike chlorproniazine, and their localization in lysosomes. So far we have not been able to demonstrate release of hydrolytic enzymes from the lysosomes ill vivo, so our findings may not be related to the pharmacological effects. We found a significant concentration of neutral red in nerveending, or synaptosome fractions of rat brain. This may be responsible for the pharmacological effects (Koenig, Gaines, Nelson, Hurlebaus and Cavender, Tram. Amer. Neurol. Ass. 90, 189, 1965). Basic dyes also inhibit cholinesterase activity. Thus there are several possible mechanisms by which a drug such as neutral red may exert its central effect.

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The Evidence that Ganglioside, a Metal, and Chemical Energy are Involved in the Serotonin Household of Synaptic Vesicles from Brain D. H . D E U L * , J A N N Y A. H A I S M A

AND

J. F. L. V A N B R E E M E N

Biochemistry Deppurtiiierit, Itistitute of Psychiairy, State University, Grotiiiigeii, The Netherlands

In an attempt to gain some knowledge about the physiological function of serotonin, we wished to study its binding to synaptic vesicles. The evidence that this binding might be mediated by gangliosides is the following: ( I ) Woolley and Gommi (1964) needed a treatment with the enzyme neuraminidase to inhibit the contraction of smooth muscles by serotonin. Neuraminidase is capable of splitting off the neuraminic acid moieties of gangliosides. (2) Marchbanks et a/. (1964) could inhibit the complex formation between serotonin and synaptosomes (formerly called pinched-off nerve ending particles) by LSD. The binding of LSD could be inhibited by neuraminidase treatment. (3) Albers and Koval (1962) found that serotonin can be bound by isolated gangliosides. (4) Eichberg et a/. ( 1964) showed that synaptic vesicles contain gangliosides. The evidence that chemical energy might be involved is provided by the observations of: (1) Marchbanks et a/. (1964), that 2-Br-LSD could be bound to synaptosomes only in the presence of phosphate ions, (2) Whittaker and Sheridan (1969, that synaptic vesicles undergo a morphological change on treatment with phosphate ions, (3) Hosie (1969, that synaptic vesicles possesss an adenosine triphosphatase that can be distinguished from a similar enzyme in membranes which is stimulated by sodium and potassium ions and from one in mitochondria which is stimulated by dini trophenol. We were able to provide more evidence by the following experiments, which will be published in full details elsewhere. By hypo-osmotic treatment of a crude mitochondria1 fraction from rat brain, an Mg-fraction was prepared according to de Robertis et a / . (1963). This fraction was centrifuged on a sucrose gradient, and the synaptic vesicles were isolated from the layer just below the water-0.4 M sucrose boundary (Whittaker et al., 1964). Photo-

* Present address: N.V. Philips-Duphar, Refercwrs p. 129-130

P.O. Box 2, Weesp, The Netherlands.

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graphs of the preparations ‘obtained by electronmicroscopy’ were analysed with a Zeiss particle-size analyzer TGZ-3. The statistical analysis by the method of Hald (1952) of the distribution-curves obtained showed the presence of particles of 420 A (n = 2830; SD = 87 A) and of 71 3 8, (n = 378; SD = 130 A). These results indicate the presence of synaptic vesicles (see also Janiik and Glees, 1966). The particles mentioned represented 95-99% of the particles present, the contamination consisting of membrane fragments. The binding of serotonin to synaptic vesicles or isolated gangliosides was indicated by increased values of the polarization of the fluorescence light of serotonin at 337 mp (Laurence, 1952, 1957). Such polarization values, P,were recorded for each ratio, n = moles serotonin/mole ganglioside used. The gangliosides were determined by the neuraminic acid content according to Warren’s method ( 1 963). Results as obtained with synaptic vesicles from brain in 0.1 M citrate buffer pH 6.0 are shown in Fig. 1. Three maxima at n-values 4.5, 6, and 9 are superimposed on a

420 0.19 0.18 0.1 7 p

016 0.1 5 0.14 0.1 3

012

0 1 2 3 4 5 6 7 8 91011 n

Fig. 1 . The change in fluorescence polarization of varying mixtures of serotonin and synaptic vesicles from brain in 0.1 M-citrate buffer pH 6.0. The abscis n denotes the ratio moles serotonin/rnole synaptic vesicle ganglioside.

general curve. The three maxima indicate the existence of three complexes between serotonin and vesicles. The general curve under the maxima could be explained by the turbidity of the suspension of vesicles, making any interpretation of the P-values impossible. A clear solution of isolated gangliosides provided a very similar

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picture. The maxima were found at n = 4, 6.5, and 9, while the “turbidity-curve” was absent. Treatment of both types of preparations with neuraminidase resulted in a general decrease of the n-values for maximal P-values by 4-1 unit. This was explained by a general decrease rather than a total loss of binding capacity. The similarity in these results supports the evidence that gangliosides form at least a part of the vesicles’ binding sites for serotonin. The effect of phosphate on the binding of serotonin to synaptic vesicles was investigated, and also the effect of pH. Whittaker, Michaelson and Kirkland (1964) observed that the vesicles aggregate at pH 4 with a concomitant rapid release of acetylcholine. By producing results, as represented in Fig. I , at various pH’s in the presence and absence of phosphate we could represent the n-values for maximum polarization for the three maxima under these conditions in Fig. 2.

l3 12 ‘brnax)

11

t

0

9

a a 7 “(Prnax) 6

5 4

Fig. 2. The influence of pH on the accessibility of serotonin to the binding sites of synaptic vesicles from brain in the presence and absence of 0.1 M-phosphate ions. The unit n(Pmax)denotes the ratio I t = moles serotonin/mole ganglioside for which a relative maximum as demonstrated in Fig. 1 is obtained. Straight lines were drawn through the experimental points, the lines being calculated by the least squares method. 0:without phosphate ions; A:with phosphate ions. The dashed lines approaching the limit n(Pnlax)= 0 at pH 3.2 indicate the impossibility of synaptic vesicles to bind serotonin at that pH. R ~ ~ f i i ~ v 1). i c ~129-130 s

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It can be seen that the three maxima were observed at every pH tested from 4 to 10. The vesicles could thus bind serotonin at pH 4. However, by light-microscopy with dark field illumination and by measurement of the light that can be scattered by the particles we could confirm that the vesicles aggregated maximally at that pH. It was quite astonishing though that pH 3.2 was a second condition for aggregation of the particles. We could show that the aggregation is a reversible process. A subsequent experiment showed that a preparation which could bindlserotonin at pH 4, in the way indicated in Fig. 2, could not do so at pH 3.2, as indicated also, whether phosphate was present or not. It follows from Fig. 2 that in the absence of phosphate the binding capacity is increased by decreasing the pH, while this capacity is not influenced by pH in the presence of phosphate. It thus seems that phosphate protects the binding capacity of synaptic vesicles from changes in pH of the environment. The opposite could be equally important of course. Similar experiments with isolated gangliosides produced results which could best be interpreted as effects of the ionic strength of the salt or buffer being used on the variability of the micelle formation of gangliosides (Trams and Lauter, 1962). Another difference between isolated gangliosides and vesicles was demonstrated by the use of 0.1 M arsenate buffer instead of phosphate. While serotonin could be bound to the gangliosides in the usual manner, a picture like Fig. I could not be obtained with synaptic vesicles under this condition. Instead, the underlying curve from the light-scattering was obtained without any indication of complex-formation. It seems likely, therefore, that the effect of phosphate described is rather specific for the synaptic vesicles. It thus became interesting to test the hypothesis that the binding of serotonin to the vesicles is influenced by the energy household of the cell or vesicle. We could confirm the results of Hosie (1965) with respect to the distinction of the adenosine triphosphatase of the vesicles from other similar enzymes. Besides, we found that this particular enzyme had its pH-optimum at 6.5, i.e., in a H+-ion concentration area where phosphate has no influence on the serotonin-binding capacity. In addition we observed the presence of an adenosine triphosphate: creatine phosphotransferase (E.C. 2.7.3.2.) (“creatine phosphokinase”) in the vesicles. Although this enzyme is considered to be freely dissolved in the cytoplasm, it follows from the preparation procedure that it is also inherent to the vesicles. The next step was to see whether the reactions catalyzed by these two enzymes were significantly influenced by serotonin or acetylcholine. Concentrations of 1-3 x 10-8 M of these amines (the ratios n = 4.5,6, and 9, were used) produced about a doubling of the ATPase activity and a 20-30 % inhibition of phosphocreatine production by the creatine phosphokinase. Although these results favour the availability of ATP and thus of phosphate for the binding of serotonin, they appeared to be hardly reproducible with various preparations. As similar inconsistencies were observed by Hosie, we think that further support for the evidence provided might be produced when the preservation of the synaptic vesicles and their enzymes is understood and can be controlled better.

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Our tentative conclusion from these results is that the binding of serotonin to synaptic vesicles might be regulated by the consumption or production of energy by either the cell or the vesicle. The evidence that a metal might participate in the binding of serotonin to synaptic vesicles was provided by Colburn and Maas (1965), Maas and Colburn (1965) and Roberts (1966). Colburn and Maas demonstrated that in the centrifugation procedure copper is enriched to the greatest extent in a fraction which they call synaptic vesicles but which is in fact De Robertis’ fraction M2, i.e. a mixture of vesicles, synaptosome ghosts, and mitochondria (Whittaker, Michaelson and Kirkland, 1964). In this respect we observed that the three maxima as represented in Fig. 1 could be produced with isolated gangliosides in the presence of 10 mM-Naz EDTA, pH 5.0. With synaptic vesicles, however, these usual three maxima could not be obtained under theseconditions. In the presence of 5 p M EDTA, the vesicles showed a decreased binding capacity for serotonin as the n-values for maximum polarization were decreased. Therefore, on the basis of our experiments, we are also inclined to think that a metal atom is involved somehow in the binding of serotonin by synaptic vesicles.

REFERENCES AND KOVAL, G . J. (1962) The interaction of gangliosides with cationic molecules. Biochint. Biophys. Acra, 60,359-365. COLBURN, R. W. A N D MAAS,J. W. (1965) Adenosine triphosphate-metal-norepinephrineternary complexes and catecholaniine binding. Nature, 208, 37-41. EICHBERG, JuN., J., WHITTAKER, V. P. AND DAWSON, R. M. (1964) Distribution of lipids in subcellular particles of guinea-pig brain. Biocheni. J., 92, 91-100. HALD,A. (1952) in Statisrical Theory with Engineering Applications, Chap. 6.10. New York. Wiley and Sons, Inc. HOSE, R. AND JEANETTE, A. (1965) The localization of adenosine triphosphatases in morphologically characterized subcellular fractions from guines-pig brain. Biochem. J., 96, 404-412. JAN~IK H., AND GLEES,P. (1966) Dens? and clear synaptic vesicles in the central nervous system. Naturiviss., 53, 385. LAURENCE,D. J. R. (1952) A study of the adsorption of dyes on bovine serum albumin by the method of the polarization of fluorescence. Biochent. J., 51, 168-180. - (1957) Fluorescence techniques for the enzymologist, in: Merhocls in Enzymology, IV, Colowick, S. P. and Kaplan, N . 0. (Eds.), 174-212. New York, Academic Press, Inc. MAAS,J. W. AND COLBURN, R. W. (1965) Co-ordination chemistry and membrane function with particular reference to the synapse and catecholamine transport. Narure, 208, 41-46. MARCHB~NK R.S , M., ROSENBLATT, F. AND O’ERIEN,R. D. (1964) Serotonin binding to nerveending particles of the rat brain and its inhibition by lysergic acid diethylamide. Science, 141, 1135-1 137. ROBERTIS, E. DE, ARNAIZ, G. R. DE L., SALGANICOFF, L., IRALDI, A. P. DE AND ZIEHER. L. M . (1963) Isolation of synaptic vesicles and structural organization of the acetylcholine system with brain nerve-endings. J. Neurochenr., 10, 225-235. ROBERTS, G . C. K. (1966) The formation of complexes between 5-hydroxytryptamine, adenosine triphosphate and divalent cations in vitro. Biocheni. J., 100, 30 P. TRAMS, E. G. AND LAUTER, C. J. (1962) On the isolation and characterization of gangliosides. Biochini. Biophys. Acra, 60,350-358. WARREN, L. (1963) Thiobarbituric acid assay of sialic acids, in: Methods iti Enzymology, VI, Colowick. S . P. and Kaplan, N. 0. (Eds.), 463-465. New York, Academic Press, Inc.

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WHITTAKER, V. P., MICHAELSON, I. A., KIRKLAND, R. AND JEANETTE, A. (1964) The separation of synaptic vesicles from nerve-ending particles (“Synaptosomes”). Biochem. J., 90,293-303. WHITTAKER, V. P. AND SHERIDAN, M. N. (1965) The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles. J. Neurocheni., 12, 363-372. WOOLLEY, D. W. AND GOMMI,B. W. (1964) Serotonin receptors: V, Selective destruction by neuraminidase plus EDTA and reactivation with tissue lipids. Naiure, 202, 1074-1075.

D 1SC U S S l ON MANDEL: I would like to make a general comment about vesicles. Whenever we find a destruction in a cell or destruction of membranes in particular, the result is the formation of some vesicles. Therefore, we have to be quite careful that all vesicles are synaptosomes. DEUL: If one is not familiar with the sort of particles mentioned in the paper, a confusion of the nomenclature is likely to occur. A “mitochondria1 fraction” from brain is usually called “crude” because it is, a.o., contaminated with synaptosomes. This can be considered to consist of a part of the cell membrane containing the synapse. On disruption of the cell membrane this is thought to close by some sort of artificial process, therewith forming a closed bag, the synaptosome. Such a synaptosome contains synaptic vesicles and mitochondria, which can be isolated therefrom by hypo-osmotic disruption, followed by differential centrifugation and centrifugation on a sucrose gradient. The latter technique provides the separation of synaptic vesicles from membrane fragments and mitochondria. The conviction that we worked with synaptic vesicles is based on three observations: 1. The average size of the particles was observed to be in agreement with statements in the literature. 2. In agreement with Hosie (1965) we could distinguish the ATP-ase of the vesicles from the mitochondrial ATP-ase which is stimulated by dinitrophenol and from the membrane ATP-ase which is stimulated by Na+- and K+-ions. 3. The enzyme creatine phosphokinase is considered to be located mainly in the cytoplasm. We could also detect this enzyme in our preparations of synaptic vesicles.

KOENIG:Could you tell us how you obtained your synaptic vesicles? Most of the published methods result in a very substantial contamination of this fraction by membranes. We find that morphologically pure preparations contain very little ganglioside. The gangliosides seem to be associated almost exclusively with the membranous portion of the synaptosomes rather than with the vesicles themselves (Koenig, Gaines, Nelson, Hurlebaus and Cavender, Some Studies of Synaptic Vesicles, Trans. Anrer. Neurol. Assoc., 90, 189,1965).

DEUL:De Robertis’ fraction MZ (synaptosome ghosts and fragments, mitochondria and synaptic vesicles) was placed on top of the discontinuous sucrose gradient as used by Whittaker. The synaptic vesicles such as used in our experiments were found at about the interphase between the water and the first sucrose layer (of 0.4 M concentration), let us say at about 0.34.35 M sucrose. Synaptic vesicles can be found in the layers with higher sucrose concentrations as well but there they are progressively contaminated with so called membrane fragments. Therefore we usually almost filled the centrifuge tube with 0.4 M sucrose and placed the fraction MZ on top. The use of this procedure gives a clearer separation between the pure and contaminated synaptic vesicles while the latter are much better forced towards the bottom of the tube. KOENIG:By using 0.4 M sucrose, according to Whittaker you still have a large membranous contamination. Almost pure synaptic vesicles devoid of gangliosides can be collected by spinning osmotically ruptured nerve endings at a 0-0.2 M interphase (Koenig et a/., unpublished). DEUL:In the paper I stated three reasons to believe that in the synaptic vesicles gangliosides might be involved in the binding of serotonin. So, instead of using the protein content we expressed our observations in terms of the neuraminic acid content. We simply called that the ganglioside content ( I .57 mole neuraminic acid per mole ganglioside with an average molecular weight of 1700). In this way we neglected the possibility that the acid might be bound to non-gangliosidic constituents and

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that not all synaptic vesicles do have to contain neuraminic acid or ganglioside. It is a pity that we did not demonstrate any gangliosid-s in the vesicles, for example by thin layer chromatography. The three evidences mentioned were strengthened by the striking resemblance between our results obtained with synaptic vesicles and isolated gangliosides. We found an average value of 100 pmole “ganglioside”/g protein. Let us now suppose that this were totally present in the contaminating membrane fragments (assume a 10% contamination instead of the 5 % found, and an equal protein distribution), then these fragments should have I mmole “ganglioside”/g protein. This order of magnitude is rather unlikely. Values of pmoles neuraminic acid/g protein can be calculated from data collected by McIlwain (1963) for a preparation from brain tissue rich in membrane fragments (Table 7,p. 86 for correction for differences in centrifugal forces used and the last line of Table 9, p. 90). Similar values were found by Eichberg er al. (1964), Wallach and Eylar (1961) and Patterson andTouster (1962), who worked with synaptic vesicles, membranes from Ehrlich ascites-carcinoms cells and membranes from liver cells respectively. We therefore feel quite sure that our findings were caused by the synaptic vesicles and not solely by the few membrane fragments present. Our measuremmts of the light scattered by the synaptic vesicles at varying pH gave reproducible results qualitatively. However, if the turbid layer of vesicles was isolated from the centrifuge tube in two parts (e.g. an upper and a lower half) slight differences between the light-scattering properties of these parts were observed under various conditions. In the light of Dr. Koenig’s remark I now wonder whether this sort of variation correlates with the results with vesicles that are purified with density gradients of greater resolution. LITERATURE: MCILWAIN, H. (1963) Chemical exploration of the brain. Amsterdam, Elsevier, Publ. Co. PATTERSON JR., M. K. AND TOUSTER, 0. (1962) Intracellular distribution of sialic acid and its relationship to membranes. Biochim. Biophys. Acta, 56,626-628. D. F. H. AND EYLAR,E. H. (1961) Sialic acid in the cellular membranes of Ehrlich ascitesWALLACH, carcinoma cells. Biochim. Biophys. Acta, 52,594-596. CSAKY:The fact that you have a varying amount of ATPase activity also suggests that you may have a membrane contamination. This could be checked by measuring the ion requirement of your ATPase. If you have a sodium-potassium dependent ATPase, which, in turn, you can completely eliminate by strophantine or ouabain, then you have a good indication of a possible membrane contamination.

DEUL: I agree with you that any sodiurn/potassium-dependency should be a measure for membrane contamination. I mentioned before already that we could distinguish the ATP-ase of the vesicles from similar enzymes from membranes or mitochondria by its independency of stimulation by either sodium- and potassium ions or dinitrophenol. However, I am inclined to think that the variability of the vesicles’ ATP-ase activity that we observed might be due to factors such as the preservation of the quality of the vesicles which are not understood and therefore can not be controlled a t the present time. The evidence for it is the fact that the enzyme is liable in quite a varying way to inhibition by excess of the substrate ATP, suggesting a variable quality of the enzyme. MANDEL: Did you see what was the effect of pyrophosphate? Was it the same as that of ATP? DEUL:I have not tried that.

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GENERAL DISCUSSION

FORD: Dr. Pappas as well as many others have shown quite clearly a series of anatomical membranes, starting with the endothelial cell and culminating with the membrane of the neuron which certainly seemed to keep out at least methylene blue, and of course, many other dyes as well. However, I know that Dr. Koenig has managed to get neutral red into the lysosome following an in viva technique, which brings us to the question of whether these membranes constitute a barrier or whether we are dealing with ii great deal of selectivity. If one uses some of our traditional dyes which go back t o Ehrlich, we have no difficulty showing a barrier, but with other substrates which look different we begin to see perivascular spaces, perhaps many different kinds of channels, and then when one uses even more physiological substrates one wonders what the situation is. After all, the nerve cells utilize metabolites at tremendous rates. I wonder if these anatomical membranes can really be shown to inhibit amino acid entry. PAPPAS: I agree with you and with others in this conference in saying that there is not a single barrier here and that the membrane selectivity is a very important phenomenon. We have tried to measure, for example, with Evans blue, a local breakdown in the barrier by freezing certain portions of the cortex, or applying a droplet of chloroform-methanol to the cortical surface. Certainly those damaged areas could be seen to turn practically blue. However, examinations of these areas showed that what happened was that small blood vessels have actually ruptured; so that these methods which werr: supposed to measure a localized lesion in the blood brain barrier really measured a localized lesion in the blood vessels. K O E N ~ GIt: should be emphasized that basic dyes differ markedly from acidic dyes. The brain is fairly permeable to basic dyes, while the acidic dyes such as Trypan blue do not penetrate the central nervous system. One reason may be that acid dyes bind to serum proteins. If one injects acid dyes intrathecally, some penetration occurs. As contrasted to the basic dyes which stain lysosomes, acid dyes selectively stain cell nuclei. Acid dyes perhaps stain histones or the nucleohistone complex. it is interesting in this respect that Trypan blue can cause malformations and genetic defects, pointing out its possible complexing with histones. STEINWALL: As regards the apparent difference in blood-brain permeability of acid and basic dyes this is not primarily a matter of negative or positive charge but more dependent on the degree of ionization and lipid solubility. Both basic and acid dyes are unable to permeate into the brain provided they are fully dissociated and thusexist in the blood as lipid-insoluble ions. The blood-brain permeation of certain basic dyes is adequately explained by the fact that they have a considerable non-ionized lipid-soluble fraction. This was illustrated by Dr. Koenig's work with tetrazolium salts and neutral red, the formx being much more ionized than the latter.

KLATZO:Generally, at the present time there is some disinclination to become involved in discussions of the morphological substrate of the Blood-Brain Barrier. This lack of enthusiasmmight be due partly to increasing realization of the complexity of transfer mechanisms which can possibly involve a great variety of structural components, and due partly to prudent hesitancy to draw conclusions about function from the static morphological pictures. However, using morphologically traceable indicators certain definite conclusions seem to be justified. On the basis of fluorescent microscopic observations there is no doubt in my mind that under normal conditions the crido/he/iurn, especially at its luminal surface, constitutes the essential barrier to proteins where this barrier function is present in the brain and the peripheral nerve. In the latter location, very recently Olsson's elegant demonstration ( A c t a Neuropath., 7, 1-15, 1966) of the barrier to proteins in blood vessels of the endoneurium and its absence in those of the external nerve sheaths indicates again that it is errdotheliunr and not the frequently implicated tight basement membrane-glia complex which is responsible for the phenomenon. KATZMAN: I want to recall an article which was published in the 1938 ARNMD by King (King: Some Aspects of Hematoencephalic Barrier. A. Resenrch Nerv. Ment. Dis. Proc., 18: 150-177, 1938.) who postulated that the barrier effect is due to the absence of connective tissue in the capillary walls in the central nervous system. You discussed 200 Angstrom spaces in which particles can move quite freely. I wonder if you could comment on some more recent reports which said that the size of these spaces is a fixation artifact, and that in freeze substitution methods the spaces are much larger.

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PAPPAS:The freeze substitution methods, the type that Van Harreveld and others have used, show a rather poor preservation of the cytoplasm of both neurons and glia, and therefore 1 would call these large spaces artifacts which are produced by the methods used. After all, one of the criteria we may have is to look how well the cells themselves are preserved. There was a paper published by Larsen and Schultz at UCLA who claim that there was no extracellular space after perfusing with glutaraldehyde in the rat brain. Again, in these preparations, the organelles in the cytoplasm are completely washed oL;t, so we have to watch not only what spaces appear but also what structures are preserved and what structures are destroyed or altered. These results would make me believe that perhaps the more nearly correct picture is the one of the 200 Angstrom spaces that I have discussed.

ELLIOTT:I am interested in the endophilic coat, the glycocates. It seems to me that there is a continuum of transport mechanisms from electron transport to particle transport all the way up to the large bunch of things transported by pinocytosis. If this is so, I wonder if they would all haveenzyme substrate relationships. If this is so, it seems to me these glycocates should have certain specificity. Do you have any picture of what kind of substrates stick to these glycocates? PAPPAS:In terms of the endothelium, I do not have any knowledge, but some years ago while working with Dr. Brandt on the extraneous coat with living amoebae, we found that certain things, such as proteins, would be absorbed on this fuzzy coat and reach a critical amount, but would not increase beyond that, and would not block the binding on the surface of other materials. This showed that there seemed to be specific binding sites. This is shown by morphological studies that we have made on the amoeba; but others observed similar things with autoradiographic methods with various ions and compounds. So one substance seems not to inhibit the binding of another substance.

RALL:When we discuss the penetration of a compound, or lack of penetration of this compound in the nervous system, we should really mention and measure the physical and chemical characteristics of these compounds, among them the pKa, lipid solubility, plasma protein binding. May I ask what Dr. Pappas thinks the size of the extracellular space actually is in the brain? PAPPAS:This will depend on the presence of cellular elements in the particular brain area, of course. If you have very small processes, which you have in the molecular layer, for example, there would be much more surface and therefore much more extracellular space, still maintaining the 200 Angstrom relationship of the opposing membranes. If you have large cellular elements, then the extracellular space would be limited for that particular area.

TOWER: Dr. Pappas brought up a new point here. In the past we have heard mostly about 150 Angstrom spaces. I am delighted to hear the value is up to 200 Angstrom and I wonder what it will be next year. I want to emphasize, however, his statement that it also depends on the presence of cellular elements and the size of these elements because these factors vary, not only from one area of the brain to another area, but also from one species to another. Certainly, cellular elements are not the same size in mouse and chimpanzee, for example. Moreover, cortical extracellular space is not the same as thalamic extracellular space.

I would also like to emphasize that all we were seeing and talking about right now was the BAKAY: cortex, and by implication, gray matter. The white matter is a quite different substance. The intercellular cleft in the white matter is quite a bit larger and we have seen even 600 Angstrom clefts occasionally, which has to be taken into consideration when one calculates the total size of extracellular space in the brain from electronmicroscopic pictures. The calculations of Meves and Horstmenn were perfectly straightforward. Nevertheless, the margin of error is quite large. But the question can also be raised whether it is necessary to postulate this very large extracellular space, because Kuffler’s experiments showed very beautifully that fairly large molecules can move in very small spaces. Because the elements are so heterogeneous, I don’t think one can make any really meaningful calculations by just observing these 200 Angstrom clefts in the electron-microscope. TENNYSON: We know from electron microscopic studies that we can producelargespacesin thenervous system from either rupture or swelling of glial tissue. In fact, that is the easiest artifact we can produce. One of the explanations, therefore, for the discrepancy in the size of the spaces as seen by electron microscopy and that measured biochemically may be the swelling and rupture of astrocyte processes,

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CUTLER: We have studied the extracellular space of about 20 areas of cat brain, by a technique suggested by Davson. Equivalent concentrations of 3% sulfate were produced by combined intravenous injection and continuous ventriculo-cisternal perfusion of isotope. In areas well-bathed by perfusate, thercgional sulfate spaces were all about 15 percent.

Cerebrospinal Fluid

Cerebrospinal Fluid Transport R. V. COXON Ut1iver.vir.v Laboratory of Physiology, Oxford, England.

In a recent symposium (Brooks, Kao and Lloyd, 1965) concerned with the cerebrospinal fluid (CSF) two consecutive papers opened with two contrasting statements. The first, emanating from Drs. Hugh Davson and Michael Bradbury, and referring to the origin and fate of the CSF, was as follows: “Essentially the present viewpoints differ very little from those elaborated by Weed between 1914 and 1935”. The second came from Dr Edgar Bering, who asserted that the results of recent investigations of CSF physiology have provided information which substantially modifies the old concept of the CSF circulation proposed by Weed (1914). Clearly it all depends on what one means by “essentially” and “substantially”. However, both authors quoted take Weed’s (1914) concepts as their starting point and I shall follow suit. Weed’s view was that the CSF is formed very largely in the choroid plexus and passes thence into the subarachnoid space from which it is absorbed into the blood through the arachnoid villi*. The first modification of this view, which one may or may not choose to regard as substantial, is imposed by the likelihood that only about two-thirds of the CSF is formed by the choroid plexus. By perfusing on the one hand the ventricle and on the other the subarachnoid space with solutions of inulin and using the inulin clearance as a measure of the rate of absorption, and hence deducing from outflow-inflow differences the rate of addition of newly formed CSF, Bering and Sato(1963) found in the dog that about 40% of the CSFis produced extraventricularly. The same appears to be true of the cat, while studies in hydrocephalic infants suggest it may also hold for man. Some results (quoted from Bering, 1.965) are given in Table I . The exact site of intraventricular production of CSF is itself also somewhat unclear. There is no doubt that fluid is secreted by the choroid plexus, since it can be directly sampled from the surface of the plexus exposed in an oil-filled ventricle, but on the other hand, CSF formation can take place in a lateral ventricle from which the-choroid plexus has been surgically removed (Bering 1965). Moreover, the claim has:been made by Jacobi and Magnus (1925) that fluid can actually be seen seeping from the ependymal wall into the ventricle. A second modification of the classical concept, which may be considered substantial, results from the demonstration by Welch and Friedman (1960) of channels

* Weed in 1917 recognised a “small addendum” to the choroidal secretion from the perivascular spaces on the surface of the brain and also mentions such a source in his 1914 paper. The change of view in recent years is thus really of a quantitative nature. Rcfcrmcrs p . 142-144

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penetrating the arachnoid villi in the monkey which have valve-like properties being patent when the subarachnoid pressure exceeds that in the sagittal sinus, but closing down when the pressure gradient is reversed. The substitution of channels (of capillary diameter) for the semi-permeable membrane which was previously assumed to separate CSF from blood provides a ready explanation of the otherwise very puzzling observation of Simonds (1953) that labelled erythrocytes can pass from the CSF into the TABLE I O R I G I N O F C S F (From Bering, 1965)

Species

Man Dog Cat

Rabbit

yh front ventricles 55 58 60 78

blood. It is thus no longer necessary, as Davson was constrained to do in 1956, to “attribute the rapid disappearance of blood from the subarachnoid space to a special facility of the erythrocyte for squeezing its way through cellular membranes, presumably through intercellular cement”. Although the existence of the channels identified by Welch and Friedman seems now to be widely accepted, some doubt remains as to their quantitative importance in the normal process of absorption of CSF The timehonoured argument against the importance of the arachnoid villi as a drainage route based on their absence in certain species of animal and in many young animals must still carry some weight, and to this must be added the demonstration by Bering and Sato (1963) that absorption of perfused artificial CSF can take place from the ventricles of dogs when these have been artificially isolated from the subarachnoid space. Thus it seems proper to think of the flow of CSF as taking place in a channel, which, while it may begin at the choroid plexus and end at the arachnoid villi, is bounded by very leaky surfaces. In considering the general hydrodynamic aspects of CSF production and drainage some reference should be made to the effects of varying hydrostatic and osmotic pressure in the ventriculocisternal system. These effects have been studied in detail by the Pappenheimer group (Heisey, et al., 1962) utilizing their elegant method for perfusing these regions in the goat. It turns out that the rate of formation is virtually independent of the perfusion pressure, but the rate of bulk removal of fluid is very sensitive to pressure. The crystalloid osmotic pressure of the perfusing fluid affects predominantly the rate of formation of CSF, a perfusion fluid hypertonic to plasma leading to an increased output of CSF; however, CSF continues to be formed even in the presence of a hypotonic perfusion fluid. As might be expected the diffusion of tritiated water out of the perfusion fluid is unaffected by the perfusion pressure. Now it has long been realised that by virtue of the differences in composition between

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it and an ultrafiltrate of plasma, CSF must be regarded as a secretion elaborated as a

result of selective action on the part of certain cells, and the demonstration that substances such as diodrast and P.A.H. can be actively transported out of the CSF into the blood against a concentration gradient (Pappenheimer, Heisey and Jordan, 1961) means that the ultimate composition of the CSF may well result from the combined contributions of secretory and reabsorptive processes in much the same way as the composition of urine is determined by the secretory and reabsorptive activity of different parts of the nephron. (Severinghaus (1965) has in fact referred to theCSF as “neural urine”). Nevertheless it would clearly be an oversimplification to regard the choroid plexus as analogous to the renal glomerulus. This is evident from the studies of Ames, Sakanoue and Endo (1964). In the case of Mg21 and Ca2+ there are clearly departures in the choroidal secretion from the concentrations which would be expected for a simple ultrafiltrate. Moreover the fact that these divergences are in opposite directions for two ions of the same sign rules out any simple effect of the electrical potential difference between CSF and blood. Such a potential difference (p.d.) is, however, itself of some interest, although its nature is not by any means fully elucidated. Early reports regarding this potential were conflicting not only concerning its magnitude but also its sign. Thus Tschirgi andTaylor (1958) recorded the CSF as 1-5 mV negative with respect to blood, while Mitchell, Loeschcke, Massion and Severinghaus (1963) recorded a p.d. of +5 to +12 mV with respect to an indifferent electrode in the extracranial tissues. The explanation of these discrepancies probably lies partly in the differing characteristics of the recording systems used and partly in the fact that the potential varies under different conditions; for example, it is markedly sensitive to the pH of the arterial blood. Moreover ouabain has the effect of greatly reducing the slope of the dPd/dpH line. The exact origin of the potential is obscure but Held, Fencl and Pappenheimer (1964) have emphasized that its presence must, as they say, “contribute to the steady-state concentration ratios of all charged particles which exchange between CSF and plasma”. Comparison of the actual concentration of the principal ions undergoing such exchange with the calculated concentration assuming passive distribution in the electric field resulting from the observed normal potential of +5 mV reveals that only CI- and Ca2f could be passively controlled in this way. K + and HC03- appear to be actively transported from CSF to blood, while Na+ and Mg2+ are actively moved from blood to CSF However, as a result of the bulk flow of the fluid the final concentration of all ions in the cisternal outflow will depend both on the flux rate across the actively transporting epithelia and the rate of removal of the fluid in contact with their surfaces. The bicarbonateion has assumed particular significance in the last fifteen years or so as a result of the implication of the CSF in the control of respiration (see Leusen, 1950), and it is also of special interest in relation to the effect of carbonic anhydrase inhibitors on the formation of CSF From the viewpoint of the study of the CSF the importance of the experiments made in recent years by the respiratory physiologists is that they demonstrate the ability of animals (including man) to maintain the level of bicarbonate in the CSF at a different value from that in the blood plasma over a period of hours and even days (Swanson and Rosengren, 1962). Steep gradients in bicarbonate Concentration have Rrfrrmces p. 142-144

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also been observed between blood and artificial CSF in perfusion experiments (Pappenheimer, Fencl, Heisey and Held, 1965). The effects of carbonic anhydrase inhibitors on CSF production and composition have been extensively studied and are well documented. In the cat, for example, the administration of such drugs leads to a rise in bicarbonate concentration of 4.4 meq./l and a fall in chloride concentration (Davson and Luck, 1957), while it also apparently produces a 50% reduction in the rate of production of CSF in the normal rabbit. Furthermore it brings about a reduction of CSF pressure in hydrocephalic infants. Curiously enough no altogether satisfactory detailed explanation has been advanced to account for the effect on fluid production. The general tendency seems to be to relate this to reduction in chloride movement with presumably a secondary effect on water transport but since the reduction in chloride is accompanied by an almost equivalent increase in bicarbonate any simple osmotic effect is hard to visualise. However, the turnover of z4Na is also affected (Davson and Luck, 1957). This seems a suitable point at which to refer to some studies on the passage of 14Clabelled bicarbonate from blood to CSF which were carried out a few years ago in my laboratory in collaboration with Dr. A. G. Swanson (Coxon and Swanson, 1965). Such studies, of course, give no indication of the form (i.e. whether as C02 or HC03-) in which the administered material passes into the CSF but they d o provide information on the readiness with which overall exchange can take place between the two fluids. In a number of experiments [14C]bicarbonate was administered intravenously to cats and the specific activity of the total COZin the blood and CSF was followed. The specific activity became equal in the two fluids after a mean interval of some 10

I 0

5

10

15

20

min

Fig. 1 . Change in the ratio of specific activity of total C02 of blood to that of CSF following an intravenous injection of NaH14C03 in the cat. 0 = mean crossover time.

CSF

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TRANSPORT

min, indicated in Fig. I , where the times of attainment of equal specific activity in a series of experiments are indicated. Since we were interested in the penetration of isotope into the brain as well as into the CSF the animals were sacrificed at various times following the administration of the labelled bicarbonate, but it is possible to build up a comprehensive picture of the time-course of isotope distribution by combining the data from a number of animals. For ease of presentation the specific activities have been expressed in arbitrary units related to the actual value at the time when the specific activities in blood and CSF were the same. The composite results are shown in Fig. 2, from which it will be seen that the specific activity in the CSF, once it has risen to equal that in the blood, then

15~1

0

i,

10

20

30

40

50

60

70

00

90

Id0 min

Fig. 2. Composite curves showing the change of specific activity with time of the total C 0 2 of blood and CSF (and brain) following an intravenous injection of NaH14C03 in the cat. 0 = blood; A = CSF; 0 = brain.

decays with it in a more or less parallel fashion with a time lag of about 10 min over the steep part of the curve. It will be seen too that the values of brain specific activity lieveryclose to the blood curve and well separated from the CSF curve, due, no doubt, to the intimate relationship of the brain cells to the blood capillaries in contrast to the situation in the CSF where a considerable pool of rather stagnant liquid is in only very limited contact with the circulating blood. When labelled bicarbonate was injected into the cisterna magna some counts rapidly appeared in the blood but isotopic equilibrium was attained very much later than in experiments where the isotope was injected into the blood stream. I should like now to turn to a consideration of some non-electrolytes, whose rate of penetration into the CSF and brain tissue seems remarkably slow by comparison with References p . 142-144

140

R. V. C O X O N

their rate of entry into other compartments of the body water. These are, first, urea and secondly glycerol. In the case of urea, even at normal blood levels, there is a nonuniform distribution between blood plasma and c.s.f. Thus in the cat, Bradbury and I (Bradbury and Coxon, 1962) found the ratio of the concentration of CSF to that in blood to be 0.6 and figures varying from 0.58 to 0.82 have been reported for man. In the case of the dog the deviation from diffusional equilibrium is much less and a figure for the ratio in this species of 0.945 has been reported (Gad Andresen, 1921). At the time when Bradbury and I carried out our experiments we were principally interested in the penetration of urea into the brain but we made some incidental observations on the movement of urea into CSF when the blood concentrations were kept high (about 275 mg/100 rnl) by a continuous intravenous infusion. We were struck by the difference in the rate of movement of urea into the brain and CSF of the cat on the one hand and the dog on the other, and recently Mr. B. S. MacLean in my laboratory has made a more detailed study of the differences between these two species. Fig. 3 shows a comparison of the rates of movement of urea from plasma into the CSF in the cat and the dog. Kleeman, Davson and Levin (1962) showed that in the rabbit passage of urea into the brain and CSF was much slower than into muscle. However, in the steady-state condition the urea concentration appeared to be higher in the brain than in the CSF.

Fig. 3. The penetration of urea into the CSF of cat and dog indicated by the ratios (R)of the concentration in CSF to that in plasma at raised plasma levels maintained by intravenous infusion. The times are those from the start of the infusion.

In some later experiments in which the kinetics of entry and exit of urea from the ventriculocisternal system were studied Bradbury and Davson (I 964) came to the conclusion that “the transport of urea between blood, brain and CSF may be completely described in terms of diffusion and entrainment with streams of fluid” and they hint that previous reports of a greater concentration of urea in brain than in blood were probably in error. Bradbury and Davson suggested that urea is “entrained”,

CSF

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possibly by solvent drag, into fluid entering the ventricular system; the urea molecules must then move more sluggishly than the water and so appear in lower concentration in emerging fluid. Alternatively, but less probably, there may exist an active reabsorption mechanism for urea similar to that described in the kidney of certain frogs. Whichever type of process underlies the relatively low concentrations of urea in the CSF in the cat and some other species it is much less prominent in the dog. A t about the time when we were completing our first series of experiments with urea some clinical ophthalmologists (Casey and Trevor-Roper, 1963) began to take a lively interest in glycerol as an osmotically active agent capable of reducing the intraocular pressure. The efficacy of glycerol in achieving this seems to be clearly established and suggested to us that glycerol might share with urea the property of penetrating the water of nervous structures less readily than that of other tissues. The same idea evidently occurred to others and reports have recently appeared showing that a high concentration of glycerol in the blood acts in much the same way as a high concentration of urea in reducing intracranial pressure (Cantore et al., 1964). Moreover glycerol is effective in this way when given by mouth, whereas a continuous intravenous infusion is required in the case of urea, and, in addition, the glycerol effect is relatively free from rebound. Like urea, glycerol is present in small quantitites in the blood of many mammals. Someestimations by Britton (Britton, 1962) reveal that about 0.1 to 0.2 pM/I is usually found. In the dog this blood glycerol turns over very rapidly, figures of 2 to 3 min being quoted for its half-life (Hagen, 1963). Some early experiments by Holst (1944) and some later ones by Larsen (1963) have demonstrated that removal of glycerol from the blood takes place virtually exclusively in the liver and kidneys. These experiments were done on cats and led us to believe that very little, if any, metabolism of glycerol was to be expected in the nervous system. This belief was strengthened by the results of a survey of the tissues of the rat for the occurrence of the enzyme glycerokinase published from Wieland's laboratory (Wieland and Suyter, 1957) which revealed

"'1

04

Time (h)

Fig. 4. Concentration of glycerol in muscle and in CSF expressed as a fraction of that in plasma (R) at different times after establishing a high plasma level. (A = CSF, 0 = muscle). Refircnrrr p . 142-144

142

R. V. C O X O N

its absence from the brain, and by a statement by Elliott and Wolfe (1962) that glycerol is not metabolised by brain. Moreover, attempts to resuscitate animals from hypoglycaemia by the administration of glycerol have given results which suggest that any effect that glycerol may exert in this condition probably results from its conversion to glucose (Voegtlin et al., 1925). Possibly the incorporation of [14C]-glycerol into brain lipids reported by Davison and Dobbing (1960) may be similarly explained. However, despite its lack of glycerokinase activity, Toews (1966) has found that rat muscle is capable of oxidising small quantities of glycerol and he attributes this capability to the presence in muscle of a NADP-linked dehydrogenase converting glycerol to glyceraldehyde which then condenses with dihydroxyacetone to yield fructose-phosphate. In the light of Toews’ findings it is clearly necessary to re-check the metabolic fate of glycerol in brain preparations. We have not yet done this but I mention the problem by way of a cautionary note before embarking on a consideration of the penetration of glycerol into the CSF*. In the cat the volume of distribution of glycerol is some 65% of the body weight which means that it enters very nearly the whole of the body water. For eas: of comparison with our most recent data on urea we have studied glycerol principally in the dog, but it seems clear from indirect evidence that glycerol is also distributed widely in the body-water of that species also. The results of some experiments on glycerol penetration carried out in collaboration with Mr. B. s. MacLean are shown in Fig. 4. The blood concentration was maintained steady at about 25 mM in those experiments by means of a constant intravenous infusion. It is evident from these results that glycerol accumulates in relatively low concentrations in CSF as compared with the water of skeletal muscle. We have also found that accumulation in the brain itself proceeded slowly, at about the same rate in fact as in CSF, and if it turns out that this low degree of accumulation is due to poor penetration rather than a high rate of utilization, glycerol will qualify with urea for inclusion in a group of non-electrolytes to which the water of the CNS and CSF is much less accessible than the rest of the body water. ACKNOWLEDGEMNT

Figures 1 and 2 are reproduced by courtesy of the editors of the Journal of Physiology. REFERENCES AMES, A., SAKANOUE, M. AND ENDO,S. (1964) Na, K, Ca, Mg and C1 concentration in choroid plexus fluid and cisternal fluid compared with plasma ultrafiltration. J . Neurophysiol., 27, 672-681. BERING, E. (1965) In: Cerebrospinalfluidand the regulation of ventilation, p. 396. C. McC. Brooks, F. F. Kao and B. B. Lloyd (Eds.)., Oxford. Blackwell.

* Since the present paper was presented a report by Sloviter, Shimkin and Suhara (1966) has come to the writer’s notice. These authors have found that slices of guinea-pig brain, when exposed to a concentration of glycerol similar to that employed in the penetration experiments described in the present paper, do oxidise the glycerol as revealed by the production of W02from 14C-labelled substrate. They also found an effect of glycerol on the EEG of hypoglycaemic rabbits.

CSF

TRANSPORT

I43

BERING, E. A. JR. A N D SATO,0. (1963) Hydrocephalus: changes in the formation and absorption of cerebrospinal Ruid within the cerebral ventricles. J. Neurosurg., 20, 1050-1063. BRADRURY, M. W. B. AND COXON.R. V. (1962) The penetration of urea into the central nervous system at high blood levels. J. Physiol., 163, 423-435. BHADBURY,M. W. B. AND DAVSON,H. (1964) The transport of urea,creatinineandcertain monosaccharides between blood and fluid perfusing the cerebral ventricular system of rabbits. J. Physiol., 170, 195-21 I . BROOKS,C. McC., KAO, F. F. A N D LLOYD,B. €3. (1965) Cerebrospirial fluid and the regulation of ventilation. Oxford. Blackwell. BRITTON, H. G . (1962) Some non-reducing carbohydrates in animal tissues and fluids. Biocheni. J., 85, 402407. CASEY,T. A. A N D TREVOR-ROPER, P. D. (1963) Oral glycerol in glaucoma. Brit. Mecl. J.,ii 851-852. CANTORE, G., GUIDETTI, B. A N D VIRNO,M. (1964) Oral glycerol for the reduction of intracranial pressure. J. Nerrrosrtrg., 21, 278-283. COXON,R. V. AND SWANSON,A. G. (1965) Movement of ['T]bicarbonate from blood to cerebrospinal fluid. J. Physiol., 181, 712-727. DAVISON, A. N. A N D DOBRING, J. (1960) Phospholipid metabolism in nervous tissue. 2. Metabolic stability. Biocheni. J . , 75, 565-570. DAVSON,H. (1956) Physiology of the ocular and cerebrospinal fluid.s. London. Churchill. DAVSON,H. A N D BRADBURY,M. (1965) In: Cerebrospinal fluid and the regirlation of ventilation, p. 385. C. McC. BROOKS, F. F. KAOAND B. B. LLOYD(Eds.), Oxford. Blackwell. DAVSON, H. AND LUCK,C. P. (1957) The effect of acetazoledmide on the chemical composition of the aqueous humour and cerebrospinal fluid of some mammalian spxies and on the rate of turnover of "Na in these fluids. J . Physiol., 137, 279-293. ELLIOTT, K. A. C. A N D WOLFE,L. S. (1962) Brain tissue respiration and glycolysis. In : Neurocheniistry, p. 177. K. A. C. Elliott, I. H. Page and J. H. Quastel (Eds.). Springfield. Thomas. GADANDRESEN, K. L. (1921) Verteilung des Harnstoffs im Organismus. Biocheni. Z., 116,266-302. HAGEN,J. H. (1963) The turnover of glycerol in plasma. Life Sci., 3, 170-174. HEISEY. S. R., HELD,D. AND PAPPENHEIMER, J. (1962) Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Anier. J. Pliysiol., 203, 775-781. HELD,D., FENCL,V. AND PAPPENHEIMER, J. R. (1964) Electrical potential of cerebrospinal fluid. J . Neurophysiol., 27, 942-959. HOLST,R. J. (1944) Glycerol oxidation in the animal organism. Acta Physiol. Scancl., 7 , 69-79. JACOBI,W. AND MAGNUS,G . (1925) Gefass und Liquorstudien am Hirn des lebenden Hundes. Arch. P.sychiat. Nervenkratrkh., 73, 126- 138. KLEEMAN, C. R., DAVSON,H. AND LEVIN,E. (1962) Urea transport in the central nervox system. Anier. J. Physiol., 203. 739-747. LARSEN, J. A. (1963) Elimination of glycerol as a rnztsure of the hepatic blood flow in the cat. Acta Physiol. S c a d . , 57, 224-234. LEUSEN, 1. (1950) Influence du pH du liquide ciphdo-rachidien sur la respiration. Experietrfia, 6, 272. MITCHELL, R. A,, LOESCHCKE, H. H., MASSION, W. H. A N D SEVERINGHAUS, J. W. (1963) Respiratory responses mediated through superficial chemnosensitive areas on the medulla. J. Appl. Physiol., 18, 523-533. PAPPENHEIMER, J. R., FENCL,V., HEISEY, S. R. AND HELD,D. (1965) Role of the cerebral fluids in control of respirtaion as studied in unanesthetized goats. Atpier. J. Physiol., 208,436450. PAPPENHEIMER, J. R., HEISEY,S. R. AND JORDAN,E. F. (1961) Active transport of diodrast and phenolsulfonphthalein from cerebrospinal fluid to blood. Anier. J. Physiol., 200, 1-10. SEVERINGHAUS, J. W. (1965) I n : Cerebrospinal flirid and the regulation of ventilation, p. 248. C. McC. Brooks, F. F. Kao and B. B. Lloyd (Eds.). Oxford. Blackwell. SIMMONDS, W. J. (1953) The absorption of labelled erythrocytes from the subarachnoid space in rabbits. Airsir. J. Exptl. Biol. Med. Sci., 31, 77-83. SLOVITER, H. A,, SHIMKIN, P. AND SUHARA, K. (1966) Glycerol as a substrate for brain metabolism. Nature, 210, 1334-1336. SWANSON, A. G. A N D ROSENGREN, H. (1962) Cerebrospinal fluid buffering during acute experimental respiratory acidosis. J. Appl. Physiol., 17, 812-8 14. TOEWS,C. J. (1966) Evidence for the metabolism of glycerol by skeletal muscle and the presence of a muscle nicotinaniide-adenine dinucleotide phosphate-dependent glycerol dehydrogenase. Biochenr. J., 98, 27C.

I44

R. V. C O X O N

TSCHIRGI, R. D. AND TAYLOR, J . L. (1958) Slowly chsnging bioelectric potentials associated with the blood-brain barrier. Anier. J. Physiol., 195, 7-22. VOEGTLIN, C., DUNN,E. R. AND THOMPSON, J. W. (1925) The antagonistic action of certain sugars, amino acids and alcohols on insulin intoxication. Amer. J. Physiol., 71, 574-582. WEED,L. H. (1914) The pAthways of escape from the subarachnoid spaces with particular reference to the arachnoid villi. J. Med. Res., 31, 51-91. -, (1917) An anatomical consideration of the cerebrospinal fluid. Anat. Rec., 12, 461496. WIELAND, 0. A N D SUYTER, M. (1957) Glycerokinase: Isolierung und Eigenschaften des Enzyms. Biochem. Z., 329, 32C331. V. (1960) The cerebrospinal fluid valves. Brain, 83, 454-469. WELCH,K. AND FRIEDMAN,

DISCUSSION D. B. TOWER: I would like to ask Dr. Coxon about thedifferencein bicarbonateconcentration between the CSF and the capillary blood. It has been reported that this difference is not as great, or may actually be in the opposite direction from what you found. I find it a little confusing, and I wonder if you have any comment on this point. Is this simply a technical problem? R. V. COXON:I am afraid that I cannot make any authoritative pronouncement on this point. Our own work on bicarbonate has been confined to studying fluxes of tracer. As regards concentration in capillary blood there certainly is a technical problem in that capillary blood is rarely sampled and its composition has been inferred from measurements made on arterial and venous samples. In connection with tissue concentrations, which is a related problem, we have found some of the published techniques based on frozen brain very difficult to replicate. I think they are subject to some reservations. H. M. ADAM:I would like to ask Dr. Coxon if he has studied the distribution and fluxes of ammonium ions between the CSF and brain. R. V. COXON:I have not done that myself, but some other people whose names I can’t recall, have in fact started looking into this, partly in connection with hepatic coma. (See Rall’s comment below.) It seems from the short accounts that I have read so far, that one probably has the same situation there that one has in relation to COa; that the unionized NH3 seems to equilibrate, while the ammonium ion has not equilibrated nearly so rapidly.

D. P. RALL:With respect to ammonia: we did study this some six to seven years ago (Rall, D. P., 1959, J . Clin. Invest. 38 : 373). As soon as anybody can tell us how to measure ammonia in the blood, I think this is a wonderful problem to study. The CSF/blood ammonia ratio was around 0.3 or 0.4, and this seemed to be entirely artefactual. You cannot measure ammonia in the blood without also measuring some free amine from other sources. It would be a very interesting study if it could be performed, technically.

W. W. TOURTELOTTE: Dr. Coxon, is glycerol metabolized by the brain? R. V. COXON:I am glad you brought this point up. I had intended to refer to it, but time ran rather short. We have not yet made the crucial experiments (which 1 think we will have to do) of incubating nervous tissue with glycerol, and certainly preferably labeled glycerol. However, I think the indirect evidence suggests very strongly that it is not metabolized in the nervous system. It is metabolized very rapidly in the liver and in the kidneys. In some of the work by Holst (1944, Ac/a physiol. Scaud., 7 : 69-79; the Scandinavian work that I mentioned) some years ago it was shown that if one injects glycerol into a cat, and then removes its liver and its kidneys, the blood level does not change substantially after a matter of 24 hours. We have made rough calculations as to how rapidly the glycerol would have to be metabolized in order to account for the low concentrations that we found in the nervous tissue. I think it would have to be metabolized a t a rate which Holst would have seen.

Dr. Coxon, I don’t know whether these are appropriate questions to address W. W. TOURTELOTTE: to you now, but have you measured the concentration of glycerol in the cerebrospinal fluid, the cerebrospinal fluid pressure values at various times after glycerol and is there a rebound phenomenon

CSF

TRANSPORT

145

here as there is with many other of these agents that dehydrate the brain? Clinically we are very impressed with the effect that glycerol has on lowering increased intracranial pressure due to many different causes. It is the best medical treatment we have for keeping the pressure low in the brain, without a rebound phenomenon. However, we have to give the medication every six hours (Buckell, M. and Walsh, L., 1964, Effect of glycerol by mouth on raised intracranial pressure in man. Lancer, 11, 1151-1 152; Cantose. G., Guidetti, B., and Virno, M., 1964, Oral glycerol for the reduction of intracranial pressure. J. Neurosurg., 21 : 278-283). R. V. COXON:We have not ourselves made any measurement of this, but I have seen publications in clinical journals which seem to show that the rebound is much less brisk than has been found with urea. I don't know why this should be so.

W. W. TOURTELOTTE: Have you checked the cerebrospinal fluid pressure a t those times after glycerol administration that you determined the glycerol in the various tissues? R. V. COXON: Not yet, no.

K. A. C. ELLIOTT:I think I can answer the glycerol question. In the old days glycerol appeared to be virtually unmetabolized by brain tissue in vitro. Quite recently, however, it has been reported that, with radioactive glycerol, one can show a slight metabolism by brain. Metabolism of glycerol is definite, but slight and has been shown only in vifro. W. W. TOURTELLOTTE: Therefore, it would appear that there exists a Blood-Brain Barrier t o glycerol.

R. V. COXON:I think that it is an entry problem. It may, of course, have some interest for those people who are trying to freeze animals, because glycerol is one of the anti-freezes they use. J. DOBEING: What kind of evidence is there for the substantial formation of cerebrospinal fluid which occurs away from the choroid plexus which you mentioned at the very early part of your talk? Were these workers sufficiently aware, that the composition of the CSF is modified by the exchange of substances between the brain and the CSF-ventricular surface? This is not necessarily the same thing as formation at the other sites? What is the evidence for that? R. V. COXON:I think on the one hand that there is the evidence with experimental animals, where the system has been blocked at various points. In particular Behring and Sato perfused the subarachnoid space from one point to another, having (they said) completely isolated it from the entry to the system. There is also evidence, again quoted by them in hydrocephalic infants. Now you may not feel this is very reliable evidence, but again the story is that where you have a non-communicative hydrocephalus you can in fact detect formation of CSF in the subarachnoid space. Thes: are the two main bits ofevidence that have been found. J. DOBBING: Dr. Behring's figures were reasonably precise about the relative amounts formed in various species, and I wonder on what basis these calculations were made.

R. V. COXON:They were made on a rather different basis in three of the four species that I quoted. Behring's own experimental work is mainly on the dog, and this was essentially based on separate perfusions of the ventricles on the one hand, and the subarachnoid space on theother. Thecalculations for man were based on a series of hydrocephalic infants. The figure for rabbit was indirectly calculated from some data of Davson's, for the cat, that again was Behring's own perfusion work, I believe.

C. CRONE:It is very interesting to hear that glycerol has been proposed as a therapeutic agent for dehydration of the brain. Being a strongly hydrophilic substance it is to be expected that it penetrates only slowly into the brain. In this respect it resembles sucrose or mannitol. The advantage of glycerol is that it is eliminated rather slowly from the blood, while sucrose and mannitol are excreted rapidly due to their high renal clearances. Rebound phenomena are liable to be less pronounced when the concentration in the blood of the dehydrating substance falls slowly, therefore glycerol should be more advantageous.

146

R. V. C O X O N

The efficiency of glycerol as a dehydrating agent depends undoubtedly on the presence of OHgroups in the molecule. 1 undertook investigations to study the effect of introducing OH-groups in organic non-electrolytes on the rate of passage from blood to brain tissue. With the “Indicator Diffusion’’ technique (Crone, 1963) the loss of test substances during one passage through the brain was studied. The effect on the ability of a test substance to penetrate into the brain of introducing two OH-groups is clearly shown: the propanol curve is very low because the material has penetrated into the brain, while the glycerol curve closely follows the curve of the non-diffusable reference substance (Evans Blue Dye), (Crone, 1965). CRONE,C.: The permeability of capillaries in various organs as determined by use of the “Indicator Diffusion” method. Acfaphysio/.scand. 1963,58 : 292-305. CRONE,C. : The permeability of brain capillaries to non-electrolytes. Acta physiol. scand. 1965; 64 : 401417. P. MANDEL:But you can not explain all your hydrophilic phenomena, because in glucose you have the same hydrophilic particles as in glycerol. C: CRONE:That is true. Glucose is an exception to a general rule. May I ask Dr. Coxon another question? You showed a distribution ratio of 0.2 after some hours for glycerol. Did you keep the blood level constant in your experiments?

R. V. COXON:Yes, it did not vary by more than 1 or 2 mM/liter. D. B. TOWER:Dr. Crone, I think, has implied that there seems to be evidence for an inward facilitated transport of glucose, and one wonders whether this might be the other way round for something like glycerol. I don’t know that it is just simply the polarity. This is a question which we still have not answered. R. V. COXON:The red cell data suggest that there may be a special carrier for glycerol in them.

147

The Choroid Plexus as a Glucose Barrier

Dcparttiierit of Ptiarniacology, University of Ker;tucky College of Medicitre, Lexirigton, Ketitrtcky ( U.S.A.)

The choroid plexus is composed of strongly vascularized connective tissue lined with an epithelial layer. It has long been considered a “barrier” between the blood and the cerebrospinal space. The luminal membrane of the epithelial lining shows a typical microvillus structure similar to that of the epithelium of the small intestine or kidney tubule (Millen and Woollam, 1962). As the latter organs are involved in the biological transport of sugars, by analogy a similar role could be presumed for the choroid plexus. The present paper summarizes the results of preliminary experiments aimed at clarifying the role of the choroid plexus in the maintenance of glucose concentration between the blood and cerebrospinal fluid (CSF). I t has been known for some time that CSF contains less glucose than blood does (Davson, 1956). The relationship is roughly 1 : 2. Moreover, a change in the glucose concentration of the blood plasma is followed, after some delay, by a corresponding change in the sugar concentration of the CSF,still maintaining the approximate 1 : 2 concentration ratio. It is clear that such an uneven equilibrium cannot be maintained by a passive barrier alone. Theoretically, three possibilities could be proposed for such a distribution : ( I ) Glucose could be filtered by the plexus and then metabolized in the CSF. (2) Glucose could be actively secreted from the blood into the cerebrospinal space by the choroid plexus. (3) Glucose could continually be filtered somewhere else and then actively reabsorbed by the choroid plexus. The first possibility would require a specific mechanism that would regulate the metabolic disappearance of glucose at a rate depending on the concentration of the sugar in the blood plasma. Although the existence of such metabolic regulatory re:eptors is theoretically not impossible, in actuality it has not been demonstrated. With the second possibility, it is somewhat difficult to envision a layer of epithelium that would secrete a solute from the blood into the CSF and maintain it at half its concentration level in the blood. There are glands, like the salivary glands, that produce secreta hypertonic to blood; however, this is done not by a simple transport across an epithelial layer, but by a process of filtration followed by selective reabsorption at a different site. The third possibility, namely that a plasma filtrate is continually produced and gluR
T. Z . C S k K Y , B. M. R I G O R

148

coseis thenreabsorbed from it by the epithelium of the choroid plexus, would requirea low-capacity sugar pump in the latter. The function of such a pump would be limited by the concentration gradient against which it would have to transport. Thus it is quite possible that the limit of the gradient would be I : 2, which would result in the maintenance of the very uneven equilibrium between the blood and CSF as it was actually observed. In the following, evidence is presented that there exists a sugar pump in the choroid plexus, that this pump is of low capacity, and that it actively transports sugar from the CSF into the blood stream. Sugar-concentrating transport mechanism in the choroid plexus The following experimental procedure was performed (Csaky and Rigor, 1964). Choroid plexuses were removed from pentobarbital-anesthetized dogs and placed in a Krebs-Henseleit bicarbonate Ringer's solution containing a very small amount (7 pM)of 14C-labeled sugar. After gassing with 95 % 0 2 5 % COz, the vessels were shaken in a Dubnoff metabolic shaker for 1 h at 37" C. At the end of this period the plexuses were removed, gently blotted, and placed on a small piece of carefully preweighed filter paper. They were dried to obtain the total water content. The dry residue was combusted in a closed oxygen atmosphere, and the resulting W02was determined in a liquid-scintillation spectrometer using the scintillation liquid as described earlier (Kalberer and Rutschman, 1961). I n a number of experiments, radio-iodinated serum-albumin was also added to the Krebs-Ringer solution, and the distribution of radioiodine was determined in the choroid plexus. Assuming that this protein occupies only the extracellular space, the intracellular space of the choroid plexus was computed and the results calculated in such a fashion that the distribution ratio of radioactivity between the intracellular water of the choroid plexus and the incubating medium was

+

TABLE I R A T I O O F C O N C E N T R A T I O N O F R A D I O A C T I V I T Y I N T H E I N T R A C E L L U L A R W A T E R OVER T H E MEDIUM ( I / M ) I N T H E C H O R O I D P L E X U S F O L L O W I N G I N C U B A T I O N W I T H T H E L A B E L E D SUGARS

Sugar

Glucose Galactose 3-0-Methylglucose

Number of experinrents

(mean 4 standard deviation)

26 35 25

21.34 & 3.01 19.70 t 3.36 0.70 +. 0.32

I/

obtained. Table I shows that the radioactivity in experiments with glucose and galactose was concentrated more than twenty-fold in the intracellular space of the choroid plexus. However, it is interesting that 3-0-methylglucose (3-methylglucose) was not concentrated, although in other transport systems, such as the intestine, this sugarether behaves very much like glucose or galactose (Cshky, 1942, 1958). Glucose and galactose were metabolized by most tissues, whereas 3-methylglucose is not metabo-

C H O R O I D P L E X U S AS G L U C O S E B A R R I E R

I49

lized in animal ticsue (Csaky and Wilson, 1956). It was therefore of interest to examine whether or not the uneven distribution of the radioactivity between the intracellular water of the choroid plexus and the surrounding nutrient medium in the case of glucose and galactose was simply the result of transformation of the sugar to labeled metabolites, and not of true accumulation. For this reason choroid plexuses, having been incubated with radioactiveglucose or galactose, were extracted with hot distilled water. The protein-free filtrate was concentrated and chromatographed on filter paper, using either pyridine-water-butanol or acetic acid-water-butanol solvent mixtures (Tyszkiewicz, 1962). In the case of galactose it was found that about 70-75 % of the total radioactivity could be recovered in one spot, the migration of which was identical with that of the reference galactose. About 5 % of the radioactivity was found in another spot, the migration of which was identical with a reference galactose-I-phosphate. The rest of the radioactivity was distributed in various spots, the identity of which was not established. In the case of glucose, 60-70% of unchanged glucose was found in the extract. Also, glucose phosphate and unknown spots were located. These results indicate that even when making corrections for a possible slight metabolism there is a considerable concentration of true unchanged glucose and galactose in the choroid plexus. It is known that the intestinal sugar pump is inhibited by a number of factors, such as the lack of sodium in the medium; a low concentration of digitalis, 2,4-dinitrophenol, or phlorizin; or the lack of oxygen. In order to ascertain whether the choroid T A B L E 11 I N H I B I T I O N O F A C C U M U L A T I O N O F S U G A R S B Y L A C K O F S O D I U M , B Y I N H I B I T O R S A N D BY A N O X I A . F I G U R E S R E P R E S E N T T H E PER C E N T I N H I B I T I O N I N P A I R E D E X P E R I M E N T S

Glucose

Potassium Ringer

Lithium Ringer

Mannitol Ringer

Ouabain lO-4M

Phlorizin 10-3M

Dinitrophenol 10-3~

Anoxia

37 51 48 48

30 54 59 65 66 36

38 63 51 74 83

22 41 40 28 21

57 47 56 38 34

93 96 98 96

89 90 95 93

-

-

-

-

-

-

-

Average

46.0

51.7

61.8

30.4

46.4

96.3

91.7

Galactose

87 73 57 47 32

29 37 51 27 33

50 40 45 39

32 47 40 42

72 75 76 60 80 49

96 95 87 98

80 92 93 87

57

22

Average ~~

Rrfermces p . I54

-

__

-

-

-

-

-

53.6

35.4

43.5

40.3

68.7

94.0

88.0

T. Z. C S A K Y , B. M. R I G O R

150

plexus sugar pump is similarly inhibited, the following experimental procedure was developed :two lateral ventricle choroid plexuses were removed from dog and compared in pairs. One was incubated as a control in a normal Krebs-Ringer solution; the other, in a modified solution or in the presence of the inhibitor. The results were then expressed as percentage of inhibition versus the control. Table 11 shows that the choroid plexus sugar pump is inhibited by the same factors as the intestinal sugar pump, however, to a different degree. Two facts deserve specific mention: the lack of absolute dependence ofthechoroid plexus sugar pump on sodium in the incubating medium and the very strong dependence on the presence of oxygen. The former is in contrast with what one finds in the intestine (Csaky, 1960). The possibility that sodium leaks out of the tissue into the medium is of little importance because the plexuses were incubated in a relatively large volume of the medium (the tissue water diluted at least 1 : 50). Moreover, it was found that all sodium-dependent transports are readily inhibited with relatively low concentration of digitalis (Csaky, 1963). On the one hand, the sugar pump of the choroid plexus is only partially depressed by the presence of the cardioglucoside. On the other hand, the strong dependence on oxygen and the complete inhibition of the pump by low concentration of 2,4-dinitrophenol suggest that the transport mechanism derives its energy mainly from a direct oxidative process. The capacity of the concentrating pump was examined by incubating the tissue with increasing amounts of the sugar. As can be seen in Table 111, the galactose-accumulating system became saturated between the concentrations of 0.5-1.1 mM. By analogy it can be assumed that the glucose pump is also a low capacity pump.

T A B L E 111 I N H I B I T I O N OF T H E S U G A R C O N C E N T R A T I O N BY L A R G E A M O U N T OF S U G A R I N T H E M E D I U M . F I G U R E S R E P R E S E N T THE PER C E N T I N H I B I T I O N IN P A I R E D E X P E R I M E N T S ( C O N T R O L A N D E X P E R I M E N T A L P L E X U S FROM T H E SAME D O G )

Non-labeled sugars (30 m M )

Labeled sugars (7P M ) Glucose

Glucose

Galaciose 3-0-Meiliylglucose

99 96 98 98

55 75 49 77

97.8

61.2

-_

71 43 83 69

98 98 99 98

30 22 57 43

50

Average

Average

-

-

66.5

98.3

54 53 69 49 56.3

__

38.0

CHOROID PLEXUS AS GLUCOSE BARRIER

151

T A B L E 1V SATURATION O F THE GALACTOSE CONCENTRATING

Galactose in the Mediuni ltrert Radioactive Total (I'M)

0 56 111

560 1120

(PM)

(ItM)

7

7 63 118 567 1127

7 7 7 7

SYSTEM IN T H E C H O R O I D P L E X U S

Mean 11M

20.73 13.95 6.47 5.04 0.58

The possible active transport of 3-methylglucose in the choroid plexus merits further comment. As was seen, this sugar-ether is not concentrated in the plexus in vitro; however, the presence of a large amount of 3-methylglucose is capable of inhibiting the concentration of both glucose and galactose in the plexus (Table IV), as if the sugarether would compete with the other sugars for the transporting sites. Such behavior is not uncommon. In our laboratory we found that 3-methylglucose is most likely actively absorbed in the renal tubulues, yet in vitro it is not accumulated by the kidney slides*. Thus it is quite possible that, in spite of the lack of measurable accumulation, 3-methylglucose would still be actively transported across the epithelial layer of the plexus.

Direction of the sugar pump in dog choroid plexus in vivo The experiments described above demonstrate the presence of a concentrating pump in the choroid plexus but do not suggest anything about its direction. In other epithelial cells (kidney tubule or intestine), sugar is transported from the direction of the brush border towards the direction of the basal membrane. By analogy, then, the morphology of the choroid plexus epithelium suggests an active reabsorption of sugars from the CSF into the blood. To test this possibility, the following experiments were performed : dogs were anesthetized with sodium pentobarbital, and both lateral ventricles were tapped and cannulated, and an artificial CSF (Pappenheimer et al., 1961) was recirculated with a velocity of approximately 5 ml/min. The glucose content of the artificial CSF was less thanin the blood. From time to time, the glucose concentration in the perfusate was determined by the glgcostat technique (Teller, 1956). At the same time arterial blood samples were taken and the glucose concentration measured in the plasma by using the same method. Results of a set of typical experiments are reproduced in Fig. 1, which shows that there was a steady disappearapce of glucose from the cerebrospinal space into the blood. Because it probably proceeds up-hill, by definition the transport is active. This active transport is completely eliminated, however, if in the artificial CSF sodium was replaced by potassium or if digitalis or phlorizin was added to the medium.

* Unpublished Refcrenccs p . 154

observations.

T. Z. C S A K Y , B. M. R I G O R

152

HR.

HR.

HR.

nn.

Fig. 1 . Perfusion of the lateral ventricles in the dog. Solid line (CSF): glucose concentration in the perfusate. Broken line: glucose concentration in the arterial blood plasma. First column : control; 2nd col.: Na+ was replaced by K+ in the perfusing medium; 3rd col.: Digitoxin (IO-4M) added to the perfusate; 4th col.: Phlorizin (lO-4M) added to the perfusate.

Sugar transport in the isolated choroid plexus of the horse

A more direct evidence regarding the direction of active sugar transport in the choroid plexus was obtained from experiments on the isolated horse choroid plexus in vitro. Healthy horses (obtained through the courtesy of the Department of Animal Pathology) were anesthetized with sodium pentobarbital, their skulls were opened, and the lateral plexus with some surrounding brain tissue was quickly removed, placed on ice, and carried rapidly to the laboratory, where the anterior and posterior choroidal arteries and the main choroidal vein were cannulated with small polyethylene tubings. The procedure between the removal of the plexus and the start of the experiment did not take longer than 10-20 min. The choroid plexus from the ventricle side was bathed in a Krebs-Ringer bicarbonated solution containing 50 mg% of galactose. An identical solution was infused slowly into the choroid artery. The outflow from the choroid vein was collected and analyzed for galactose by a method utilizing specific galactose oxidase*. The entire preparation was kept in a 37°C moist chamber, and oxygencarbon dioxide (95-5 %) was continuously bubbled through the bath. At the end ofthe experiment a dye was introduced into the artery. If the dye appeared in the bath, indicating a leak between the blood and the cerebral spinal space, the preparation was discarded. The galactose concentration of the venous outflow increased continuously during the experiment, as shown in Fig. 2. Such results would be possible only if the galactose was actually transported from the outside into the blood by active transport, or if the blood was losing water. In order to check the latter, in a few experiments

* Pamphlet 6-65

“Galactostat”, Worthington Biochem. Corp.

CHOROID PLEXUS AS GLUCOSE BARRIER

153

Fig. 2. Horse choroid plexus perfused in vifro. Broken line: galactose concentration in the arteria input. Solid line: galactose concentration in the venous outflow. First column: perfusion with KrebsRinger: 2nd col.: Na+ replaced by K+ in the perfusing Krebs-Ringer: 3rd col.: perfused with KrebsRinger, gassed with N2.

radioactive inulin was added to the fluid which was pumped into the artery. The inulin concentration did not change in the venous outflow, indicating that there was no appreciable loss of water from the blood in the preparation. Consequently, one must conclude that in this preparation galactose was actually transported from the outside into the bloodstream against a concentration gradient by active transport. It is also noteworthy that this active transport was eliminated by the absence of sodium or by anoxia. CONCLUSIONS

Evidence is presented of an accumulating low-capacity sugar pump in the choroid plexus that transports glucose or galactose from the ventricle into the bloodstream. The experimental results would be in accord with the following working hypothesis: the CSF is essentially a blood filtrate in which glucose is originally present in the same concentration as in the plasma. The choroid plexus possesses an actively transporting pump system which continuously reabsorbs glucose from the cerebral spinal fluid into the blood. Because the pump is of low capactiy, the net transport will be determined by the gradient against which the transport has to take place. As a result, the glucose content in the CSF is always below that in the plasma but is in rough accord with the concentration fluctuations in the latter. As it happens, the pump is regulated so that a ratio of roughly 1 : 2 is maintained. Rrfercnces p. 154

154

T. Z. C S A K Y , B. M. R l G O R ACKNOWLEDGEMENT

This work was supported in part by research grants from the U.S.P.H.S. and the American Heart Association. REFERENCES CSAKY,T. Z. (1942) Hoppe-Seyler’s Z . Physiol. Chem., 277, 47. CSAKY, T. Z. (1958) Intern. Abstr. Biol. Sci. Suppl., 6 , 79. CSAKY, T. Z. (1963) Biochim. Biophys. Acta, 74, 160. CSAKY,T. Z. AND RIGOR,B. M. (1964) Li/e Sci., 3, 931. CSAKY, T. Z. AND THALE, M. (1960) J . Physiol. (Lond.), 151, 59. CSAKY,T. Z. AND WILSON, J. E. (1956) Biochim. Biophys. Acta, 22, 185. DAVSON, H. (1956) Physiology of the Ocular ana Cerebrospinal Fluids. Boston, Little Brown. KALBERER, F. AND RUTSCHMAN, J . (1961) Helv. Chim. Acta, 44, 1956. MILLEN, J. W. AND WOOLLAM, D. H. M. (1962) The Anatomy of the Cerebrospinal F h i d Londcn, Oxford University Press. J. R., HEISEY, S . R. AND JORDAN, E. F. (1961) Amer. J . Physiol., 200, 1. PAPPENHEIMER, TELLER, J. D. (1956) Abstract of Papers, 130th Meeting, Amer. Chem. SOC.,69c. TYSZKIEWICZ, E. (1962) Anal. Biochem., 3, 164. D I S C U S S I ON D. B. TOWER: I think this is very interesting work of Dr. Csaky’s, and I would like to ask several related questions. It has been known for a good many years in clinical practice that when the blood glucose level rises above a certain point, and if my memory serves me correctly it is about 300 mg%, then the cerebrospinal fluid glucose level no longer rises. One has the impression in looking at these figures that the pump is saturated in the opposite direction. In other words, the glucose is pumped into the central nervous system, and if one increases the glucose level on the side from which the .glucose is being pumped, the pump can be saturated. This obviously does not agree with your data; so I am wondering if you have an explanation for this clinical observation. The second point relates t o some experiments that Dr. Fishman did in the dog. Unfortunately I don’t think he published the data, but he told me that he studied the effects of phlorhizin, both given into the blood and injected into the cerebrospinal fluid. I think he used up to 1 0 - 3 M concentrations, and got no effect on the entry of glucose into the central nervous system. I just wonder how you would interpret this observation of Dr. Fishman’s on the studies in vivo in the dog. Finally, I wonder what is the role of glucose metabolism in the central nervous system in your system; since glucose is the major substrate being used by the brain and a t a very substantial rate (about 65% of the glucose consumed by the body per unit time is consumed by the central nervous system). I find it a little difficult to visualize how one may conceive of an outward pump for glucose together with this type of metabolic system.

T. Z. CSAKY:For the first question, regarding the effect of phlorizin, I can provide only a partial answer. In our experiments we obtained an inhibition with this glucoside both in vitro and in vivo, but only a partial one. So it is possible that the sugar carriers are different in different anatomical entities. Concerning sugar transport into the brain at very high sugar concentration: we did not study such a phenomenon directly. However, one can speculate about this problem by analogy. E.g., it is known that if the metabolic pattern of glucose is changed, its transport rate also changes in some tissues. Thus, semi-starvation leads t o an increase in the rate of glucose absorption from the intestine. Perhaps it would be conceiveable that the high glucose containing blood “overfeeds” the brain cells and thereby decreases the rate of transport. With regard to your third point about the slices: I do not think that the brain cells are fed through the choroid plexus, but that glucose enters the brain from the capillaries via the mediation of the glia. In connection with this point I may mention that in our laboratory we recently found an active sugar pump in the isolated glial cells grown in tissue culture.

C H O R O I D P L E X U S A S GLUCOSE B A R R I E R

1 S5

D. P. RALL:In which direction? T. Z. CSAKY:Working essentially with a cell suspension we cannot at present answer this question. We could only demonstrate an energy dependent accumulation of the sugar in the cell. But it is not inconceivable that the glia pumps glucose from the blood into the brain. Of course, in brain slices the glia c:lls are also included. I think the choroid plexus is different. Its epithelium morphologically and perhaps also functionally seems to be like that of the kidney tubule or intestinal mucosa.

D . B. TOWER:Could I just make a comment then? I a m glad that we put this in about the glia, because it would make it much easier t o understand the total picture. I think it would also make it easier to understand Fishman's results, because if I remember his experimental set-up, he was following the influx of glucose into the central nervous system. If you will allow this for the sake of argument, that the glia more or less overpower the pump in the choroid plexus, then you would not see the plexus effect in an overall result. I think this may make it easier t o put the whole picture together.

D. M. WOODBURY: HOWd o you know that it is pumped into the glia? T. Z. CSAKY:We incubated cultured glia cells with radioactive sugars and found an accumulation wiiich was inhibited by cooling, anoxia, metabolic poisons and by digitalis.

D. P. RALL:What about neurons? T. Z. CSAKY: We have not cultured neuron cells. D. P. RALL:Just one question: You perfused dogs ventricles at a rate of 15 ml per minute? T. Z. CSAKY:Yes.

D. P. RALL:And you perfused them with a sodium-free potassium Ringer? T. Z. CSAKY:Yes. D. P. RALL:Are those dogs alive?

T. Z. CSAKY: Yes, they are. D. P. RALL:Seriously, I am sure they are, because you are doing it from one ventricle t o the other, but I think you must destroy the tissue adjacent to your perfusions. And I would assume that the fact that the glucose is increasing there is because you have tissue breakdown. Definitely. We d o not claim that the choroid plexus alone has contributed t o the glucose T. Z. CSAKY: ontflux. We claim only that in these experiments the choroid plexus does not function the same way as in normal Ringer. D. P. RALL:You think the choroid plexus is still alive? T. Z. CSAKY: The animal is, judged from blood pressure. D. P. RALL:Can you d o this from ventricle to cisterna? That is the critical thing.

T. Z. CSAKY:We were not interested in examining the effect of potassium as such, only in demonstrating, that in vivo, as irr vitro, the choroid plexus does not pump sugar if sodium ions are lacking from the perfusate. As far as the active sugar transport is concerned the plexus is dead. This is my only point.

I56

T. 2. C S A K Y , B. M. R I G O R

D. P. RALL:Is the level of potassium as high as the sodium level? T. Z. CSAKY:It was a complete Ringer solution, potassium replacing sodium iso-osmotically. Other similar epithelial cells, e.g., in the kidney or in the gut can be exposed reversibly to such solutions.

R. CUTLER: But the brain is different, I hope! T. Z. CSAKY:Not necessarily. R. CUTLER: It has a zero-membrane potential.

T. Z. CSAKY:We are discussing now the epithelium of the choroid plexus and not necessarily the "brain". In other epithelial tissues zero potential can be readily obtained, reversibly, by changing the ionic environment. J. FOLCH-PI: You have not mentioned exchange between the cerebrospinal fluid and the brain parenchyma. The brain parenchyma has about 1/5 of the concentration of glucose that you find in the blood, and the cerebrospinal fluid seems to be in between the two. So, if you assume as a logical consequence that a steady state will be reached, you would have an ultrafiltrate of plasma entering the cerebrospinal fluid, and from there your ultrafiltrate glucose would be remove4 by the sink. An injection into the brain parenchyma produces a sink into which glucose can be definitely forced. Can you gather such evidence by studying the point of the possible exchange by whatever mechanism it is, between CSF and the whole brain parenchyma?

T. Z. CSAKY:Not in such experiments as I have just described. Here we demonstrated only the presence of the sugar pump in the choroid plexus and its direction. We cannot at present assess the quantitative contribution of the choroid plexus sugar pump to the steady state level of glucose in the brain. J. FOLCH-PI: Now a general question to anybody: Does anybody know if there is any evidence concerning a possible exchange of glucose between the cerebrospinal fluid and brain parenchyma in general?

R. CUTLER: There is the evidence of Tschergi, I believe it was, who has perfused the CSF with glucose, which did not maintain the living brain.

J. FOLCH-PI: No, that is different. The amount of glucose you need to maintain a living brain is phenomenal. You would have to inject syrup which you could not possibly provide for the brain from the cerebrospinal fluid. K. A. C. ELLIOTT: What is the relation of the accumulation of the sugar by the choroid plexus t o the transport of sugar from the cerebrospinal fluid to the blood? The accumulation within the choroid plexus does not constitute sufficient evidence of transport through the choroid because glucose must be taken up from the one side and passed on t o the other side. I went along with the idea that substituting potassium for sodium is the same as leaving out sodium. Are you satisfied that the effect of substitution of potassium for sodium is indeed due to lack of sodium and not to the presence of the extra potassium? T. 2.CSAKY:Let me answer the two points separately. About the substitution: in vitro, as we had shown, the pump was inhibited if the sodium was replaced, regardless whether with potassium, mannitol or lithium. We have not used choline, but we could have and probably would have obtained the same results. Tris and magnesium have been used by us and by others as sodium substitute and we could probably find fifty other cations in the Merck index which could be used for the same purpose. The point which our experiment stresses is, that the sugar pump in the choroid plexus, like sugar pumps in other tissues, requires sodium. Your other question refers to the relationship between intracellular accumulation and transcellular transport in the epithelial cell. The general concept is that cross-cellular up-hill transport is preceeded by an intracellular accumulation. The sugar pump is most likely localized in the brush

C H O R O I D PLEXUS AS GLUCOSE BARRIER

157

border and is responsible forcreatinga concentration difference between the two sidesofthemembrane. The basal membrane is probably rather freely permeable for the substrate. Consequently the actual degree of intracellular accumulation depends on the ratio between the pumping and the leakage on the o t h x side. I f the leak is great the intracellular accumulation may besosmall that it may escape our analytical skill. Thus, I fully agree with you that a clear-cut demonstration of intracellular accumulation is not an absolute requirement for a transcellular up-hill transport; but it is quite different the other way around: if one can demonstrate a true intracellular accumulation of the free substrate it is a proof of the pump. In our experiments this was the case with glucose and galactose so that we can be certain that these sugars are actively transported. The failure in demonstrating the accumulation of 3-methylglucose does not necessarily disprove active transport. 1 hope this answers your question.

K. A. C. ELLIOTT:I agree immediately that there is a pump involved in the accumulation, but I would tend to use the word “transport” in the case of movement through a cell, as transport could include two membranes: the entrance and theexit. T. Z. CSAKY:The word “transport” means only translocation, active or non-active alike. In case of active transport energy is invested and the free energy of the transported solute increases. For such transport the expression “pump” is an excellent choice.

K. A. C . ELLIOTT: But I am wondering how does the sugar get out again. T. Z. CSAKY: I t leaks out passively.

K. A. C . ELLIOTT: But if it is all in the same cell, how can it be pumping in actively, and leaking out at the same time? T. Z. CSAKY: Such a combination is very common occurrence in nature. In the red blood cell there is a steady state equilibrium of sodium and potassium caused by the pump-leak ratio. If the pump is inhibited by digitalis, the leak can be beautifully demonstrated.

K. A. C. ELLIOTT: In the same place? T. Z. CSAKY: Probably not, but indeed in the same membrane.

D. M. WOODBURY: Certainly, in the choroid plexus, if it pumps potassium out, you can measure the ratio, whether it is lower in the CSF than in the plasma. It concentrates in the choroid plexus so that this pump is on on: side, and it just leaks out passively on the other side. The same situation can apply here. T. Z. CSAKY:This would fit admirably with the postulate that there must be a sodium pump in the choroid plexus. I t has been amply demonstrated in other tissues that a functioning sodium pump is a prerequisite for the functioning of the sugar pump.

D. M. WOODBURY: Yes, and the sodium is pumped of course in the other direction. D. P. RALL:I am a bad mathematician, but as I calculate it I mM glucose is about 20 mg%. If that is what it saturates, then it seems to me this system has essentially no biological usefulness, because there is generally around 50 or 60 mgo4 glucose in the spinal fluid. And if it was saturated back down at 20 mg‘:”! T. Z. CSAKY: Your arithmetic is almost correct (actually 18 nigO/;) but 1 am afraid your conclusion regarding the possible biological significance of the choroid plexus sugar pump is too hasty. First, you should remember that I showed that the in v i m saturation of the galactose (not glucose) pump occurs at about 1 mM. Furthermore, there is a difference between in virro and in vivo. In the former case both brush border and basal membrane of the epithelial cells are exposed to the same solution while iri vivo the polarity of the cell is maintained. From similar experiments conducted in other tis-

158

T. 2. C S A K Y , B. M. R I G O R

sues we know that the steady state accumulations in the two preparations are only roughly comparable and, if the polarity of the cell is maintained, the tissue-to-medium values are somewhat higher. Thus, we should only make an intelligent guess that the sugar pump is of low capacity but whether the in vivo limit in case ofglucose is 1,5, or 10 mM cannot be said at this moment. Davson 1967, (Physiology of the Cerebrospinal Fluid, Little, Brown and ComW. W. TOURTELLOTTE: pany, Boston, Table XX, p. 188) quoting other investigators’ data stated that the concentration of glucose was higher in the ventricles than in the cisterna magna, and least concentrated in the lumbar sac. Have you conducted experiments which were designed to explain the concentration gradient of glucose in the cerebrospinal fluid axis? T. Z. CSIIKY:As you may recall Pappenheimer suggested that various choroid plexuses may have different transport properties not unlike the epithelial cells in the different areas of the kidney tubule. We compared sugar pumps in plexuses obtained from different regions but could not detect any gross differences in vitro. We have not yet reached the point where we perfuse the individual ventricles and measure glucose fluxes. Such experiments may yield the information you are asking for.

159

Transport through the Ependymal Linings D A V I D P. RALL Depurtnient of Experinieirtal Therapeutics, Natiorial Cancer Institute, National Institutes of Health, Bethesda, Maryland (U.S.A.)

I n a consideration of the problems involved in the transport of compounds across the ependyma, 1 wish to cover three main areas. The first concerns techniques of potential use in studying transport with particular reference to the problems involved in transport across the ependyma. The second concerns an attempt to characterize, in so far as possible, the ependymal lining of mammalian brain as a membrane. Lastly, I wish to comment briefly on functional aspects of the relationship between cerebrospinal fluid and the brain. At the outset it is important to consider what we mean when we talk about transport and particularly active transport. It must be emphasized that there are a variety of mechanisms which can yield a concentration ratio of a solute across a membrane significantly different from unity that are not dependent upon an active transport process, but are completely consistent with passive diffusion (Table I). Let us consider TABLE I FACTORS C A U S I N G U N E Q U A L DISTRIBUTION O F A C O M P O U N D ACROSS A BIOLOGICAL M E M B R A N E NOT R E L A T E D r o A C T I V E TRANSPORT

Unsteady State Conditions Macromolecular Binding Potential Differences p H Gradients Bulk Flow Metabolism

a two compartment system in which a typical lipoid-like biological membrane separates the two compartments. There are no active transport mechanisms for the solute in this membrane and the solute must move solely by passive diffusion. We shall be concerned with the concentration ratio of this solute on the two sides of the membrane at a steady state. The importance of steady state conditions, easy to achieve in vitro or in theory, but often difficult to achieve in vivo, must be emphasized. This point is illustrated in an experiment in which the entry of sulfanilamide into the cerebrospinal fluid was followed following a single intravenous injection in the dog. If samples had been taken only at 3 to 1 h, it could have been concluded that this drug was relatively excluded from the cerebrospinal fluid (Fig. 1). If samples were Rcfermcrs p. 167

160

D. P. R A L L

1

-

I

3

J 4h

TIME

Fig. 1. After a single intravenous injection of sulfanilamide in the dog, the plasma concentration (CPI.)falls and the CSF concentration (CCSF)rises. If only one sample had been taken at ‘/z or at 3 h, a “barrier” or “transport” might have been postulated.

taken at 3 h, the opposite conclusion might have been reached, i.e. that sulfanilamide was concentrated in the cerebrospinal fluid. In fact, the steady state concentration ratio of sulfanilamide between cerebrospinal fluid and plasma is unity (Rall et al., 1959). If the solute is bound, reversibly or irreversibly, to a component such as plasma protein which is not in equal concentration in both compartments, then the total concentration of the solute (bound and free) will be different in the two compartments, although the concentration of free or unbound solute can be identical. Ifthe solute is partially ionized at the pH of either of both compartments and the membrane like most biological membranes is more permeable to the nonionized moiety, an unequal distribution ratio will occur if the pH of one compartment is different from that of the other. This is of particular importance in the study of the distribution of compounds in the central nervous system, since the pH of cerebrospinal fluid and likely of the extracellular fluid of the brain is normally different from that of plasma pH and further under conditions of altered acid base metabolism, these pH’s can diverge in apparently paradoxical ways (Rall et al., 1959). Similarly, a difference in electrical potential between the two compartments will alter the distribution of ionized solutes. If in one compartment only there is a constant replenishment of relatively solute free solution and removal of solute containing solution in bulk, a steady state inequality of solute concentration will occur. There is ample evidence that such bulk flow does occur with respect to the cerebrospinal fluid (Rothman et al., 1961). A very common problem involves the situation in which the solute in question undergoes a metabolic alteration on one side of the membrane. This metabolic trap or sink serves to reduce the concentration of the solute inonecompartment and results in an unequal distribution. Not only are metabolizable compounds sometimes trapped in this manner, but also occasionally a physiologist may be trapped and sunk. Thus there are a variety of conditions which can yield concentration gradients different from one which are not really involved in the active transport of that par-

TRANSPORT THROUGH EPENDYMAL LININGS

161

ticular solute, and it becomes incumbent upon all of us to critically examine our experimental details to be certain that we are not invoking active transport processes when simple passive diffusion in a less than simple situation may not adequately explain the observations. Let us now specifically consider the ependyma as a barrier to transport. Fig. 2 Ventricular Fluid

Ependyma

Brnin

Neuronal C e l l s G l i a l Cells

0

a

Capillaries Extracellular Fluid

Blood Brain Barrier

Fig. 2.

points out the fact that the ependyma as a membrane system is complex. On one side there is ventricular or cerebrospinal fluid. The composition of this fluid is relatively well known. The pH relationships and the electrical potential differences between cerebrospinal fluid and other body tissues are also, in part, at least understood. I t is clear that bulk flow of cerebrospinal fluid also occurs. This, however, is probably the simplest part of the system. On the brain side of the barrier, we are involved in a variety of challenging questions. What is the nature of the blood brain barrier, the extent of the extracellular space, the nature of the extracellular fluid, and the characteristics of the glia and neuronal cells? These all become critical problems tending to confuse the study ofthe ependymal barrier itself. There are a number of techniques which are of use in an effort to elucidate the nature of the ependyma. In view of the complexity of the brain, the neuronal cells and the neuropil and their extreme susceptibility to anoxia, it seems to me that in vitro techniques are not likely to be useful and I shall concentrate on in vivo techniques. The basic in vivo technique is to establish a concentration of a tracer substance either by injection into the ventricular system or ventriculocisternal perfusion or by injection into the blood stream. The concentration of this substance at some point or points in time thereafter, is determined in brain tissue and in cerebrospinal fluid and blood and assumptions concerning the barrier are made on the basis of these concentration differences. A common technique for measuring these substances has been to use substances which either are fluorescent or are highly colored. The lack of ability to quantitate either of these should be a great concern to us in the late 1960s. I feel that we have reached a stage where quantitative studies are necessary. As a pilot experiment and as an exploratory technique, such methods are quick and simple and have considerable usefulness. One problem which exists in the use of fluorescent compounds should be considered. This is the fact that the relationship between the intensity of the fluorescence and a concentration of the fluorophore is not a simple linear relationship, Rc:fircncvs p. 167

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but is biphasic. With high concentrations of fluorophore quenching, an apparent decrease in the intensity of the fluorescence occurs. For the use of radioactive tracer compounds, radioautography is intriguing. 1 am still concerned that it is not adequately quantitative because of differential absorption of the various tissues within the brain. Similarly, problems of translocation of the labeled solute still exists. However, techniques are now being evolved which should greatly increase the ability to quantitate radioautographs and decrease the translocation of the labelled solute (Stumpf and Roth, 1965). Thus the utility of radioautography will become increasingly apparent. The only technique that can yield today unequivocal quantitative results is that of either chemical or direct radioisotopic analysis of the tissue on one side of the ependyma and of the cerebrospinal fluid on the other. All of the problems of metabolism, metabolic sinks, pH and potential differences, bulk flow, steady state relationships, and so forth still exist and must be solved in any meaningful experiment. Using the technique of radioisotopic analysis, we have recently described a method in which we study the diffusion profile of substances moving from the cerebrospinal fluid across the ependyma into the brain substance (Rall et al., 1962; Rall, 1964). The exact nature of the diffusion profile is related to the duration oftheexperiment, the volume of the extracellular fluid, the diffusion coefficient of the solute being studied, the permeability of the ependyma, the presence of bulk flow across the tissue, the binding of metabolism of the compound being studied as well as other factors. In general, the effect of each of these factors can be estimated and to a greater or lesser extent, quantitated. Thus one factor may be isolated and studied in appropriate experiments. We first used this technique to explore the question of the extracellular space of the brain. Integral to this was an understanding of the movement of compounds in a quantitative way across the ependymal barrier. This is shown schematically in Fig. 3. Details of the technique have been described as have the pertinent T h e o r e t i c a l nude1 Ependyma

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mathematical considerations (Rall et al., 1962). Briefly we established a constant concentration of the substance under study in the ventricular fluid by ventriculocisternal perfusion. After a specified length of time we sacrificed the animal and re-

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moved small blocks of tissue sequentially on an axis perpendicular to the ependymal surface. The quantity of the tracer in each small block of tissue was determined by appropriate counting techniques. Each block of tissue represented only a one to two millimeter increment in distance from the ependyma. We were then able to study the diffusion profile of any compound across the ependyma and deep into the brain tissue provided the compound under study did not freely pass the blood brain barrier from brain into blood. As a convenience, we plotted the ratio of the concentration at any point in brain tissue over the concentration outside the ependyma against the distance into the brain tissue from the ependymal surface. If this is plotted on a complementary error function paper, simple diffusion in an aqueous medium will yield a straight line. Some of the questions one asks about the nature of the membrane are as follows: First is there a potential difference across this membrane? We do not have this answer. A potential difference exists between ventricular fluid and the extracellular space of a body (Held et al., 1964). The exact locus of this potential difference is however, unknown. What is the permeability of the ependyma? We have used the movement of inulin across this membrane in an attempt to answer this particular question. If inulin is diffusing into a constant unstirred space within the brain, it is clear that if there is a significant barrier at the ependyma, the amount of inulin in the space will increase as time is allowed for more complete attainment of a steady state. In other words, the extrapolated extracellular space should increase as a function of the duration of the experiment if there is significant impermeability to inulin. T A B L E I1 DURATION O F PERFUSION A N D APPARENT INULIN SPACE: A N INDICATION OF EPENDYMAL IMPERMEABILITY?

Duration of perfitsion hours

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I n Table I 1 are shown data related to inulin space as a function of time in both the caudate nucleus and midbrain. There is some indication that the space increases in the caudate nucleus, but does not significantly increase in midbrain. This might suggest that the ependymal linings of the caudate nucleus, composed largely of gray matter, might possess some relative impermeability to inulin but that the ependyma aqueduct above the midbrain is rather permeable to this large polysaccharide. The exact definition of the permeability must, therefore, wait further studies. It is clear, however, that the ependyma is not a tight barrier. The possible presence of active transport processes across the ependyma has been R c f ~ r c ~ r rp~. s167

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considered. Let us examine therefore, the diffusion profile of a weak organic acid, paraaminohippuric acid, from ventricular fluid across the ependyma into the brain substance. Paraaminohippurate (PAH) is typical of the class of compounds which are actively transported, not only by the choroid plexus, but also by the proximal renal tubule. The experimental design is similar to that described for inulin. If an active transport process were present at the ependymal layer, one would anticipate an increased concentration of this substance in the subependymal layer. This is not seen; the diffusion profile is perfectly straight and the space is similar to that space occupied by sucrose (Fig. 4).Obviously one does not rule out active transport on the basis of

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experiments with a single compound and studies must be made with other organic acids and particularly with the organic bases, since Schanker and his group (Schanker eta/., 1962) have shown the importance ofthe transport of organic bases in thechoroid plexus. Let us now consider the possibility of bulk flow moving across the ependyma, either into the ventricular fluid from the brain or vice versa. The question is related of course in part to the origin of the cerebrospinal fluid. If cerebrospinal fluid is produced by the capillaries of the brain and percolates through the brain tissue and flows out the ependyma into the ventricular system, there will be bulk flow from brain to ventricular fluid. Alternatively, cerebrospinal fluid may move through the ependymal lining percolating again through the intercellular space and be absorbed at the capillaries of the brain. Movement in either direction would alter the shape of the diffusion profile of inulin or other such substances. Movement from brain to ventricular fluid would tend to bend the curve in a concave fashion shown schematically in the left

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Fig. 5. The effect of bulk flow on the diffusion profile of inulin into the brain - hypothetical curves. For details, see text.

part of Fig. 5. Movement from the ventricular surface into the brain would move the curve to the right and make it convex. Considering the limits ofprecision ofthemethod and the experimental results obtained, deviation from linearity of the experimental curves would allow no more than a 25 "/, production or absorption of CSF by the brain tissue resulting in bulk flow moving across the ventricle (C. S. Patlak, personal communication). Thus, there is, at most, relatively little bulk flow of fluid across the ependymal barrier. One particularly vexing problem concerns the possibility of pinocytosis at epithelial borders such as the ependyma. Let us consider a situation in which a constant concentration of the tracer substance has been maintained in the ventricle for a reasonable length of time. The concentration gradient profile is then established. If the concentration of the tracer in the ventricular fluid is at this time suddenly reduced to zero by perfusion with tracer free fluid, then there will be back diffusion from the extracellular fluid of the brain into the ventricular system across the ependyma. This back diffusion may be measured experimentally if one perfuses with the tracer for a period of time such as four hours then rapidly exchanges the inulin containing ventricular fluid for inulin free fluid for two hours. It is possible to calculate the diffusion profile that should result from such an experiment if only diffusion were involved in the movement of the tracer compound. The results of this experiment are shown in Figure 6. The solid line is the calculated theoretical curve on the basis of the freediffusion of inulin. The points are the experimental data. These have been normalized with reference to the first millimeter section. It may be seen that in the five experiments there was good agreement between the experimental points and the solid theoretical curve. This agreement lends considerable strength t o the conclusion that diffusion alone can account for quantitatively, the movement of inulin across the Refermcrs p . 167

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ependyma. If pinocytosis does occur it must occur with the same rate as the free diffusion of inulin and must be bidirectional. This seems to me inherently unlikely and therefore I suggest that this is rather good evidence that pinocytosis is not quantitatively important in the movement of inulin across the ependymal barrier. These results are only preliminary and tentative. Yet they help us in a beginning attempt to characterize the ependyma. The important points it seems to me, are that there is relatively little barrier for molecules as large as inulin. There may not be active transport of compounds at this barrier. There is not likely to be significant bulk flow of fluid in either direction across the ependyma, and it seems that pinocytosis probably has little quantitative importance at this barrier. These studies taken in conjunction with the bulk of knowledge of the blood brain barrier, and the blood cerebrospinal fluid barrier, suggest certain characteristics which are important in relationship to the function of the cerebrospinal fluid system, the ependyma, and the brain. Since there is relatively little restriction of the movement of a compound as large as inulin, it seems likely that for the smaller and more diffusable ions the fluid outside the ependyma and the fluid within the brain are very nearly identical in ionic composition. This suggests that if one is interested in estimating the chemical composition of the extracellular fluid of the brain, the appropriate model is the ventricular fluid. Further it seems likely that lipid insoluble compounds which may be products of metabolism from neural or glial cells should be able to move freely and diffuse from the brain into the cerebrospinal fluid. Once in the cerebrospinal fluid they may be flushed away by bulk flow or actively removed by the active transport process known to be located in the choroid plexus. Since no barrier is absolute, even the blood brain barrier with respect to large lipid insoluble molecules, this pathway may offer an opportunity for the quantitatively small amount of such compounds as plasma proteins to move out into ventricular fluid and be removed from the central nervous system. Many other problems exist. For instance, the nature of the junction between the ependyma and the choroid plexus should be interesting. I have treated the

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ependyma as a homogeneous membrane and this is a n over simplification. The various kinds of ependymal cells which Fleischhauer (1960) has described, must be studied separately. What is the origin of the potential difference between CSF and blood? I look forward to the next symposium on barriers within the central nervous system.

R E F ERENCES FLHSCHHAUER, K . (1960) Fluorescenzmikroskopische Untersuchungen an der Faserglia. 1. Beobachtungen an den Wandungen der Hirnventrikel der Katze (Seitenventrikel, 111. Ventrikel). Z. Zellforsch., 51, 467-496. HELD,D., FENCL,V. A N D PAPPENHEIMER, J. R. (1964) Electric potential of cerebrospinal fluid. J. Nertrophysiol., 21, 942- 959. RALL,D. P. (1964) The structure and function of the cerebrospinal fluid. The Cellular Functions of Meriihrarie Trarisport. J. Hoffman (Ed.). New Jersey, Prentice-Hall (p. 269). RALL,D. P., OPPELT,W. W. AND PATLAK, C. S. (1962) Extracellular space of brain as determined by diffusion of inulin from the ventricular system. Life Sci.,1, 43-48. RALL,D. P., STABENAU, J. R. AND ZUBROD, C. G. (1959) Distribution of drugs between blood and cerebrospinal fluid: General methodology and effect of pH gradients. J . Pliarniacol. Exptl. Therap., 125, 185-193. ROTHMAN, A. R., FREIREICH, E. J., GASKINS, J. R., PATLAK, C. S. A N D RALL,D. P. (1961) Exchange of inulin and dextran between CSF and blood. Anier. J . Physiol., 201, 1145-1 149. SCHANKER, L. S., PROCKOP, L. D., SHOU,J. AND SisomA, P. (1962) Rapid efflux of some quarternary ammonium compounds from cerebrospinal fluid. Lve Sci., 10, 515-521. STUMPF,W. E. AND ROTH, L. J. (1965) Dry-rnounting high-resolution autoradiography. Isotopes irr Experinienlal Pharniacology. L. J. Roth (Ed.). New York, The University of Chicago Press (p. 133).

DISCUSSION K. A. C. ELLIOTT:A crazy question: Those first pictures of yours, showing the slow movement of substances through the brain tissue: have you any idea what the rate of flow of those substances, the PIH and the inulin, would be through ice?

D. P. RALL: A very interesting idea, and it certainly could be done, could it not? W. W. TOURTELLOTTE: My question is along the same line. Does the concentration of agar used, or the salt and water concentration of the gelled agar have any significant effect on the movement of substances through the gel? I t would seem to me that you could get a variety of diffusion rates depending on the “stiffness” of the gel? Have you determined if the substance you are testing reacts with supporting media? D. P. RALL:It is interesting that the change in concentration of agar does not change the sort of curve 1 showed. I t changes the place where you extrapolate the origin of the curve, the entry. A high concentration of agar will give you only something like 60% space left over for your inulin or PIH. N. M. VAN GELDER: It is not quite clear to me, but do not these data depend to some extent on the rate of diffusion, or the rate of perfusion, through the ventricles as well as the concentration, or is this completely independent of that?

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D. P. RALL:Except for the initial situation in which you are trying to establish your constant concentration of tracer, it is independent of rate. If you perfuse it at a very low rate, essentially all of your material will move into the brain, and you will have a great drop in concentration, but it is essentially independent. N. M. VAN GELDER: But does not the perfusion rate approximate the natural flow-through of the ventricular fluid?

D. P. RALL:It is about 3 or 4 times the natural rate of CSF production in our typical experiment. D. H. FORD:I am not quite sure if I recall the figures on this, but doesn't your diffusion rate with inulin correspond somewhat to what I would imagine one would get from radioautographic studies with RISA, when injected intraventricularly? Perhaps Dr. Bakay could comment o n this point. L. BAKAY: No, not with RISA. I doubt that you would get such a steadily changing concentration rate with RISA. But you will get a very similar steadily decreasing concentration rate from the ependyma1 lining all the way across the brain with radioactive phosphate. With the absorption of phosphate one would expect some other mechanism except passive diffusion, and yet the curve is very similar. There is one question I wanted to ask: Was there any difference between grey and white matter? Because Feldberg's perfusions showed that for a great number of substances (and some of them are probably not metabolized) there is a fast rate of diffusion through the ventricular wall into the periventricular grey. It comes to an abrupt stop when it reaches the white matter. I found this also, e.g.. with radioactive tetracycline which I don't think is metabolized particularly at this early stage of absorption. Considering the great molecular differences in these two types of tissue, it seems to me to be almost inconceivable that an inert, very large molecule would not show just S O I ~ Pkind of a change when it passes the grey-white interphase.

D. H. FORD:If one injects labeled thyroid hormone, [13'l]triiodothronine, into the ventricular system or into the subarachnoid space, one finds (perhaps paradoxically, perhaps not) that it does not go through the ependyma; it simply appears to adhere to it, and this is quite different from something like phosphate. Then, if one injects the labeled thyroid hormone into the vascular system, one finds that the amount of labeled material present in the brain depends on the density of the nerve cell population and that it is largely present as the injected hormone. The time course of the distribution of the hormone and its metabolites in the plasma, then in the brain, and finally in the cerebrospinal fluid, directs one to conclude that the CSF, as you were suggesting, is acting almost as a type of a lymphatic drainage for the brain. Thus, in this instance the thyroid hormone comes in through the vascular system, passes through the brain parenchyma, and then diffuses across the ependymal lining into the CSF as thyroidal hormone or the metabolized hormonal break-down products. D. P. RALL:In answer to Dr. Bakay's question: I am just too bad a neuroanatomist to know where would be a good area to determine a grey-white interface. L. BAKAY: I t is the junction of the grey and the white matter. D. P. RALL:Yes, but it must be where the geometry is good for our diffusion studies. L. BAKAY: There is no such place! You cannot get any length of tissue from the ventricular surface passing in the direction of the cortex where there would be only white, or only grey matter. The periventricular grey ends after a while and is followed by white matter; if the periventricular substance is essentially white matter, sooner or later within a short distance one finds the basal ganglia or the thalamic nuclei, and then it is grey matter again, In other words: There is no significant cross section of tissue which will not be mixed.

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D. P. RALL:In that case then I think it is the same for a block of tissue, not onlyat the place I showed you. There is no change in the curve there, very clearly. We also looked in the midbrain, down near the pons, and the curve still seemed very much like this. Let me point out that most of the studies, showing a block at the beginning of the white matter, were done by judging the intensity of the dye. But didn't you get a difference between the spaces that you calculated in the caudate D. B. TOWER: area and in the pons?

D. P. RALL:Not much. D. B. TOWER: I thought you got 12 to 14% for the caudate area, and 7 to 8 % for the pons? D. P. RALL:A little higher. We tried to take some sections from the thalamus, but we got into great geometric problems. I think this is what you are referring to. Where the geometry of the system was just completely inadequate, we have a very great technical problem. M a y I just add that anyone who says there are such great differences between grey and white matter, I suggest that they use this technique and demonstrate it. If the difference is there, it will be unequivocal. You are suggesting the differences: you show them. I can't find them! D I S C U S S I O N B Y C. C R O N E When discussing blood-brain barrier problems it is important to keep in mind whether one is dealing with static phenomena, such as spaces, or whether one is dealing with dynaniic phenomena such as rates of transport. When dealing with the rate of transport from blood into brain tissue there are two principal ways in which such a rate can be determined: ( I ) By establishing a constant concentration in the blood of a particular substance and then measure its presence in the brain by cutting out brain tissue at different time intervals and analyze for the content of the test substance. When dealing with fast processes this is obviously a difficult approach. (2) One can measure the loss from the blood of a test substance when it passes through the brain. What is lost from the blood is gained by the brain. This last principle can be applied in the following way: A solution containing a non-diffusable reference substance (e.g., Evans Blue Dye) and a test substance is injected quickly intracarotidly and immediately afterwards blood samples are collected from the superior sagittal sinus. The extraction of the test substance by the brain (= the relative loss) is determined in each of the samples collected within the first 10 to 30 seconds.

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Fig. I . Ordinate. Extraction of [14C]glucoseduring the initial passage through the brain. Abscissa. Concentration of glucose in plasma. Every point on the figure represents the results from one experiment in which 5-9 samples were collected.

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DISCUSSION

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For passively transferred substances the extraction will be constant despite variations in blood concentration because the flux is proportional to the intracapillary concentration. Various extractions were found for a series of non-electrolytes, but for each substance the extraction was constant (Crone, 1965). For glucose, however, the extraction by the brain varied with the concentration of glucose, being higher at low concentrations. Thus, the fraction of glucose lost from the blood during one passage depends on the concentration of glucose in the blood (Fig. I). This finding led to the idea that the major part of the glucose which is taken up by the brain is transferred by some other mechanism then simple diffusion (Crone, 1965b). From knowledge of blood flow, blood glucose concentration and extraction, a maximal transport rate can be calculated (Fig. 2). What happens to glucose once it has passed the first hindrance on its way from the blood to the cells in the central nervous system? I t has been claimed that glucose is taken up by the glia cells which in turn nourish the neurons. This theory, I find, is uninteresting as long as it has not been put to a rigorous test. In collaboration with Dr. W. F. Agnew (Pasadena) we tried to localize radioactive glucose by autoradiography to see whether there was glucose in the glia cells before it was found in

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the neurons (rat experiments). Brain tissue was cut out immediately after intravenous injection of D-glucose-6-T and dropped into isopentane cooled in liquid nitrogen. After sectioning and freezedrying at -3OC, emulsion was applied on the tissue sections. We always found glucose distributed uniformly over the brain and there was no evidence of any specific accumulation in the glia cells. In control experiments with radioactive mannitol we found that this substance was limited to the vascular space. This was taken as evidence that spread by diffusion after cutting out the tissue slice did not occur. I suggest that we should give up the idea that material passes to the neurons via the glia cells until somebody has demonstrated material in glia cells before it reaches the neurons. Nicholls and Kuffler (1964) have presented evidence that the speed of diffusion in the small intercellular space is fast enough to explain transport rates of small molecules and ions in the brain tissue. The figures are published with the permission of the Editors of J. of Physiology, London.

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REFERENCES CRONE,C. (1965a) The permeability of brain capillaries to non-electrolytes. Acta Physiol. Sca~id. 61 :407-417. CRONE,C. (1965b) Facilitated transfer of glucose from blood into brain tissue. 1. Physiol. ( L o n h n ) , 181 : 103-1 13. NICHOLLS, J. G. AND KUFFLER, S. W. (1964) Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neirrophysiol., 27 : 645-67 I . GENERAL DISCUSSION R. V. COXON:I would like to ask Dr. Crone whether he had any comparable measurements on other tissues. I have a vague recollection that Fisher (Bleehan and Fisher, 1954, J. Physiol., 123 : 260), when he was perfusing the rat heart, also found that there was a distinct limitation on the amount of glucose uptake, and that this restriction related to the work that the heart was doing. In other words: if he raised the pressure through which it is working, the restriction went up. C. CRONE:There is no doubt that there is a limited uptake of glucose in heart muscle and in striated muscle, but here the system which limits uptake is in the cell membrane.

R. V. COXON:What is the evidence that in the brain it is at an earlier stage? C. CRONE:It is quite true that there is no evidence that the Blood-Brain Barrier may be situated “before” the neurons, if I may say so. But this question is the very essence of our meeting here, and I am of course, not able to say where this barrier is located. 0. STEINWALL: Dr. Crone has just shown that glucose uptake shows a very nice Tm comparable to that seen in the tubules of the kidney. I would like to ask: Did you ever try to load it up, and find an overloading effect, where the uptake really went down, more or less? C. CRONE: I made some studies on diabetic dogs where the blood glucose level went up to 350 mg%. But H e did not find any evidence that the transport system wasaffected by thoseglucoseconcentrations.

T. 2. CSAKY: I would not share your pessimism about the impossibility of showing the effect of sugar on the glia. As you know, there is a carrier system which is always shared with a number of other sugars. Some of them are not metabolized. As you know, e.g., you can methylate the glucose, and free 3-methyl glucose behaves like glucose in many systems. Galactose shares all the characters. K. A. C. ELLIOTT:I would like to say three disconnected things. I am not worried about hypotheses: Surely we have got to have hypotheses and until we know more of what is going on, we must not throw them out just because we have not been able to prove them yet. But once you can do something about proving them the hypotheses become either good or bad theories. The next thing I want to say concerns the remark you made about 50% of the radioactivity in the brain after injected labeled glucose being amino acids. The point is that if you give radioactive glucose intracisternally, within 5 minutes 50% of the radioactivity in the brain is in amino acids. But this does not mean that 5076 of the glucose is being metabolized via amino acids. I t does not say anything about how much radioactivity is going into the brain and going out, and also it means primarily that the u-ketoglutarate, produced from radioactive glucose has equilibrated with the large pool of glutamate at a very rapid rate. The third point, on a different subject, is about a very simple experiment that I did about 20 years ago, which shows a very clear relation of sugar to brain tissue. I say “relation” deliberately and ambiguously. If you take a brain slice and just drop it into water, it will swell up like a large mass of jelly. If you take the brain slice and drop it into isotonic glucose or 4 times isotonic glucose, it will still swell up like jelly. But if you put a little salt in with the glucose, it will shrink the slice. This indicates that the glucose behaves as if it was completely permeable and exerts no osmotic action as long as there is no salt. When it is salted, it behaves as if it exerts an osmotic effect and is evidently not readily permeable.

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C. CRONE: Just a short comment to Dr. Elliott about theories. Of course we cannot work without theories. But what puzzles me is that (and especially in this field of blood-brain barrier) you will find that theories very quickly pass from the desk in the laboratory into text books. You can find the theory about preferential uptake by glial cells and subsequent delivery to the neurons in several textbooks. If anybody here can give me any evidence for this theory I would be very happy indeed. H. KOENIG:Dr. Crone’s demonstration of a carrier substance and a limited transport of glucose from the blood stream into the brain correlates well with the clinical observation that one can temporarily shrink the brain with hypertonic glucose. Together with Dr. Elliott’s point it places the barrier, if you will, or the carrier, at the blood-brain barrier level, and not at the level of the nerve cell. K. A. C. ELLiorr: Not the blood-brain barrier but a tissue fluid barrier in tissue slices. There is no blood-barrier there.

H. KOENIG:Though glucose permeates freely into a slice this does not occur irr v i v a That would tend to place the transport system somewhere between the circulation and the brain, wherever the “mystic” Blood-Brain Barrier is. When you inject glucose intracisternally there is no barrier restriction to diffusion. A. LAJTHA:May 1 again emphasize here that there is not a single “mystic” barrier that determines

the distribution of a substance between blood and brain, but there are a number of membranes and a number of mechanisms that all play a part in influencing the final distribution ratio. Undoubtedly, a number of these mechanisms are altered in slices, but I would not say that the barrier system is not present in slices at all, nor that it may play no role when glucose is administered intracisternally. Of course, there are a number of reasons for intracisternally administered compounds penetrating the brain in a different manner from those administered intravenously. B. D. WYKE:I was just wondering if Dr. Crone or somebody else might react to two clinical observations that are pertinent to this kind of experimental study. One is that everybody who deals with the treatment of hyper-and hypoglycaemic patients is familiar with the fact that if the arterial glucose concentration is chronically depressed to establish in a patient what you call a “steady state hypoglycaemia” and then you rapidly increase the blood glucose level, the patient’s behavior, or his EEG. or any other criterion you can use, only slowly shows significant reactions to theelevated bloodglucose concentration (in contrast to the rapid reactions of actutely hypoglyceamic patients). The point is: where is the glucose going to (perhaps to the liver), if it may take up to half an hour to wake up a chronically hypoglycaemic patient? Secondly, if I may continue to make things more complicated, Dr. Crone has shown that you can fully saturate whatever cerebral transport system for glucose there is at a blood concentration of 90 mg per 100 ml. How does he react to the situation in relative cerebral hypoglycaemia (Wyke, 1959, Electroenceph. din. Neirrophysiol., 11 : 602), that I mentioned the other day, where in order to make the brain function normally, you have to increase the arterial blood glucose concentration to nearly 200 mg per 100 ml? C. CRONE:If the brain cells have been “damaged” by a hypoglyceamia of long duration, it might well take half an hour for the brain to recover. Second, if the mechanism which transports glucose from blood to brain is less effective than in normal subjects, one way of “normalizing” the transport could consist of increasing the “load”, i.e., increasing the blood glucose concentration.

K. A. C. ELLIOTT:I have seen a patient in hypoglycaemic stupor, talking to the physician before he finished injecting.

Substrates for Brain Metabolism

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S. CLAYMAN

McGill Utiiversity C w c e r Research, M c l t i t ) ~Medical Scietices BiiilditiK, Utiit, 3655 Drimitiiotirl Street, Motitreal, Quebec (Cutiadu)

For several years now our interest in metabolite transport has been focussed on movement of amino acids into and out of preparations of various tissues. The amino acids most frequently employed are those which are not metabolized within the tissue under investigation e.g. a-amino isobutyric acid (a-AIB) and I-aminocyclopentane carboxylic acid (ACPC) which are not known to break down in any of the tissues thus far investigated. In addition, there is usually a group of amino acids which are of use for each tissue because they are relatively stable within that tissue. Certain properties of the amino acids have put them into the position of being the metabolite of choice in studies of transport phenomena - most of them are very well concentrated by tissues in general; many of them, as pointed out above, are relatively inert in tissues; most of them are now available labeled with radioactive isotopes to facilitate assay; they are a naturally occurring metabolite; many analogues are easily obtained and structural relationships may be studied through the use of the stereoisomers. Two types of measurement have been used in the study of amino acid uptake by tissues. These are: ( I ) measurement of the initial velocity of uptake of the amino acid and (2) measurement of the steady state level attained within the tissue. In most of our previous approaches to the study of transport phenomena, we have chosen to measure the steady state levels obtained because of the difficulties inherent in the accurate measurement of initial velocities. Let us now consider some of the factors which determine these steady state levels. Fig. I illustrates a scheme which was proposed (Heinz and Walsh, 1958), as a working model for the transport of amino acids into ascites tumor cells. This scheme illustrates the three basic factors which may be involved in the movement of a Inetabolite across a cell membrane. They are: ( I ) transport of metabolites into and perhaps also out of the cell, (2) diffusion of metabolites across the cell membrane and (3) exchange phenomena whereby metabolites on one side of the membrane may change places with a similar metabolite on the other side of the membrane. These factors may be summarized as in Fig. 2 which indicates that the total movement is the sum of transport plus diffusion plus exchange diffusion. Net transport is usually simply the facilitated entry of a metabolite into a cell. There are, however, cases where serious consideration has had to be given to the possibility of transport processes which lead to the exit of metabolites from cells.These have usually beenconcerned with bacterial systems (Horecker et a/., 1960), although more recently this phenomenon RtY?,rciicivp . 181

P. G . S C H O L E F I E L D , S. C L A Y M A N

174

appears to have been encountered in studies of exit of amino acids from brain slices (Levi et a/., 1966). Diffusion is usually considered only in terms of exit of metabolites from cells but it may be more correctly expressed as a net diffusion process resulting from the balance between diffusion of materials into, and diffusion of materials out of, the cell. The individual diffusion processes will depend upon the concentrations on each side of the membrane and the net process will depend upon the concentration gradient. However, no consideration appears to have been given to the possibility that Medium

Cell

Transport region

+ A

lc I

I I

I I I

I L-- - - - - --

Free dlffuslon

- - ---

- - -I

Fig. 1. The scheme of Heinz and Walsh (1958).

exchange diffusion may also modify the steady state level attained by a metabolite within the cell. If a mixture of two amino acids is present inside and outside a cell membrane, then there are four possibilities for exchange diffusion. If exchange of a labeled metabolite such as A is being studied, then exchange of A with itself will have no effect upon levels of radioactivity or upon the chemical level of A on either side of the membrane. Effectively, therefore, this factor does not contribute to net movement. Similarly, exchange of B with itself will not be measured if only A is radioactive and as pointed out above homo-exchange can not alter the level on either side of the membrane. On the other hand, exchange of A inside the cell with B on the outside of the cell is a unidirectional process, the extent of which will be governed by a function involving the concentration of A inside the cell and of B outside of the cell. Similarly the exchange of B inside the cell with A outside the cell is another unidirectional process whose magnitude must depend upon a function involving the concentration of B inside the cell and of A outside the cell. There is no reason to believe that these two functions should be equal and opposite. If they are not, then this third factor of ex-

MECHANISMS OF METABOLITE T R A N S P O R T

175

change diffusion should be taken into account in all studies in which steady state levels are involved. It is the purpose of the present communication to point out that steady state levels may be modified by exchange processes, to indicate the nature of the relationship between the exchange process and the concentrations of amino acids on either side of the membrane and to discuss the relation between the processes of transport and exchange diffusion. In a recent re-examination of the interactions between amino acids during transport into Ehrlich ascites cells certain trends were observed which indicated that the

Movement Net T Net D Net E.D.

= ~

-

+ +

Fig. 2.

T D E.D. Tin - Tout Din - Dollt f(Ai, Ao) i- f(Bi, Bo)

+ f(Ai, Bo)

t f(Bi, Ao)

exchange diffusion between amino acids during transport into these cells couldalter the steady state level (Gillespie and Scholefield, unpublished). One such trend was a small but progressive change in K I values as the concentration of inhibitory amino acid was increased. Further, quite striking differences were noted between the affinity of amino acids for the methionine transport system in these cells and that responsible for the transport of ethionine, two amino acids which by other tests seem to share the same transport system. This was not observed in studies concerned with the interactions between amino acids during transport into preparations of mouse pancreas. The phenomenon could be explained either by an exchange diffusion system in pancreas which has a rather limited ability to cause movement of amino acids or by an exchange of amino acids into pancreas which is nearly balanced by exchange of the same amino acid out of the tissue. We have therefore directed our efforts towards obtaining evidence for the process of exchange diffusion in preparations of mouse pancreas, predicting that it should be much slower than in Ehrlich ascites cells, and to determining some of the factors which control the rate at which this process takes place. During studies of influx by exchange, the pancreatic tissue was incubated at 37" with or without non-radioactive amino acids to obtain a tissue loaded with amino acids to a predetermined level. Tissue was then removed from the incubation medium, washed with cold isotonic saline, freed from excess moisture, transferred to a second medium containing labeled amino acid and incubated at a temperature of 15". The uptake of radioactive amino acid by the preloaded and the nonloaded tissues was then determined. In studies of efflux by exchange, a similar procedure was adopted except that the preincubation at 37" was conducted in the presence of a radioactive amino acid and the efflux into media with or without unlabeled amino acid present was studied at 15". In preliminary studies involving only transport of amino acids into the pancreas, it had been noted that the interactions between tryptophan and methionine could not be accurately represented by the standard Lineweaver and Burke (Lineweaver and Burke, 1934), type of analysis. The initial experiments on the exchange process were therefore conducted with this pair of amino acids since it was felt that exchange difRelerrnrrs p. 181

P. c. S C H O L E F I E L D ,

176

S.

CLAYMAN

fusion phenomena might be the cause of this discrepancy. The results obtained are shown in Table I. It is quite apparent from these results that the presenceof methionine inside the tissue can lead to an increased extent of tryptophan uptake (in this case amounting to more than 30 %) and also that the presence oftryptophan in the medium can lead to a marked increase in the efflux of prepacked methionine from the pancreas TABLE I INTERACTIONS BETWEEN METHIONINE A N D T R Y P T O P H A N LEADING T O INCREASED AMINO A C I D E F F L U X F R O M M O U S E P A N C R E A S S L I C E ] (In each case the values presented in columns (a) refer to movement of amino acid when no added or prepacked amino acid is present on the other side of the membrane. The values presented in columns (b) refer to the observed fluxes when tissue prepacked with methionine is incubated in a medium containing tryptophan).

T = 15" Time (min)

fbl 1.57 2.30 2.80

(a)

5 10

15

*

I n i M Tryptophan moving I N

5.4 m M Methionine moving OUT

0.82* 1.13 1.36

(a) 1.45 1.95 2.40

(bl 1.85 2.60 3.27

pmoles amino acid/g tissue water.

approximately double in this example). It is also apparent from these results that the fluxes attributable to exchange diffusion are of the same order of magnitude and that the total fluxes in and out are approximately equal. It should be noted that more accurate observations can be made when efflux by exchange is being measured and the technique employed in the study of efflux has the further great advantage that, in time course studies, successive aliquots of the medium may be taken for analysis while in studies of influx by exchange, individual samples of tissues have to be utilized. Stimulation of efflux of one amino acid through the presence of a second one in the incubation medium may also be interpreted in terms of an inhibition of reconcentrationof the amino acid lost from the cell into the medium.This possibilityseems to be disproved in the present studies by the demonstration that on incubation of pancreas, TABLE I I EFFECTSOF~~MU-AA IB ND

1 mM

METHIONINE O N T R A N S P O R T A N D E X C H A N G E DIFFUSION

OF

ACPC* Additions

Movenient

Nil

5 mM u-AIB

I m M Mrthionine

Uptake ( I mM) ( I h, 37")

9.35*

6.35

6.02

Exchange (5.4 mM) 5 min, 15" 15 min, 15"

0.73 1.35

0.73 1.51

1.28 2.4 1

*

pmoles amino acid/g tissue water.

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MECHANISMS O F METABOLITE TRANSPORT

prepacked with methionine, the efflux of methionine is stimulated by the presence of tryptophan in the incubation medium whilst the presence of methionine inside the cells increases the flux of tryptophan into the tissue i.e. that the increased flux occurs in both directions. Even more conclusive proof that the process is one of exchange diffusion is apparent from the results presented in Table 11. The effects of 1 mM methionine and of 5 mM a-AIB on the transport of ACPC, and the ability of these amino acids to exchange with ACPC previously accumulated by the pancreas, are compared. The concentrations of a-AIB and methionine employed were so chosen that the two amino acids inhibited the uptake of ACPC to about the same extent. If the effects quoted in Table I were obtained simply because of an inhibition of reconcentration, then these two amino acids at these concentrations should have exactly the same effect on the rate of loss of ACPC from prepacked tissue. However, as shown in the second part of this table, it is apparent that 5 mM a-AIB had almost no effect on the efflux of prepacked ACPC but methionine at a concentration of 1 mM produced an increased efflux amounting to more than 75 %. It seems safe to conclude that the increased flux of amino acid studied in these experiments is due to exchange diffusion and that the process has its own specificity which is not necessarily the same as the specificity of the carrier system involved in the transport of amino acids. This specificity is further exemplified by the results presented in Table I11 T A B L E I11 E F F L U X OF L - M E T H I O N I N E - M E - ' * C

Atnino Acid added ( 5 mM)

Nil

Taurine U-AIB Glycine GABA DL-u-amino-ri-butyric

ACPC Methionine

acid

FROM P A N C R E A S I N

tration 5.4 m M )

I5

M I N U T E S AT

15" (Initid

Efpux pmoleslg tissue water

Percentage of Control

1.41 1.41 1.57 1.82 2.34 2.92 3.53 4.19

100

COnCen-

100 111

129 166 207 25 1 291

which concerns the ability of various amino acids to stimulate the efflux of methionine from pancreas. Taurine, which proved to be quite an effective inhibitor of the accumulation of most amino acids by pancreas, was completely unable to stimulate the efflux of methionine and the ability of a-AIB to exchange with methionine, as noted above, was minimal. a-Amino-n-butyric acid, the straight chain compound corresponding to a-AIB, was investigated and it was found that it more than doubled the efflux of methionine. The effect of the physiological isomer, GABA, was more than that of a-AIB and less than that of a-amino-n-butyric acid. Glycine had a relatively small effect, ACPC increased the flux two and one half fold and homo-exchange as measured by the effect of methionine in the outside medium increased the flux nearly References p. 181

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threefold. The effects of GABA are particularly intriguing because, in other studies with radioactive GABA, it has been shown that this compound is concentrated poorly if at all by the pancreas. In other words, GABA seems to have little or no affinity for any transport system functioning in mouse pancreas and yet to have a significant affinity for the system involved in the exchange process leading to efflux of methionine. In view of these anomalies, it seemed of interest to determine whether the concentration effects controlling exchange differed in any way from those controlling transport. The system investigated first was that in which ACPC inside the pancreas was caused to exchange with methionine in the incubation medium (Fig. 3). In the first set of INSIDE. OUTSIDE

ACPC

25r

*

10.5mM ACPC+ METHlONlNE

I r n M METHlONlNE 25

2.0

1.5

1.c

a! -- Ot I 5 10 15 20 Concentration (mM )

Fig. 3. The effects of variation in the concentration of amino acid (a) inside the tissue (ACPC) or (b) in the external medium (rnethionine) on the increased flux of ACPC from pancreatic tissue into an incubation medium containing methionine.

experiments, pancreas was preloaded with ACPC to different levels and caused to exchange with methionine at a concentration of 1 mM in the incubation medium. The results obtained are shown in Fig. 3a. It is apparent immediately that the system would only be termed saturated at extremely high levels of ACPC inside the cell. On the other hand, when the concentration of ACPC inside the cell was maintained at 10.5 mM and the concentration of methionine in the outside medium was varied, the results shown in Fig. 3b were obtained. The external amino acid is able to saturate the exchange system at quite low concentrations and approximate estimates of an affinity constant have been obtained. It should be noted that these are only approximate values since they involve data in which large blank values have been subtracted from the overall flux i.e. the rate of movement of ACPC into a n amino acid free medium has been subtracted from the total flux observed in the presence of methionine. The value obtained for the affinity constant depended on the period of time over which the rates were calculated but they were always of the same order. A summary of some of the

179

MECHANISMS O F M E I'ABOLITE T R A N S P O R T

experimental values obtained is presented in Table IV. In the examples where the concentration of methionine present in the outside medium was varied, the affinity constant seems to be of the order of 2 mM and it is worth noting that the affinity constant characterizing the transport of methionine into pancreas has previously been shown to be 2.9 mM (BCgin and Scholefield, 1965). Similarly the uptake of ACPC is characterized by an affinity constant of 5.8 mM (BCgin and Scholefield, 1965), which is of the T A B L E 1V Itisicle

Oiirside

Variahle

KexfnrM)

ACPC

Meth

ACPCi

>30

ACPC ( 5.4 m M ) ACPC (10.5 m M ) Meth ( 5.5 mM) Meth ( 6.0 mM) ACPC ( 5.4 mM)

Meth Meth Meth ACPC ACPC

Metho Metho Meth, ACPCo ACPG

1.5 2.2 3.0 7.1 5.0

same order as the constant characterizing the affinity of ACPC present in the medium for the system responsible for exchanging it with the prepacked methionine. The similarity of these values strongly suggests that exchange systems may have much in common with transport systems and that competition or interaction between amino acids for the exchange system might occur just as it does in the case of the transport system. In Table V, the fluxes of ACPC, prepacked into the pancreas, have been studied in media containing non-radioactive ACPC, methionine or a mixture of these TABLE V T H E EFFECTS O F M I X T U R E S OF A C P C A N D M E T H I O N I N E I N T H E E X T E R N A L M E D I U M O N E F F L U X O F A C P C FROM MOUSE P A N C R E A S S L I C E S

6

2 5 8

12 15

It1 M

ACPCo

0.71 1.73 2.46 3.24 3.71

6 tnM ACPC p l i i . ~3 ttiM Merh

3 tnM Meth,

0.78 1.74 2.41 3.19 3.58

0.71 1.53 2.21 2.63 3.04

__

ACPCi*

=

5.3 mM.

two amino acids. The concentrations employed were approxinlately those characterizing the half saturation level, to be certain that additive effects were possible. However as seen from Table V the presence in the incubation medium of the two amino RvJiwnrrs p. 181

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P. G . S C H O L E F I E L D , S. C L A Y M A N

acids gave no more effect than ACPC alone. It should also be noted that simple diffusion should only occur at a very low rate since the concentrations of ACPC inside and outside the tissue are approximately equal. In fact, rapid movement of radioactivity occurs confirming again that exchange diffusion is responsible for the observed fluxes. If competition for one exchange site had occurred through the presence of two amino acids at half saturation levels then a significant increase in the flux of ACPC should have occurred. In view of the other possibility that inhibitory effects might occur, a similar experiment was conducted in which 1 mM ACPC, T A B L E VI ACPC A N D a - A I B I N T H E E X T E R N A L ACPC FROM M O U S E P A N C R E A S S L I C E S

T H E EFFECTS O F MIXTURES OF

M E D I U M ON E F F L U X O F

Time (min)

I m M ACPCo

1 m M ACPC plus 5 m M AIB

5 mM a-AIBo

2 5 8 12 I5

0.63 1.26 1.64 2.10 2.52

0.71 1.25 1.74 2.22 2.48

0.42 0.70 0.93 1.15 1.32

ACPCI* = 5.5 mM.

5 mM a-AIB or a mixture of these amino acids at these concentrations, were present in the incubation medium. In this case the concentration of ACPC in the medium was made 1 mM in order to have the minimal possible concentration of substrate which would show a significant exchange diffusion effect. The concentration of AIB was one which caused a very marked inhibitory effect on the accumulation of ACPC from a medium containing the amino acid at a level of 1 mM. As may be seen from the results presented in Table VI there was no significant effect of a-AIB on the homo-exchange TABLE VII T H E EFFECTS O F M I X T U R E S O F T H R E O N I N E A N D M E T H I O N I N E O N E F F L U X O F M E T H I O N I N E F R O M MOUSE P A N C R E A S SLICES

Time (min)

5 mM Threonineo

1.4 m M Meth plus 5 m M Threo

1.4 m M Merho

3 6 9 12 15

1.15 1.89 2.38 2.82 3.15

1.58 2.57 3.11 3.45 3.79

1.23 1.85 2.35 2.72 3.10

Methioninel*

=

5.3 mM.

MECHANISMS O F METABOLITE T R A N S P O R T

181

of ACPC under these conditions. Additive effects have been encountered as may be seen from Table VII but even here the extents of exchange were only partially additive. TABLE VIII SUMMARY 1. Stimulation of influx and efflux 2. Stimulation = exchange diffusion 3. Characteristic specificity

4. Saturation phenomena 5. KT fi KE.D. 6. N o competition or inhibition

The results presented today are summarized in Table VLII. The differing specificities between the transport process and exchange diffusion, the interaction during transport but not during exchange diffusion, the lack of accumulation of GABA but its positive effect in causing flux by exchange - these all indicate that the factors controlling interaction of amino acids with the exchange “carrier” can not be identical with the factors controlling interaction of amino acids with the transport carrier. This is in spite of the demonstrated similarity of the respective affinity constants. One may speculate on several possible explanations -are there completely different mechanisms for transport and exchange, is there simply a different carrier system or are carriers involved at all, does the amino acid react with a simple carrier complex during transport but with a carrier-amino acid complex during exchange, is there a two step reaction involved in which one step determines the affinity constant for the amino acid being moved while the other step confers the specificity, does the involvement of ions and ATP during transport but not in exchange diffusion play some role in determining specificity and how far can generalizations be made on the basis of the present results? Needless to say, we are trying to design experiments to yield answers to to some of these perplexing questions. ACKNOWLEDGMENT

The financial support of the National Cancer Institute of Canada is gratefully acknowledged. REFERENCES BBGIN,N. AND SCHOLEFIELD, P. G. (1965) The uptake of amino acids by mouse pancreas in vitro. 11. The specificity of the carrier systems. J . Biol. Chem., 240, 332-337. HEINZ,E. AND WALSH,P. M. (1958) Exchange diffusion, transport, and intracellular level of amino acids in Ehrlich carcinoma cells. J . Eiol. Chem., 233, 1488-1493. HORECKER, B. L., THOMAS,J. AND MONOD, J. (1960) Galactose transport in Escherichia coli. 11. Characteristics of the exit process. J . Biol. Chem., 235, 1586-1590. LEVI,G., BLASBERG, R. AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vifro. Arch. Biochem. Biophys., 114, 339-351. LINEWEAVER, H. AND BURKE,D. J. (1934) The determination of enzyme dissociation constants. J . Amer. Chem. SOC.,56, 658.

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S. C L A Y M A N

DISCUSSION T. Z. CSAKY:If you performed all the experiments at IS" then probably the active accumulation was somewhat slowed down.

P. G. SCHOLEFIELD: At 15" there is very slow accumulation relative to 37". T. Z. CSAKY:When you studied the straight transport, was the concentration always higher outside than inside? In other words: was the transport always down along the concentration gradient? P. G. SCHOLEFIELD: No. When I quoted the actual transport values they were generally obained at 37", and we were looking at steady state levels. In amino acid transport, as you know, the steady state level is usually determined by the initial velocity; thus, if one steady state level is higher than another, the initial velocity will also be higher and it does not matter which you measure.

T. Z. CSAKY:Because your results really would suggest two kinds of carriers. P. G. SCHOLEFIELD: I would not say that.

J. FOLCH-PI: Is i t valid to assume that what happens at 15" will happen at 37"? In the work of Larrabee kinetics at 25" were quite different from there when he increased the temperatiire. Are you sure that a similar situation is also valid at 37"? P. G. SCHOLEFIELD: I cannot be positive. However, anything which inhibits transport at 3 7 ' also inhibits at 15". J. FOLCH-PI: But with the same temperature coefficient?

P. G. SCHOLEFIELD: 1 don't know. If you study reactions at one temperature only, you really cannot talk about the effect of temperature. T. Z. CSAKY:The temperature for active transport is always higher than for mediated simple transport. So the two temperatures cannot be directly compared with respect to transport and exchange.

If I am correct, Dr. Scholefield actually proposes six different methods of transport: A. LAJTHA: transport in, transport out, diffusion in. diffusion out, exchange in, exchange out. The possibility that each of these six mechanisms has characteristic properties is a very important one. We also had evidence for similar differences in properties at 37", for example, in measuring stereospecificity. 1 could explain perhaps a number of differences between the transport in and transport out by the operation of mechanisms that are basically the same except that the membrane is asymmetrical on the two sides. One compound being an inhibitor of uptake and an activator of exit also does not necessarily mean that uptake and exit are different, if the level or the binding of this compound is significantly different at the two sides of the membrane. But the differences between exchange and active transport in most cases, as Dr. Scholefield also showed, are more difficult to explain with the same mechanism operating and with inhomogeneity in the membrane. As far as passive diffusion is concerned, I think, at least under physiological conditions,this we can ignore. As far as we can see, it does not really quantitatively enter much. I wanted to ask Dr. Scholefield if there are any other differences in properties. Could you show any differences between active uptake and exchange as far as sodium or energy requirement, or any other property, is concerned? P. G. SCHOLEFIELD: This, I think, raises the difficulty that I had in answering Dr. Folch's question. If pancreas is cooled to 15" it hardens and tends to solidify like a piece of fat, so that there are experimental difficulties. We have looked at the effect of taking out ions, incubation in a sucrose medium, the effect of ouabain, and so on, and there is little effect on the exchange process. On the other hand, when we d o transport studies over the same short intervals of time, we again get no effect. There is always an effect on transport in vitro if pancreas is incubated long enough, but during the initial period over which we measure exchange, there is very little effect. But then again, there is very

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183

little effect on transport also, and so we had to resort to these other methods, to prove that what we are measuring is really exchange and not some manifestation of the transport phenomenon.

K. A. C. ELLIOTT:1 think some time we must know what we mean by “in” and “out”. Perhaps Dr. Scholefield can help us. We know of various compartments in the cell. Does “in” mean just inside the outer membrane or inside mitochondria or Golgi apparatus or lysosomes, vesicles, or other particles, or adsorbed on membranes? Inside and outside what? Are all transported substances inside or outside the same compartment?

P. G. SCHOLEFIELD: I just want to make one point clear. If one studies uptake of amino acids into ascites tumor cells, whcre the nucleus is approximately 50”i of the total volume of the cell, one can get an average concentration of amino acid of up to 40 mM. If it does not go into the nucleus at all then it has to be present at an average concentration of 80 mM in the cytoplasm. If it only goes into the mitochondria, then it would have to be present at a fantastic concentration. So it is believed that in ascites cells, and by extrapolation, also in other tissues, that “in” really means inside the cell, and in every large water space within the cell. G. LEVI:I would like to make t w o brief comments. The first one concerns the stimulation of amino acid fluxes that Dr. Scholefield described as determined by ”a flow driving counter flow” effect. In a recent publication (Levi, Blasberg and Lajtha, 1966, Arch. Biocheni. Biophys., 114 : 339) we have shown that the exit of amino acids from preloaded brain slices into an amino acid-free medium can bc stimulated by the intracellular presence of large neutral amino acids with a non-polar side chain (such as leucine and phenylalanine). This stimulation showed some substrate specificity, in that the effect was different with amino acids belonging to different classes. The effect could not be explained as a “flow driving counter flow” phenomenon and was tentatively interpreted as due to an “activation” of the membrane exit carrier(s) by leucine and phenylalanine. My second comment supports Dr. Elliott’s suggestion of a possible compartmentation of amino acids in the tissue as well as the exit of several amino acids from preloaded brain slices. In fact, in this same publication we showed evidence for the compartmentation of the free amino acid pool in brain slices, as we noted that the exit rates depend not only on the intracellular concentration of an amino acid, but also on the experimental time period during which exit was measured. I could cite as an example the behavior of lysine exit. In these experiments brain slices were preloaded with different lysine concentrations and then the exit into an amino acid-free medium was determined. We found that at a given intracellular concentration of lysine the exit rate was progressively lower if measured in the 0-10, 10-20, and 2 0 4 0 min time periods respectively. Exit seems to occur from different intracellular pools having different exit rates, which is consistent with an amino acid compartmentation in the tissue. D. H. FORD:I would like to add a comment in relation to Dr. Elliott’s question about what is “inside” or “where” a labeled compound is inside in relation to a cell after previous injection of a labeled compound. Droz (1965, Fate of newly synthesized proteins in neurons. In The Use of Radioautography in Investigating Protein Synthesis. Leblond, C . P. and Warren, K. B. (Eds.), p. 159, Academic Press, New York, 1965) has recently published a report in which he has studied the accumulation of [3H]leucine by electronmicroscopic radioautography in neurons. At a very early phase after injection, the labeled material was associated with the endoplasmic reticulum of the Nissl substance wherein protein synthesis is believed to occur. The labeled material then becomes accumulated in the Golgi vesicles from whence it migrates to the axon hillock and down into the axon with the passage of time. Thus, to say that a labeled compound is “inside” a cell is somewhat non-specific and depends on the length of time after the initial injection of the labeled compound that is allowed to elapse before the animal is killed. P. G. SCHOLEFIELD: Yes, but that was incorporation into the protein that Leblond was studying, and I don’t think it was free amino acid. D. H. FORD:With the usual procedures used in histological processing, free amino acids are by and large lost from the tissue. Thus, what remains to be seen by radioautography would be the amino acid material either incorporated into protein or perhaps bound to it as a terminal element. N. M.

VAN

GELDER: I would like to clear something up; whenever we talk about a carrier system we

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P. G . S C H O L E F I E L D , S. C L A Y M A N

think of the binding of amino acids on the carrier because we have charged groups within the structure. We are talking about neutral amino acids, but these are not neutral a t all; they have a charged amino group and a charged carboxyl group. Perhaps the reason that some are not bound to the carrier is that thedistance between the carboxyl group and the amino acid group is toogreat. Therefore, perhaps such compounds can inhibit transport but cannot be carried. They can inhibit from carrier to carrier, in making a sort of bridge, without being themselves attached to a single carrier unit and transported into a particular tissue. I find the term “neutral amino acids” confusing. If you put them on electrophoresis they won’t migrate perhaps, but they are certainly not neutral in physiological terms.

A Comparison of the Transport Systems for Amino Acids in Brain, Intestine, Kidney and Tumour K. D. N E A M E Pliysiological Laboratory, University of Liverpool, Liverpool (England)

Amino acids enter many tissues by a process of active transport and can often be taken up into the cell against a concentration gradient (Wilbrandt and Rosenberg, 1961 ; Neame, 1962; Quastel, 1965), thus implying the existence ofa carrier mechanism. In certain tissues it seems in fact that there are different carrier mechanisms for different groups of amino acid (Beyer et al., 1947; Newey and Smyth, 1964; Blasberg and Lajtha, 1965; Christensen and Liang, 1965) and this paper is an attempt to indicate how those groups probably differ from one tissue to another. It is divided into two parts, firstly a brief review of the different transport systems that appear to exist in certain tissues (brain, small intestine, kidney and tumour), and secondly some of the relevant experimental work which has been carried out in those tissues. It is unnecessary to discuss the methods used for investigation, except to mention that the most usual involves the competition that can occur between amino acids for a common transport mechanism. If one amino acid shares the same transport system as a second amino acid, it will displace the second amino acid from the carrier. This is seen experimentally as an inhibition of the uptake of the second amino acid in the presence of the first. It may look from Figs. 1-4, in which the different transport systems are presented, as if precise answers have been obtained, but it must be appreciated that they only represent what appears at the present time to be the most likely interpretation of experimental results combined from many different sources and involving different techniques. I t is best first of all to classify the amino acids themselves. Although there are differences in transport between D- and h o m e r s and between a- and w-amino acids, it is the net ionic charge which mainly seems to determine the groupings of amino acids with respect to transport. Thus they are best divided into acidic, basic and neutral amino acids. Even this classification, however, is not truly satisfactory, as the net ionic charge may vary according to pH, as has been particularly demonstrated with respect to a,P-diaminopropionic acid (Christensen, 1964a, b), a so-called basic amino acid which may behave as a neutral amino acid at physiological pH. Amino acids have also in most cases been classified in the figures according to the length of carbon chain, which may in certain instances affect their affinity for a particular transport system. Referenres pp. 194-1 96

186

K . D. N E A M E

Fig. I . Grouping of amino acids with respect to transport systems in kidney. Regular indentations indicate overlap of groups. A patterned line related to an individual amino acid indicates a definite relationship with the group indicated. Abbreviations: DAP a, /I-diaminopropionic acid; DAB a, 7)-diaminobutyric acid; AspNHz asparaghe; GluNHz = glutamine; AIB = m-aminoisobutyric acid; GABA = y-aminobutyric acid; CYS = cystine. References : Beyer el al., 1947; Robson and Rose, 1957; Scriver et a/., 1961; Webber et a/., 1961; Rosenberg et al., 1962; Webber, 1962, 1963; Scriver and Wilson, 1964; Fox e i a/., 1964; Fox et a/., 1964; Neame, 1965, 1966. 5

~

Intestine and kidney will be the tissues discussed first, as they appear to have the simplest arrangements. With kidney (Fig. 1) the evidence for separate systems is derived mostly from investigations involving disappearance of amino acids from the renal tubule rather than by direct measurement of uptake or transport (Beyer et al., 1947; Robson and Rose, 1957; Webber et al., 1961 ; Webber, 1962, 1963) although some evidence has been obtained from tissue slices (Rosenberg et al., 1962; Fox et al., 1964; Neame, 1965, 1966). There is a transport system for neutral amino acids (Webber e t d . , 1961; Webber, 1962), another for basic amino acids (Robson and Rose, 1957; Webber et al., 1961 ; Rosenberg et al., 1962) and a third for acidic amino acids (Webber, 1962, 1963). There is some overlap between transport of basic amino acids and that of neutral amino acids (alanine, methionine and histidine) (Webber et al., 1961 ; Webber, 1962; Neame, 1966), and also between acidic amino acids and neutral amino acids (alanine and methionine) (Webber, 1962). In addition, glycine, proline, and hydroxyproline are transported not only by the system for neutral amino acids but also by a more specific system of their own (Scriver et ul., 1961). In small intestine (Fig. 2) the types of transport system appear to be similar to

A M I N O A C I D T R A N S P O R T IN V A R I O U S TISSUES

NUMBER

OF CARBON ATOMS IN CHAIN

STEREO1 SOMERS o(.AMINO

ACIDS

I

187

N O STEREOISOMERS

I

O - A M I N O ACIDS

I 2

3 4

5 6

Fig. 2. Grouping of amino acids with respect to transport systems in small intestine. Details as in legend to Fig. I . References: Wiseman, 1953, 1955; Agar, Hird and Sidhu, 1956; Finch and Hird, 1960; Nathans ef a/., 1960; Hagihira era/., 1961 ; Hagihira ef a/., 1962; Lin ef a/., 1962; Larsen er a/. 1964; Newey and Smyth, 1964; Robinson and Felber, 1964; Thier et a / . , 1964; Matthews and Laster, 1965; Neame, 1965, 1966; Reiser and Christiansen, 1965; Hindmarsh er a/., 1966.

those of kidney, except that there has been no evidence that acidic amino acids are actively transported at all (Wiseman, 1953, 1955; Finch and Hird, 1960; Hagihira et a/., 1961 ; Hagihira et a/., 1962; Newey and Smyth, 1964; Thier et a/., 1964; Matthews and Laster, 1965; Neame, 1965, 1966; Hindmarsh et a/., 1966), and in detail there are clearly differences (Figs. 6-9). Now in tumour tissue, where the Ehrlich ascites carcinoma cell is mostly involved, there is rather a different picture (Fig. 3). Overall, there are the same three main groups of transport system as described for kidney and intestine, that is, one for neutral, one for basic and one for acidic amino acids, but the groups for neutral and basic amino acids each have sub-groups (Oxender and Christensen, 1963; Christensen, 1964a; Heinz et a/., 1965). Short-chain and long-chain neutral amino acids are transported by separate systems (Oxender and Christensen, 1963) which show considerable overlap between each other. Similarly, there is a transport system for short-chain and another for long-chain basic amino acids (Christensen, 1964a) with overlap between them and the systems for neutral amino acids. There is also a separate system for acidic amino acids by which glutamic acid, but not apparently aspartic acid, is taken up, although poorly (Heinz et a/., 1965). Again there is some overlap between this system and that for neutral amino acids (Heinz et a/., 1965). In addition there is a separate system affecting mainly those neutral amino acids having a chain of Rifirmcrs pp. 194-196

K. D. N E A M E

188 NUMBER

OF CARBON A T 0 MS

IN CHAIN

1 2

3 4

5 6

Fig. 3. Grouping of amino acids with respect to transport systems in tumour. Details as in legend to Fig. I . References : Christensen et a/., 1952; Oxender and Christensen, 1963; Christensen, 1964a.b; Heinz er a/., 1965; Johnstone and Scholefield, 1965; Oxender, 1965; Neame and Ghadially, 1967.

two carbon atoms and the basic amino acid a,@-diaminopropionicacid, the latter being able to behave ionically like a neutral amino acid (Christensen, 1964b). In brain there is a somewhat similar arrangement to that seen in tumour, with three main systems overlapping to some extent (Fig. 4). Those for neutral and basic amino acids are also subdivided into a system for short-chain and a system for longchain amino acids (Blasberg and Lajtha, 1965; Neame, 1966). The system for acidic amino acids involves overlap with short-chain but not long-chain basic amino acids, and with some of the neutral amino acids (Blasberg and Lajtha, 1965; Neame, 1966). There also seems to be a separate system which involves y-aminobutyric acid and probably includes other 0)-amino acids (Blasberg and Lajtha, 1965; and see Fig. 7). Having discussed briefly the transport systems that appear to exist in the four different tissues, some examples will now be given in which there is a direct comparison, firstly, of transport of different amino acids in one tissue (brain) and, secondly, of transport in the different tissues under the same experimental conditions (Figs. 5-9). The technique involves the measurement of the uptake of an amino acid alone and in the presence, separately, of a number of other amino acids whose effects are compared one with another. In the figures the control value (uptake of amino acid when alone) has been plotted as 100 on the ordinate, and to it the other experimental values are referred. A value of less than 100 implies competition by added amino acid for a common transport system. In a number of instances the competitive nature

A M I N O A C I D T R A N S P O R T I N V A R I O U S TISSUES

189

NUMBER

OF CARBON A T 0 MS IN CHAIN

I I 2

3 4

5 6

Fig. 4. Grouping of amino acids with respect to transport systems in brain. Details as in legend to Fig 1. References : Chirigos et a/., 1960; Guroff et a/., 1961 ; Neame, 1961 ; Abadom and Scholefield, 1962; Nakamura, 1963; Smith, 1963; Tsukadaetal., 1963; Yoshidaer a/., 1963 aand b; Neame, 1964; Blasberg and Lajtha, 1965, 1966.

of the inhibition has been confirmed by reciprocal analysis (Lineweaver and Burk, 1934; Neame, 1964, 1966). In the first example (Fig. 5 ) the effect of basic amino acids on a number of other amino acids in brain is shown, the points plotted being taken directly from previously published data (Neame, 1964; Blasberg and Lajtha, 1965), showing the effect in brain slices of the presence of basic amino acids on the uptake of a number of basic, acidic and neutral amino acids. In the bottom left-hand chart of Fig. 5 the uptake of lysine and arginine (long-chain basic amino acids) in the presence ofbasic amino acids is shown. With both of them uptake is inhibited by long-chain basic amino acids (ornithine, lysine and arginine) but not by short-chain basic amino acids (DAP* and DAB*). In the top left-hand chart of Fig. 5 is shown the effect of basic amino acids on uptake of a number of neutral amino acids. There is here also a marked difference between the effect of the long-chain and of the short-chain basic amino acids; in addition, with the latter, DAB consistently produces greater inhibition than DAP, suggesting the presence of a common factor in respect of these amino acids. The effect of basic amino acids on the uptake of a number of other neutral amino acids is shown in the bottom right-hand chart. Although there is here no common

*

For abbreviations see legend to Fig. 1.

References p p . 194-196

K. D. N E A M E

190

c

Neutral

I

I

!

I

Ill DAP

2

I

I DAB

Orn

Lyr

I

I I I I

Art

I

3

4

CHAIN

5

6

2

3

LENGTH OF ADDED AMINO

4

5

6

ACID

Fig. 5. Uptake of single amino acids by brain slices in the presence of basic L-u-amino acids indicated on abscissa (adapted from data given by Neame, 1964 and Blasberg and Lajtha, 1965). The joining of data points by lines does not indicate a continuous function, but is designed merely to indicate visually the relation between different effects. Each line has been labelled with the amino acid whose uptake it represents. Neutral, basic and acidic amino acids have been grouped together for comparison as indicated, The horizontal interrupted line (control value) indicates the uptake of each amino acid when alone. The vertical interrupted line separates results obtained when using short-chain basic amino acids from those when using long-chain basic amino acids. Chain length of added amino acid calculated as number of atoms in carbon chain less one carboxyl group; abbreviations as in legend to Fig. 1. Substrate amino acid 2 rnM. Added amino acid 10 mM (but 2 mM with histidine).

factor with respect to the short-chain basic amino acids there is again a difference in the effect of the long-chain as compared with the short-chain basic amino acids. The same also applies to the effect on one acidic amino acid (top right-hand chart). Such comparisons indicate the likelihood of there being one transport system for

AMINO ACID TRANSPORT IN VARIOUS TISSUES

191

2! I 4L

5

-

KIDNEY

SARCOMA

long-chain basic amino acids and one for short-chain basic amino acids, with only the latter able to transport other types of amino acid to any extent. In the second case, results involving the four tissues already discussed (brain, intestine, kidney and tumour) are presented (Figs. 6-9). They show the effect of different amino acids on the uptake of L-histidine under identical conditions, and with the concentration of amino acids the same in all instances. Fig. 6 shows that the effect of neutral a-amino acids is somewhat similar in all four tissues, but that in no two tissues is it identical. The similarity lies, firstly, in the increase, in general, in the degree of inhibition with increase in chain length of inhibiting amino acid, and secondly, in the greater inhibitory effect of the h o m e r as compared with the D-iSOmer. Re]crrnces pp. lY4-IY6

K. D. N E A M E

192

I

BRAIN

INTESTINAL MUCOSA

Z

*-+-+-; A 0 W

u

5 2

LL

50-

Y0

z *.f 0

D

2

I -1

I -

-

50 Gly

!-Ah

I

I

1

2

GABA $-Am-val t-Am-cap I I I

3

4 CHAIN

Gly

p-Ala

GABA 6.Am-val E-Am-cap

I

I

I

1

1

I

6

I

2

3

4

5

L E N G T H OF A D D E D

AMINO

5

ACID

Fig. 7. Uptake of L-histidine by brain, intestinal mucosa, kidney and sarcoma in virro in the presence of w-amino acids indicated on abscissa (adapted from data given by Neame, 1964, 1966, and Neame and Ghadially, 1967). Details as in legend to Fig. 6.

The latter finding suggests that in all four tissues the carrier has a higher affinity for the L-isomer. Where it has been established that there are separate carrier systems for long- and short-chain amino acids, as with brain and tumour, there appears to be a dichotomy in the pattern of effect produced. The effect produced by o-amino acids (Fig. 7) is quite different from that produced by a-amino acids. In all tissues inhibition is slight or absent and indicates the importance of the relationship between amino group and carboxyl group with respect to inhibitory effect and hence to transport. A comparison between different tissues is difficult here owing to the large scatter of results compared with the relatively small amount of inhibition. With basic amino acids there are markedly different effects for different tissues (Fig. 8). With intestinal mucosa and kidney the inhibition is fairly uniform, although somewhat greater in the case of intestine. The behaviour of brain and sarcoma is, on the other hand, quite different; in both there is a definite difference in the effect of long-chain as compared with short-chain basic amino acids, but the effect in brain is

6

193

AMINO A C I D TRANSPORT I N VARIOUS TISSUES

2

2

100

L

z

0

U

.\o

a

+ Z

g 100 U

w

z

E

L

50

I

2

3

4

5

C H A I NL E N G T H

6

I

2

3

4

5

OF A D D E D AMINO A C I D

Fig. 8. Uptake of L-histidine by brain, intestinal mucosa, kidney and sarcoma in vitro in the presence of basic u-amino acids indicated on abscissa (adapted from data given by Nearne, 1964, 1966 and Neame and Ghadially, 1967). Details as in legend to Fig. 6.

the reverse of that in sarcoma. With sarcoma the long-chain amino acids produce definite inhibition of histidine uptake, but the short-chain ones produce none, whereas in brain it is only the short-chain amino acids which have any inhibitory effect. The above supports the view that in intestine and kidney there is only one system for transporting basic amino acids, whereas in brain and tumour there are two. The two systems in brain, however, are obviously very different from those in tumour. In the case of the acidic amino acids the situation is well-defined so far as the evidence goes (Fig. 9). Only in brain do all the acidic amino acids produce inhibition. In all the other tissues (apart from kidney with respect to the D-isomers) they produce no inhibition. The transport of acidic amino acids is thus markedly different in brain as compared with the other tissues in which it occurs. I t appears then as if in a number of tissues at least, when transport of amino acids can be shown, there are three main groups of transport system, one for neutral amino acids, one for basic amino acids and one for acidic amino acids, with subdivision in certain instances. But the nature of each group is different in the different tissues, and ReJiwnces pp. 194-1 96

6

K. D. N E A M E

194

CHAIN

L E NG TH O F

ADDED

AMINO

ACID

Fig. 9. Uptake of L-histidine by brain, intestinal mucosa, kidney and sarcoma in virro in the presence of acidic a-amino acids indicated on abscissa (adapted from data given by Neame, 1964, 1965 and Neame and Ghadially, 1967). Details as in legend to Fig. 6.

even where there are subdivisions, those subdivisions may havequitedifferent properties in the different tissues. What has been found in one tissue obviously must certainly not be assumed to apply to another tissue. Further, the transport systems in brain are far from identical with those in any of the other tissues investigated. Such differences between tissues imply that the proportions of amino acids taken up from the bloodstream (as opposed to differences found for uptake of single amino acids (Neame, 1962)) will vary from one tissue to another. Further, when there is an imbalance of amino acids or an excess of one amino acid in the blood, as for example in phenylketonuria, such patterns of amino acid uptake or entry into tissue will be altered in different ways according to the nature of the tissue involved. REFERENCES ABADOM,P. N. A N D SCHOLEFIELD, P. G . (1962) Amino acid transport in brain cortex slices. 11. Compctition between amino acids. C u ~ u dJ. . Eiocheni. Physiol., 40, 1591-1602. AGAR,W. T., HIRD,F. J. R. AND SIDHU,G. S. (1956) The absorption, transfer and uptake of amino

A M I N O A C I D T R A N S P O R T IN V A R I O U S T I S S U E S

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x i d s by intestinal tisstr:. Bioclrinr. Biopliys. Acra, 22, 21-30. BI-YtR, K. H., WRIGHT, L. D., SKEGGS, H. R., Russo, H. F. A N D SHANER, G . A. (1947) Renal clearance of essential amino acids: their competition for reabsorption by the renal tubules. Anlev. J. fhysiol., 151, 202-210. BLASBERG, R. A N D LAJTHA, A. (1965) Substrate speciiicity of steady-state amino acid transport in mouse brain slices. Arc.'i. Bidierri. Biophys., 112, 361-377. -(1966) H-terogeneity of the mcdiated transport systems of amino acid uptake in brain. Brain R ~ s . 1, , 86-104. CHIRIGOS, M.A., GRtENGARD, P.AND UDENFRIEND, s. (1960) Uptake of tyrosine by rat brain irr vivo. J . B i d . Clretii., 235, 2075-2079. CHRISTENSEN, H. N. (1964a) A transport system serving for mono- and diamino acids. Proc. Nail. Acatl. Sci., 51, 337-344. - (1964b) Relations in the transport of v-alanine and the ti-amino acids in the Ehrlich cell. J. Biol. C k / i i . ,239, 3584-3589. CHRISTENSEN, H. N . AND LIANC;,M. (1965) An amino acid transport system of unassigned function in the Ehrlich ascites tumor cell. J. Biol. Clierti.,210, 3601-3608. CHRISTENSEN, H. N., Ricics, T. R., FISCHER, H. AND PALATINE, 1. M . (1952) Amino acid concentration by a free cell neoplasm; relstions among amino acids. J. Biol. Chmr., 198, 1-15. FINCH, L. R. A N D HIRD.F. J. R. (1960)The uptake of amino acids by isolated segmentsofrat intestine. I I . A survey of affinity for uptake from rates of uptake and competition for uptake. Biochim. Biophys. Acta, 43, 278-287. Fox, M . , THIER, S., ROSENRERC, L., KISER,W. A N D SEGAL,S. (1964) Evidence against a single renal transport defect in cystinuria. Ncwi €rig/. J. bled, 270, 556-561. Fox, M., T H I E RS.,, ROSENRtRG, L. AND SEGAL,S. (1964) Ionic requirements for amino acid transport in the rJt kidney cortex slice. I. Influence of extracellular ions. Biochini. Biophys. Acta, 79, 167-176. GUROFF, G., KING,W. A N D UDENFRIEND, S. (1961) The uptake of tyrosine by rat brain itr vitro. 1. B i d . Cherii., 236, 1773-1777. HAGIHIRA, H . , LIN, E. C. C . . SAMIY, A. H. A N D WILSON,T. H. (1961) Active transport of lysine, ornithine, argiiiine and cystine by the intestine. Bioclterii. Biophys. Res. Conim., 4, 478481. H A G I H I R AH.. , WILSON, T. H. A N D LIN,E. C. C . (1962) Intestinal transport of certain N-substituted amino acids. A r w r . .I. f/i,wio/.,203, 637-640. , A. G. A N D PFEIFEER, B. (1965) Studies on the transport of glutamate in Ehrlich n by oiher amino acids and stimulation by H-ions. Biocherii. Z., 312, 542-552. HINDMARSH, J . T., KII.HY, D. A N D WISEMAN, G . (1966) Effect of amino acids on sugar absorption. J. f/i,v.~iti/.,186, 166-174. JOHNSTONI., R. M. A N D SCHOLWIELD, P. G . (1965) Amino acid transport in tumour cells. A h . Carrcer Res., 9, 143-226. Lwst:N, P. R., Ross, J . E. A N D T A P L ~ D. Y , F. (1964) Transport of neutral, dibasic and N-methylsubstituted amino acids by rat intestine. Biocliirv. Biophys. Acta, 88, 570-577. LIN. E. C . C., HAGIHIRA, '-1. A N D WILSON, T. H. (1962) Specificity of the transport system for neutral amino acids in the hsmster intestine. Arrier. J. Pliysiol., 202, 919-925. LINEWEAVER, H. A N D BURK,D. (1934) The determination of enzyme dissociation constants. J. Atirer. Clicwi. Soc., 55, 658-666. MATTHEWS, D. M . A N I ) LASTER, L. (1965) Competition for intestinal transport among five neutral amino acids. A ~ T J. . Pliysiol., 208, 601-606. NAKAMURA, R. (1963) The transport of histidine and methionine in rat brain slices. J. Biocheni. ( Tokyo), 53, 3 14-322. NATHANS, D., TAPLEY, D. F. A N D Ross, J. E. (1960) Intestinal transport of amino acids studied in v i / m with ~-[~:~~I]~iionoiodotyrosine. Bioclhi. Biophys. Acta, 41, 271-282. NEAME, K. D. (1961) Phenylalanineas inhibitor of transport ofamino-acids in brain. Nature (Lonclori), 192, 173-174. - ( 1962) Uptake of L-histidine, L-proline, L-tyrosine and L-ornithine by brain, intestinal mucosa, testis, kidney, spleen, liver, heart muscle, skeletal muscle and erythrocytes of the rat in vitro. J . Physiol., 162, 1-12. (1964) Effect of amino acids on uptake of L-histidine by rat brain slices. J. Neurochem., 11,67-76. -~ (1965) Effect of acidic (dicarbouylic) a-amino acids on uptake of L-histidine by intestinal mucosa, testis, spleen, and kidney iri vitro: a comp3rison with effect in brain. J . Pliysiol., 181, 114-123. (1966) EtTe:t of neutral (1- and to-amino acids and basic ci-amino acids on uptake of L-histidine

--

196

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by intestinal mucosa, testis, spleen and kidney in vitro: a comparison with effect in brain J. Physiol., 185, 627-645. NEAME, K. D. AND GHADIALLY, F. N. (1967) Uptake of L-histidine alone and in the presence of other amino acids by carcinogen-induced sarcomas of the rat itr vitro, Cancer Res., 27, 516-521. NEWEY,H. AND SMYTH,D. H. (1964) The transfer system for neutral amino acids in the rat small intestine. J. Physiol., 170, 328-343. D. L. (1965) Stereospecificity of amino acid transport for tumor cells. J. Biol. Chmr., OXENDER, 240, 2976-2982. OXENDER, D. L. AND CHRISTENSEN, H. N. (1963) Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem., 238, 3686-3699. J. H. (1965) Molecular transport at cell membranes. Proc. Roy. SOC.( B ) , 163, 169-196. QUASTEL, REISER, S. AND CHRISTIANSEN, P. A. (1965) Intestinal transport of amino acids studied with L-valine. Amer. J. Physiol., 208, 914-921. ROBINSON, I. W. L. AND FELBER, J.-P.(1964) A survey of the effect of other amino acids on the absorption of L-arginine and L-lysine by the rat intestine. Gastroenterol., 101, 330-338. ROBSON,E. B. AND ROSE,G. A. (1957) The effect of intravenous lysine on the renal clearances of cystine, arginine, and ornithine in normal subjects, in patients with cystinuria and Fanconi syndrome and in their relatives. Clin. Sci., 16, 75-93. ROSENBERG, L. E., DOWNING, S. J. AND SEGAL,S. (1962) Competitive inhibition of dibasic amino acid transport in rat kidney. J. Biol. Chenr., 237, 2265-2270. SCRIVER, c. R., SCHAFER, 1. A. AND EFRON,M. L. (1961) New renal tubular amino-acid transport system and a new hereditary disorder of amino-acid metabolism. Nature (London), 192, 672-673. SCRIVER, c.R. AND WILSON,0.H.(1964) Possible locations for a common gene product in membrane transport of imino-acids and glycine. Nature (Lotrdm), 202, 92-93. SMITH,S. E. (1963) Uptake 0f5-hydroxy[~~C]tryptophan by rat and dog brain slices. Brit. J. Pharmacol. Chemother., 20, 178-189. THIER, S., Fox, M., SEGAL, s. AND ROSENBERG, L. E. (1964) Cystinuria: in vitro demonstration of an intestinal transport defect. Science, 143, 482-484. TSUKADA, Y . , NAGATA, Y . , HIRANO, s. AND MATSUTANI, T. (1963) Active transport of amino acid into cerebral cortex slices. J. Neurochem., 10, 241-256. WEBBER, W. A. (1962) Interactions of neutral and acidic amino acids in renal tubular transport. Amer. J. Physiol., 202, 577-583. - (1963) Characteristics of acidic amino acid transport in msmmalian kidney. Canad. J. Biochem. Physiol., 41, 131-137. WEBBER, W. A., BROWN,J. L. AND PIITS, R. F. (1961) Interactions of amino acids in renal tubular transport. Amer. J. Physiol., 200, 38C386. WiLBRANDT, W. AND ROSENBERG, T. (1961) The concept of carrier transport and its corollaries in pharmacology. Pharrnacol. Rev., 13, 109-183. WISEMAN, G. (1953) Absorption of amino acids using an it1 vitro technique. J . Physiol., 120, 63-72. - (1955) Preferential transference of amino-acids from amino-acid mixtures by sacs of everted small intestine of the golden hamster (Mesocricetus aurarus). J. Physiol., 127, 414-422. YOSHIDA, H., KANIIKE, K. AND NAMBA, I. (1963a) Properties of a carrier system totransport L-dopa into brain slices. Nature (London), 198, 191-192. J., KANIIKE, K. AND IMAIZUMI, R. (1963b) Studies on active transport of L-dopa YOSHIDA, H., NAMBA, (dihydroxyphenylalanine)into brain slices. Jap. J. Pharmacol., 13, 1-9.

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197

DISCUSSION P. MANDEL: May 1 ask you: how do you explain the increase of transport by DAP and DAB for lysine, arginine, and phenylalanine? 1 do understand the decrease,but how do you explain theincrease?

K. D. NEAME: I have no specific answer to that, except to suggest that it may be some phenomenon more or less like exchange diffusion, as Dr. Scholefield was explaining. DAP and DAB may be involved with a carrier which somehow stimulates the uptake of lysine and arginine through the medium of such exchange processes. A. LAJTHA:I think Dr. Neame's is one of the possible explanations: the inhibitor also gets into the cell (if you want to call it "inhibitor") and by heteroexchange actually activates the uptake by exchange. It is interesting that the activation of efflux by intracellular analogs cannot be explained by this same mechanism. We also found that anaerobic conditions increase efflux, but not exchange, showing a complex situation. An alternate explanation may be a greater inhibition of efflux than influx. This also postulates the prior penetration of the inhibitor into the cells. May I just ask Dr. Neame one question: perhaps with the analogy of a somewhat specific acidic amino acid carrier in the brain, paralleled by high brain acidic amino acid concentration, can you draw any conclusions as to the composition of the physiologically free amino acid pools, and the specificity variations between the various tissues?

K. D. NEAME: The question of pools in the tissues, I believe, is a difficult one. I haveshown the position looking at the tissue as a whole, and not looking at the different compartments, or the different levels of amino acid in the tissue in the different compartments; this would involve further work, trying to divide the uptake or inhibition into more and more discrete parts. But at present, it is only an over-all effect in the tissue. R. BLASBERG: I would like to comment on the stimulatory effect of the small basic amino acids on the steady state uptake of arginine and lysine by brain slices. Since these data were published, the mechanism of steady state inhibition (or stimulation) was studied a little further. We have shown that the small basic amino acids inhibit the unidirectional flux of lysine and arginine both into and out of brain cells; however, the inhibition of efflux was significantly greater than the inhibition of influx. I f the steady state process is a balance between input and output and each unidirectional flux is the sum of physical diffusion, active transport, and exchange processes as discussed by Dr. Lajtha, we could expect a steady state level greater than control in situations where the inhibition of efflux was greater than the inhibition of influx. The significance of exchange mechanisms has not been fully evaluated as yet but undoubtedly will add to our understanding of steady state processes. A. LOWENTHAL: Linneweh (1963) showed that the presence of an excess of phenylalanine in serum decreased the concentration of the other amino acids in the serum of phenylketonuric patients. We could confirm this in serum but not in cerebrospinal fluid. I wonder if you have some data on the action of phenylalanine on the free amino acid content of the brain?

K. D. NEAME: Yes, it was based on work of my own, which was published in 1961, on brain slices, not in vivo unfortunately, in which the phenylalanine did inhibit the uptake of a number of amino acids into brain slices: proline, histidine, ornithine, arginine, and tyrosine. All except proline were markedly inhibited. Proline uptake was inhibited only slightly. I did not do any other tissues, only brain. J. FOLCH-PI:Histidine was inhibited by the two- and three-carbon amino acids, and not by arginine and lysine, while most of the other amino acids seemed to be inhibited by the amino acids closely alike in chemical structure. Does histidine present a special case? Is histidine a basic amino acid in its transport mechanism? Or does it behave like alanine?

K. D. NEAME: I think histidine has been considered by Christensen to act like a neutral amino acid. Whether it can sometimes act like other types of amino acids because of its structure, I don't know. It is possible that local pH changes, perhaps in the tissue or near the membrane, could cause it to act in some other way. But at present there is no way of telling.

198

K. D. N E A M E

K. A. C. ELLIOTT:Dr. Scholefield and Dr. Neame have discussed various interesting interactions of amino acids. Have they any information about interaction cf amino acids and peptides such as polypeptide hormones? K. D. NEAME: No, I have none. P. G. SCHOLEFIELD: I think efforts have been made with the ascites cells, and the results have mainly been negative, although the reports have included statements that the peptides which get into the cell are usually broken down. Certainly the extracellular peptides seem to ka\e very little effcct on the accumulation of amino acids. These were, I think, some of tke earlier results of C1:ristensen. H. M. ADAM:I would like to ask one question concerning the uptake of histidine compared with that ofotheraminoacidsrelated to biogenic amines, such as DOPA, 5-hydroxytryptophan, trqptophan, and so on. Maybe these were included in your lists, but perhaps you could be a little more explicit about this point?

K. D. NEAME: No, I am afraid I have no information on that. Unfortunately DOPA interferes nith the method of estimation for histidine, which is a colorimetric one, so its effect cannot be measured. And the amounts involved would preclude 5-hydroxytryptophan in experiments of this extent in view of the price. 1 left it out for that reason. H. M. ADAM:Nevertheless, such experiments would be interesting. The results might throw light on possible relationships in the metabolism of these amino acids, the formation of their corresponding amines in the brain, and possibly on the relative concentrations of these amines: histamine, 5-hydroxytryptamine, and catecholamines. D. B. TOWER:I would just like to ask how much good evidence there is that overloading the animal as a whole, or the patient as a whole, with one amino acid actually does interfere with some of these systems. Certainly, if you feed large quantities of certain amino acids, as we have done, you cannot see any evidence that this upsets the amino acid balance at all. And while it has always been assumed that this would happen, I just wonder what evidence we have for this, except for these rather special experimental in vitro- systems. P. G. SCHOLEFIELD: Dr. Scriver in Montreal has been doing a great deal of work on intravenous administration of amino acids. He finds, for example, that when proline is administered intravenously there is an appearance of extra glycine and hydroxyproline in human urine. These results have correlated very nicely with what has been obtained in vitro with kidney slice preparations. I don't know that it has been done for any other tissue. W. W. TOURTELLOTTE: May I speak to this point? Lloyd, et a/. at the University of Michigan (Lloyd, J. C. Jr., Fajans, S. S., Conn. J. W., Kropf, R. F. and Rull, J., 1966, J . Clin. Invest., 45 : 1487-1502) have administered large doses of essential I-amino acids to normal healthy individuals. They found that essential I-amino acids administered intravenously to healthy subjects stimulated the release of insulin, which produced a modest hypoglycemia. In addition, hepato-toxicity was noted in two individuals receiving 30 g amounts of methionine. Moreover, three subjects receiving 15 g of isoleucine. three of six subjects receiving 7.5,8.5, or 22.5 g of threonine, and one subject receiving 7.5 g of tryptophan had various symptoms (hypotension, chills and fever, leukocytosis, headaches, and backache). So it would appear that other than hypoglycemia, large doses of essential I-amino acids can be toxic. This is in answer to Dr. Tower's question in general, asking what it does to the whole body in humans. J. FOLCH-PI:Is it a primary effect of amino acids, or a secondary effect of these amino acids on the islands of Langerhans in the pancreas?

W. W. TOURTELLOTTE: They found that essential I-amino acids administered intravenously to healthy subjects stimulated primarily the release of insulin from the islands of Langerhans in the pancreas. They also established that the large increase in plasma insulin was not the result of the small increase in blood glucose that occurred secondary to gluconeogenesis.

A M I N O A C I D T R A N S P O R T I N V A R I O U S TISSUES

199

H . M C ~ L W A IIn N : regard to what Dr. Tower said, and in relation to cerebral tissue: there is quite appreciable transamination of amino acids in brain. There are several transaminating enzymes, and also there is interaction between amino acids and other metabolites. For example, glutamic acid and aspartic acid will quite rapidly change the phosphocreatine level of the tissue. I wonder to what extent you think of these interactions when trying to interpret transport measurements. K . D. NEAME: In the experiments which I did myself the measurements were entirely of net uptake inwards into the tissue. I was considering mainly the effects of what was outside the tissue, interfering with the entry of histidine. 1 did not take any measurements of the intra-tissue amino acids, but I did check on any alteration in the level of the extracellular or medium amino acids in the suspending medium. In a very few instances there were changes of more than 25%, particularly in the case of aspartate, which of course can be transaminated, and alanine appears. But only with the acidic amino acids and glutamine did I see any marked alteration. In all other cases there was no evidence of any gross metabolic change of amino acids in the medium. P. G. SCHOLEFIELD: I just want to add that if amino acids are used as the substrate, similar results are obtained whether the amino acids are readily metabolized or are non-metabolizable. So the general phenomenon seems to be the same. A. LAJTHA:We (as Dr. Tower and some others) also fed amino acids or injected amino acids into the living animal to see changes in cerebral levels. And we found (as many others found) that the changes are surprisingly small, and in a sense unexpected, from the slice-experiments. Some we could probably explain by the inhibitor's getting into the brain and inhibiting not only uptake from plasma but exit from brain as well, with, therefore, probably no net change occurring. In phenylketonuria or feeding of phenylalanine this does not hold, because phenylalanine activates efflux and inhibits influx; so you expected a great change (which we did not find). The only explanation 1 would have at least for the level of effects with phenylalanine, is that perhaps the transport mechanism in vivo is adaptive, and can react to some changes. We have some evidence that this could be the case in starvation, and in other areas, that exit slows down so that perhaps by changes in plasma level we change transport mechanisms in the brain. In the serum of phenylketonuric patients the concentration of phenylalanine is A. LOWENTHAL: high and the concentration of the other free amino acids is low. When these patients are put on a diet, free of phenylalanine, the phenylalanine concentration decreases and the concentration of the other free amino acids increases. Although during loading tests with phenylalanine in normal subjects or heterozygotes for phenylketonuria there is a marked increase of the phenylalanine in the serum, the other free amino acids do not decrease. Pathological cases behave quite differently from the normal cases. R. V. COXON:I am sure that in conditions such as starvation you get very considerable changes in the hormonal status, and both somatotropin and insulin are believed to have some effect on amino acid transport. This has been reported by a number of people (See Kipris, D. M . and Noall, M. W., 1958, Biochiti. Biophys. Acra, 28 : 226 and Riggs, T. M. and Walker, i.M., 1960, J . Biol. Cheni., 235 : 3603). A. LAJTHA:These hormones may not have effects on brain transport; their effect would have to be measured on the brain itself.

C. F. BAXTER:Some recent work of Dr. Sidney Roberts (1963, J. Neurochenz., 10, 931) should be mentioned here. Rats on a phenylalanine deficient diet had altered levels ofamino acids- in particular, threonine, lysine, valine, glycine, and histidine - in the central nervous system. The elevated levels of glycine and histidine in the brain fluid were not paralleled by similar changes in the blood plasma, and it seems to me that this altered amino acid relationship between plasma and brain reflects the possibility of changes in amino acid transport under these in vivo conditions.

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201

Transport as Control Mechanism of Cerebral Metabolite Levels ABEL LAJTHA Department of Biocheniistry, College of Physicians and Surgeons, Columbia University, and.New York State Research Institute Jor Neurocheniistry and Drug Addiction, Ward’s Island, New York, N . Y. (U.S.A.)

INTRODUCTION

During the past years, workers in our laboratories, and in a number of other laboratories, investigated mechanisms by which cerebral metabolites pass in and out of the brain. The results clearly showed that cerebral metabolites, including the components of the free amino acid pool, are in a dynamic state, that is, that continuous influx and efflux occurs (Lajtha, 1959, 1962). This work further showed that such flux occurs mostly through mediated transport mechanisms rather than diffusion (Chirigos et al., 1960; Lajtha, 1964; Lajtha and Toth, 1961, 1963). It was possible to study a number of properties of these transport mechanisms such as substrate specificity (Neame, 1961a; Abadom and Scholefield, 1962b; Blasberg and Lajtha, 1965, 1966; Levi, Blasberg and Lajtha, 1966), stereospecificity (Lajtha and Toth, 1963), energy requirements (Elliott and van Gelder, 1958; Neame, 1961b; Abadom and Scholefield, 1962a; Lajtha, 1967), kinetic constants (Blasberg and Lajtha, in preparation), and the factors that influence these mechanisms (Tsukada et al., 1963; Lahiri and Lajtha, 1964; Lajtha and Toth, 1967; Abadom and Scholefield, 1962~).The present paper discusses ths possible role that transport mechanisms of metabolites play in the nervous system. If transport not only supplies metabolites, but also determines the rate of supply and of removal, then it can have a controlling influence on the level of cerebral metabolites. Control of cerebral metabolite levels may constitute one of the major control mechanisms of cerebral metabolism. As will be discussed later, with brain slices (a model system in which transport can be measured quite accurately) fairly conclusive evidence was gathered that transport mechanisms indeed control levels of amino acids at steady state (Lajtha et al., 1966). We will discuss whether the conclusions drawn from brain slices can give us information about the role transport processes play in determining metabolic levels in the living brain. The writer is aware that calling transport processes the main control mechanisms of cerebral metabolites is only a proposal, which has not been clearly proved by experiments. Rcfcrmcps pp. 215-216

202

A. L A I T H A

RESULTS

Comparison of cerebral amino acid levels Relationships that bring attention to the idea that amino acid levels are functionally important in the nervous system appeared when the levels of cerebral amino acids in various species were compared (Table I). We measured the amino acids in the cerebrum of various species to compare these levels with amino acid transport in slices from cerebrum; the analysis in each species was done in the same laboratory by the same method (Levi et al., 1967) so that the results could be closely compared. Although there were clearly significant differences, in some cases even a two- or threefold difference, in the concentration of an amino acid in the brain of one species compared with the brains of other species, it was also clearly shown that amino acids that were at high levels in the brain, such as glutamic acid, taurine, aspartic acid, and TABLE I C O M P A R I S O N O F T H E LEVELS OF C E R E B R A L A M I N O A C I D S IN V A R I O U S SPECIES _ _ _ _ _ _ _ _ _ ~

~

/tmoles per g fresh brain Mouse

Rat

Guinea Pig

Hetc

Frog

9.9 8.3 2.2 1.8 0.91 0.55 0.19 0.13 0.08 0.08 0.08 0.05 0.04 0.04

11.6 6.6 2.6 2.3 0.68 0.65 0.21 0.11 0.07 0.05 0.07 0.05 0.05 0.04 0.02 0.02

9.5 2.4 2.0 2.1 0.71 0.60 0.09 0.13 0.10 0.08 0.04 0.05 0.05 0.03 0.03 0.03

12.2 3.8 2.8 2.7 0.42 0.70 0.18 0.13 0.07 0.06

5.4 2.7 0.86 2.5 0.83 0.20 0.14 0.22 0.09 0.17 0.08 0.08 0.09 0.07 0.03 0.04

___.____

Glutamic acid Taurine Aspartic acid GABA Glycine Alanine Lysine Arginine Valine Histidine Tyrosine Phenylalanine Leucine Methionine Ornithine Isoleucine ~.

0.05

0.02 .

0.05 0.06 0.05

0.02 0.03 0.03

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

The values shown are the averages of three experiments (Levi et a/., 1967). Extracts from single brains (cerebrum only; cerebellum, pons, and medulla were discarded) were used for each analysis (Technicon Amino Acid Analyzer).

GABA, were at high levels in all brains that we investigated, and that amino acids that were at low levels, such as methionine, ornithine, and isoleucine, were also at low levels in all brains that were investigated. The comparison was even morestriking if the amino acids were arranged in order of their concentration. The order (Table I) was very similar in mouse, rat, guinea pig, and hen, and only slightly different i n frog

203

C E R E B R A L TRANSPORT AS CONTROL MECHANISM

brain. The close similarity of the patterns of amino acid concentration not only indicates the functional significance of such patterns but also indicates the existence of mechanisms that keep the composition of the cerebral amino acid pool at a constant pattern ; as discussed further on, transport mechanisms are the most likely candidates for such a role. Mechanisms determining amino acid levels in brain slices

When brain slices are incubated in a medium containing amino acids, the slices accumulate amino acids from the medium until a concentration gradient is established and the amino acid in the intracellular fluid in the slice has a level several-fold that of the amino acid in the medium. The rate of uptake of an amino acid and the concentration gradient established at steady state between the medium and the slice depend on the concentration of the amino acid in the medium (Neame, 1964; Lahiri and Lajtha, 1964). As the incubation proceeds, and the intracellular level in the slice increases, the exit rate (efflux) of the amino acid also increases, since efflux as well as influx is concentration-dependent (Levi et al., 1965), influx depending on the concentration of amino acid in the medium, and efflux on the concentration of amino acid in the intracellular water. Net amino acid accumulation will continue until an intracellular T A B L E 11 A M I N O A C I D F L U X I N MOUSE B R A I N SLICES A T C O N D I T I O N S S I M I L A R T O S T E A D Y S T A T E

Initial flux ,uinoles/miti/ml

Coticetitratioti at steadv state ,uinoles/tiil water Aniitto acid iri

the

niediitin

ititracellular iii the slice ~ill,racelll,lar~ iriflrtx

water efflux

(1-aniinoisobutyrate

0.057

3.3

0.088

0.068

L-leucine

1.1 0.064 1.7

41 1.o

0.95 0.12 1.5

0.63 0.1 1 1.3

0.075 I .6 0.075

0.68 11 5.7 98

0.048 0.46

0.049 0.40

0.22 3.2

0.09

L-l ysine

glutam am ate

1.6

9.2

Influx was nieasurcd in short-time experiments in which slices were incubated in media containing amino acids as indicated in the first column. Efflux values are extrapolated to initial rates from experinicnts in which preloaded slices were incubated in amino acid-free media.

concentration is attained at which influx and efflux are equal. If other processes, such as amino acid metabolism or heteroexchange, can be neglected, then the steady state level of amino acids in the slices will be mainly determined by influx and efflux. As shown before (Blasberg and Lajtha, 1965), the metabolism of amino acids under the experimental conditions used was not significant enough to influence the conRcfiwncrs pp. 215-216

204

A. L A J T H A

centration of intracellular amino acid in the slice, perhaps because in most cases the concentration attained in the slice under our conditions was several-fold that of the physiological concentration. Under such conditions, therefore, the steady state level of an amino acid in the brain slice is determined by processes of influx and efflux. Supporting evidence is shown in Table 11, which summarizes the results of three types of experiments. In the first type of experiment slices were put in medium containing2.0 or 0.1 mM amino acid, respectively; they were incubated until steady state was achieved, and the concentrations of amino acid in the medium and in the slice were determined. These steady state levels in medium and in slice are shown in the first two columns. In the second and third types of experiments (Columns 3 and 4, Table II), influx and efflux were measured at concentrations corresponding to those at steady state. Influx was measured by putting fresh brain slices into a medium that corresponds to the final medium concentration at steady state and determining initial rates of uptake in short-time experiments. Efflux was measured by preloading the slices to levels corresponding to those achieved at steady state, then transferring them into a medium which did not contain amino acid. This measure of the influx and efflux of amino acids showed that although flux varied greatly with the level of amino acid, at any particular intra- and extracellular concentration corresponding to steady state levels influx and efflux values were very close. This showed experimentally what is generally assumed: that it is likely that steady state is reached when influx and efflux become equal, and that other factors such as amino acid metabolism are not the main determinants of steady state levels. If the concentration dependence of influx and the concentration dependence of effluxare measured separately, steady state levels corresponding to any medium concentration can be predicted; if influx is known for a particular medium concentration, T A B L E 111 EFFECT O F A N A L O G S O N a - A M I N O I S O B U T Y R I C A C I D T R A N S P O R T

Inhibitor Glycine Leucine

Steady state

Influx

Eflwx

-41

-5 5

-51

-34

-49 48

+

Effects are expressed as per cent alteration of control transport (prnoles per rnl water); -, inhibition; -i ,activation. The values are taken from studies of substrate specificity of steady state (Blasberg and Lajtha, 1965), influx (Blasberg and Lajtha, 1966), and efflux (Levi et a/., 1966).

the steady state will be reached at that intracellular concentration at which efflux equals this influx. When steady state levels that were predicted from influx and efflux values were also experimentally determined (Lajtha et al., 1966), good correspondence was found between predicted and measured values; this was further support for the supposition that influx and efflux, at least with brain slices, are the two dominant processes determining steady state levels.

205

C E R E B R A L T R A N S P O R T AS C O N T R O L M E C H A N I S M

Not only can steady state be estimated if influx and efflux are known, but any change i n either influx or efflux will be reflected in changes in steady state. Table 111 shows an example from a more extensive study (Levi et al., 1966), which investigated whether the effect of analogs on influx and efflux could explain the effect of these analogs on steady state. When analog effects were measured on aminoisobutyrate (AIB) uptake, glycine inhibited AIB steady state less than it inhibited influx, whereas leucine inhibited AIB steady state more than it inhibited influx (Table 111). This discrepancy between the effects on influx and steady state was explained by the effect of analogs on efflux, since, as shown, glycine inhibited AIB efflux and leucine activated it. In cases in which effluxas well as influx is inhibited, steady state should be inhibited less than would be estimated from influx inhibition alone since steady state levels decrease more if uptake alone is inhibited than they do when exit is inhibited as well. Where influx is inhibited and efflux is activated, steady state should be more inhibited than calculated from influx inhibition. These results showed not only that flux determines steady state, but that alteration in flux determines alteration in steady state. TABLE IV S T E R E O S P E C I F I C I T YO F A M I N O A C I D E X I T

Mitirites for 50 % decrease in level

Tirile interval (inin)

545 20- I20 45-240

in vivo

Leucinr

Phenylalatiine

L

D

14

28 26

L

D

27

40 32

Lysine L

D

140

190 200

From Lajtha and Toth, 1962. Cerebral levels were about doubled by subarachnoid injection of mice and then the decrease in brain in time was measured.

I t is important to emphasize at this point that mechanisms ofexit arealso important in the determination of the level of amino acids in the brain. While we know little about exit mechanisms, there is some evidence available that mechanisms other than mere passive diffusion may be involved in cerebral amino acid exit. This evidence includes the finding in the living brain of exit of some amino acids against a concentration gradient (Lajtha and Toth, 1961) and the stereospecificity of amino acid exit (Table 1V). In brain slices, the effect of metabolic inhibitors on exit (Cherayil et al., 1966) and the saturation-type kinetics of exit (Levi et a/., 1965) may be some of the supporting evidence for mechanisms other than diffusion in exit.

Energy supply of cerebral transport Lt is possible that amino acid levels in the brain are determined by the available energy, but only if the transport of amino acids at relatively high levels in the living brain has RrJ&rcnci~rpp. 215-216

206

A. L A J T H A

lower energy requirements than that of amino acids at lower levels. The effect of metabolic inhibitors on the concentrative uptake of amino acids by brain slices (Table V) was not significantly different between the various amino acids. Previous work o n substrate specificity showed evidence of several classes of amino acids, in regard to TABLE V EFFECT O F INHIBITORS O N A M I N O A C I D U P T A K E B Y BRAIN S L I C E S

Per cent inhibitioir of coticetrtrative uptake Atriitio

acid

Glycine Leucine GABA Glutamic acid Lysine

D NP

Na F

Ouahaitr

3’

90’

YO‘

90’

90‘

100 100 100 100 100

98

13 19 38 1 32

69

13

41 51

54 84 51 41

NaCN

84

99 91 87

38 23 -~

NaCN: 10 3MM, DNP: 2 x 10-5M, NaF: 2 x 10 3 M , ouabain: 10 5M. Concentrative uptake -- intracellular level-medium level. Three min experiment measures inhibition of influx; 90 min experiment, inhibition of steady state.

transport, in brain as well as i n other organs (Christensen, 1962, 1964; Oxender and Christensen, 1963; Abadom and Scholefield, 1962b; Neame, 1964; Blasberg and Lajtha, 1965). In brain, classes such as small neutral, large neutral, basic, acidic, GABA, etc., could be distinguished (Blasberg and Lajtha, 1966). Table V shows the effect of metabolic inhibitors on uptake of a representative from each of five “transport classes”. There was remarkable similarity in the inhibition of all these compounds in that strong inhibitors strongly inhibited the uptake of all the amino acids, while weak inhibitors had a lesser effect on all amino acids tried. The inhibition was dependent on the concentration of the inhibitor, but, again, increasing the concentrations of the inhibitors increased the inhibition of uptake of all amino acids tested. There were some individual differences, as shown in the values of Table V. There were also differences among the various amino acids in their sodium dependence for slice transport (Lajtha, 1967). However, it does not seem likely that the differences in the physiological concentrations of the cerebral amino acids can be explained by the energy requirements of the various carriers. In the experiments shown in Table V and discussed at greater detail elsewhere (Lajtha, 1967), cyanide was the strongest inhibitor employed. As shown previously, cyanide not only inhibited uptake but increased efflux as well (Cherayil et af., 1966). The present experiments were not designed to test whether or not a small portion of transport can be cyanide-resistant or independent of available energy, but such a possibility does exist. The compound that is the primary energy donor of transport in the brain is not known. In brain slices ATP content and uptake have been shown to be approximately parallel (Abadom and Scholefield, 1962a) in that a decrease in ATP level usually re-

207

CEREBRAL T R A N S P O R T AS C O N T R O L MECHANISM

sulted in a similar decrease in uptake. That ATP levels are not directly the limiting factor of amino acid uptake is indicated by a closer examination of the levels of ATP and amino acid uptake (Table Vl). Although as a rule a small decrease ofATPresulted in only a small decrease in ALB uptake, a number of exceptions occurred. Either replacement of glucose with ketoglutarate or inhibition by iodoacetate resulted in low TABLE VI ATP

L E V E L S A N D U - A M I N O I S O H U T Y K A T E U P T A K E BY M O U S E B R A I N S L I C E S

Per cent of control Medililn

Control (Glucose) No glucose i- Mannose N o glucose I Galactose N o glucose I Glutamate No glucose I u-Ketoglutarate Glucose I NaF Glucose -1- lodoacetate Glucose -1- ouabain Glucose -1 NaCN

Cortc. ntM

10 10 10 10

2 1

0.01 1

ATP

Uptake

100 77 12 8

100 71

16

31 32 65

35 16 47 4

24 2 20 2

ATP pmoles/nil tissue water, uptake = pmoles/ml aminoisobutyrate, intracellular-medium. Control values (with glucose): ATP, 1.14; AIB, 32.9 ,umoles/ml./Brain slices were preincubated 30 min with the medium, then [L4C]AIBwas added and the incubation was continued for 90 more minat 37". ~

levels of ATP, while uptake was very different under these two conditions (Table VI). A number of such differences were shown in a more detailed study (Lajtha, 1967). Therefore, although the possibility exists that available ATP limits transport, it is equally possible that other compounds that are in fairly rapid equilibrium with ATP are the ones that supply energy for transport. Whatever these compounds may be, it still is not likely that the physiological level of each amino acid can be controlled by the level of available energy. Comparison of cerebral amino acid levels in vivo with the uptake of amino acids by brain slices

In order to utilize a comparison of physiological levels with brain slice uptake, it would be important to know what factors determine the differences in uptake of the various amino acids by brain slices. It is not known what the rate-limiting step in uptake differences is, but as discussed previously, it is unlikely that it is energy. If the most important factor in slices is the availability or the level of the various carriers, and therefore slice uptake of an amino acid indicates the level of carrier for that amino acid, then a comparison of in vivo levels and in vitro uptake is justified. A comparison Rcfirenws pp. 215-216

208

A. L A I T H A

of amino acid flux in vivo and in vitro (Lajtha et al., 1966) revealed a considerably higher flux in brain slices than in the living brain for most amino acids. This difference would indicate that even if slice uptake measures the level of carriers, it is likely that carriers are somehow more unmasked or more active in brain slices than i n the living brain. The uptake of amino acids by slices also showed similarity in sequence among the various species that we investigated (Levi et al., 1967), in that amino acids taken up by slices to a high tissue-medium concentration ratio were taken up to a high level by TABLE VII COMPARISON O F B R A I N LEVELS W I T H B R A I N SLICE U P T A K E

Amino acid

Glutamic acid Taurine Glycine Lysine Phenylalanine Leucine Arginine Histidine

Living Brain

Brain slices

pmoles/g

Tissue/medium

-~

Mouse

Rat

Guinea Pig

Mouse

Rat

Guinea Pig

9.9 8.3 0.91 0.19 0.053 0.036 0.13 0.08

11.6 6.6 0.68 0.21 0.049 0.047 0.1 1 0.05

9.5 2.4 0.7 I 0.09 0.052 0.048 0.13 0.08

26 14 18 3.0 1.7 2.1 4.0 13

23 14 18 3.0 1.6 2.5

33 6.2 13 2.9 1.6 2.2

Brain slices were incubated 90 min in a Krebs-Ringer medium containing 2 mM amino acid. Living brain: pmoles amino acid per g fresh tissue; brain slices: pmoles amino acid per ml water tissue per medium.

slices from all spesies, and amino acids accumulated less were accumulated to a lower degree by brain slices from all species (Table VII). The sequence of slice uptake in each species was: glutamic acid > glycine > taurine > lysine > leucine > phenylalanine. This sequence, as will be discussed further on, also depended on the concentration of the amino acid in the medium and was slightly different at lower medium levels. It is of interest that amino acid uptake differs in various brain areas, while the sequence of uptake is similar in each area of the rat brain. The correspondence between slice uptake and living brain was fairly close in that those amino acids that were at high levels in the living brain were also accumulated to high levels by brain slices, whereas most of the amino acids at lower levels in the living brain were accumulated by brain slices only to lower levels. However, a number of discrepancies exist, not only that a few amino acids such as arginine, histidine, and proline, which are relatively low in the living brain, were accumulated to high degree by brain slices, but also that the order of sequence was different, taurine, for example, being much higher in the living brain than glycine although it was accumulated by brain slices to a somewhat lower degree. In these experiments (Table VII) fairly high medium

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levels were used for measuring brain slice uptake in order to minimize the effect of amino acid metabolism and the effect of possible heteroexchange with the endogenous amino acid pool. This level (2 mM) is above the Km of most amino acids, but influx is not measured under saturating conditions. However, since the high accumulation may saturate exit mechanisms, resulting in an artificially high uptake, the slice uptake was also measured at lower medium levels. The basic conclusions, however, were not altered when slices incubated at lower medium levels were compared with brain levels (Table VIlI), since, again with a few exceptions,aminoacids that were physiologically high were also accumulated by slices to a greater extent than the ones present in the living brain at lower levels. T A B L E VIII Tissuelmectium concentration ratio 1 nrM medium

Amino acid

Glutamic acid Glycine Histidine Proline Valine Lysine Leucine Phenylalanine

0.1 m M medium

Mouse

Rat

Guinea Pig

Mouse

Rat

25 23 16 8.6 5.5 3.8 3.3 2.4

21 20 13 1.6 4.0 2.9 2.4 2.2

44 14 12 8.3 3.1 3.3 2.4 2.1

29 10 15 1.4 4.0 6.1 4.4

24 9.1 10 5.9 2.8 5.8 3.6

Guinea Pig

19

7.5 11 5.3 3.1 4.3 3.5

Experimental conditions were similar to those of Table VII except the [l4C]arnino acid level in the medium was I mM or 0.1 mM instead of 2 mM.

The parallelism between physiological levels and slice uptake would indicate that those factors that are measured by the extent of brain slice uptake (perhaps among them the level of the carriers and the affinities of the amino acids to their carriers) are also the ones that significantly influence the level of amino acids in the living brain. The lack of strict parallelism between slice uptake and brain levels, on the other hand, would clearly indicate that factors in addition to those that one can observe in brain slices are operative in the living brain.

Comparison of cerebral amino acid content and amino acid uptake in different developmental stages and in different species If factors measured in slice uptake are important for determining brain levels, then the changes in physiological brain levels, such as those occurring during development, should be accompanied by similar changes in brain slice uptake. During development, the cerebral amino acid pool is altered (Agrawal et al., 1966), with most amino acids decreasing, the exceptions being glutamic acid and related compounds such as Rcfcrenres pp. 215-216

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glutamine, aspartic acid, glutathione, and GABA. These compounds, which constitute most of those at high level in the brain, increase during development; but a very sharp decrease in taurine compensates for the increase, and the free amino group levels in adults are below that of newborn (Table 1X). The changes shown here for mice (Levi et al., 1967) are similar to those observed in other species (Agrawal et a/., 1966). It is TABLE IX C E R E B R A L A M I N O A C I D C O N T E N T I N N E W B O R N A N D A D U L T MICE

prnoles per g fresh brain Amino acid

Taurine Glutamic acid Aspartic acid GABA Arginine Glycine Alanine Lysine Tyrosine Histidine Valine Phenylalanine Leucine Ornithine Met hionine Isoleucine

Newborn

Adult

18.5 4.4 1.9 1.2 0.10 2.3 0.76 0.23 0.19 0.14 0.11 0.1 1 0.08 0.06 0.05 0.05

8.3 9.9 2.2 1.8 0.13 0.91 0.55 0.19 0.08 0.08 0.08 0.05 0.04 0.05 0.04 0.02

For experimental details see legends to Table I.

interesting that arginine, which was one of the amino acids at low level in the living brain and accumulated to high degree in brain slices, is also exceptional in that it is one of the few amino acids not related to glutamic acid which increase during development. If developmental changes in in vivo brain level and brain slice uptake are compared in newborn and adult mice, only a few of the amino acids correspond. Those amino acids that are higher in the living brain in adults (glutamic acid, GABA, and arginine) are also taken up by adult brain slices more than by slices from newborn. With amino acids that decrease during development, such correspondence is not good, about half of those showing less, and the others greater, uptake in adult slices as compared to uptake by slices from newborn brain. Cerebral levels in vivo and cerebral amino acid uptake in slices can also be compared in various species. If mechanisms measured in slices are important determinants of cerebral amino acid levels, then it could be expected that if mouse brain hirs lower levels of a particular amino acid than rat brain, mouse brain slices will take up this amino

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CEREBRAL TRANSPORT AS CONTROL MECHANISM

TABLE X C O M P A R I S O N O F B R A I N LEVELS A N D B R A I N S L I C E U P T A K E I N N E W B O R N A N D A D U L T MICE

A(lult/riewborri coiiceiitratioii ratio Aniiiio acid

Glutamic acid GABA Arginine Glycine Alanine Leucine Valine Phenylalanine Lysine Histidine Taurine Ethanolamine

in vivo

in slices

2.3 1.4 1.3 0.40 0.72 0.44 0.71 0.48 0.84 0.59 0.45 0.59

I .4 I .8 1.3 1.5

Correspondence

+ + + + + -

0.83 0.78 1.1 0.69 0.87 2.3 2.6 0.42

+

+ -

-

+

/ ) I vivo: brain levels, adult/newborn; In slices: concentrative uptake, pmoles, per ml intracellular witer, adult/newborn.

TABLE XI C O M P A R I S O N O F LEVELS A N D U P T A K E B E T W E E N R A T S A N D OTHER S P E C I E S

Mouse/ Rat Amino acid

Glutsmic acid GI yci ne Taurine Lysine Leucine Phenylalanine

Guinea Pig/ Rat

HenlRat

Frog/ Rat

in vivo

slice

in vivo

slice

in vivo

slice

in vivo

slice

0.85 1.34 1.25 0.94 0.77 I .08

1.I 3 I.Ol* 1.05* 0.98** 0.86** 1.06*

0.82 1.04 0.36 0.43 1.02 1.06

1.45 0.75 0.45** 0.96 0.87* 1.00*

1.05 0.61 0.58 0.89 1.11 1.14

0.90* 0.79** 0.54** 0.76** 0.94 1.05**

0.46 1.21 0.41 0.68 1.94 1.65

1.03 1.23** 1.40 0.90* 0.90 0.99

in vivo: brain level of amino acid in species/in rat; slice: tissue medium concentration ratio in specieslin rat.; * Denotes correspondence between in vivo and in vitro comparisons; * * Closc correspondence.

acid also to a lower extent than rat brain slices. The comparison of rat with four other species (mouse, guinea pig, hen, and frog) shows 2. number of similarities. The correspondence between mouse and rat is quite good with the six amino acids tried; the correspondence is not good with glutamic acid, but it is fairly close with the other five compounds, in that compounds higher in rat brain in vivo are taken up more by rat brain slices and compounds at lower level are taken up less (Levi et al., 1967). Similarly, such comparisons are good between the rat and hen, where among Rrfircnrcs pp. 215-216

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the six compounds tested five show good correspondence, whereas in a comparison of guinea pig and rat, three compounds of the six show good correspondence of levels and uptake (Table XI). The comparison between rat and frog is not good in most cases; for example, glutamic acid, which is about half in frog brain as in rat, is taken up by frog brain slices to the same extent, while leucine is twice as high in frog brain but is not taken up more by frog brain slices. Alterations in cerebral amino acid transport and the cerebral amino acid pool

If transport processes are responsible not only for the determination of cerebral metabolite levels, but also for alterations in such levels, it can be expected that under conditions in which the cerebral amino acid pool is affected, cerebral amino acid transport will show a similar change. Initial experiments in progress in our laboratory seem to indicate that such is in fact the case. Protein-free diet (mice, for 30 days) resulted in some alteration of the amino acid pool (Lajtha and Toth, in preparation). Among other effects, there was a significant increase in histidine, a decrease in lysine, and a very small decrease in leucine levels in the free amino acid pool of animals on the diet. The following aspects of transport were measured in such animals: (a) amino acid exchange, by administering tracer amounts of labeled amino acid in the plasma and measuring the appearance of the label in the brain, and (b) cerebral amino acid uptake, after intraperitoneal administration of large doses of amino acids. Exchange and uptake seemed to change in the same direction as the composition of the pool, T A B L E XI1 EFFECT OF D I E T O N T H E CO M P O S I T I O N A N D F L U X OF C ER EBR A L A M I N O A C I D POOL

Per cent change Amino acid Composition

Histidine Lysine Leucine

+

690 - 23 - 6

Exchange

+

41 4 2

-30

Uptake

+

40 -26 0

Swiss mice were put on a protein-free diet for 30 days. Cerebral composition was measured with the amino acid analyzer; exchange was measured by the appearance of [14C]amino acid in the brain 5 min after the administration of an intraperitoneal tracer dose; uptake was measured after intraperitoneal injection of amino acid sufficient to increase plasma levels about 20 fold (Lajtha and Toth, in preparation).

in that histidine exchange and uptake were greater in the animals on the diet than in the control animals, while lysine exchange and uptake wereless in the diet-animals than in the controls (Table XII). These results point out the possibility that the effect of the diet may be primarily an effect on transport processes, histidine transport being increased and lysine transport being decreased, with a subsequent increase in histidine levels in the brain and a decrease in levels of lysine. Leucine levels and transport do not seem to be greatly altered under these conditions.

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213

The composition of the free amino acid pool of the brain is characteristic for this organ and also shows significant differences from one species to another (Roberts and Simonsen, 1962). Indeed, it could be considered that the amino acid pool of some organs is as characteristic as a fingerprint. This pool in the brain has been shown to be fairly stable, being altered under only extreme conditions. A comparison of the amino acid compositions of the brains of several species (Table I), however, shows striking similarities when the amino acids are arranged in the sequence of their concentrations. Not only are amino acids at high level present at high levels in all the brains, but the sequence of concentrations (with few exceptions) also seems to be the same in all brains. This, in addition to the stability of the composition, indicates the existence of mechanisms with high selectivity, which keep such a composition constant, and also suggests the possible important role that the composition of such a pool may play in the function of the nervous system. Evidence was found that alteration in the cerebral amino acid pool results in alterations of cerebral amino acid and protein metabolism(Roberts and Zomzely, 1965). A comparison of amino acid uptake by brain slices (Levi et al., 1967) showed somewhat similar behavior, in that the sequence of the extent of uptake of amino acids was the same in the various species, and not only were the amino acids that were taken up to a high degree taken up to such a degree by all species, but the sequence of uptake with those compounds tried was the same in all species. These observations pointed out the possibility that those transport processes that can be observed in brain slices may have the determining role in controlling the levels of cerebral metabolites, especially since it i s likely that uptake and exit determine the level of amino acids when brain slices are incubated in media containing amino acids. It seems not only that influx and efflux determine the steady state level of amino acids in brain slices, but that any alteration in either flux will correspondingly alter the steady state levels. It has to be emphasized that not only influx, but also efflux, is an important determinant, and that the possibility exists that these two fluxes can be independently influenced. It does not seem likely that the levels and availability of energy would constitute an important determinant of the pattern of the amino acid pool, since it seems that the sensitivity of amino acid uptake, as measured with brain slices, is similar with all amino acids, in that strong inhibitors seem to inhibit all amino acid uptake strongly, while weak inhibitors have less influence on all amino acids (Table V). Thus, the wide differences between the levels of amino acids cannot be due to such differences in energy requirements for their transport. To investigate the possible role of transport processes, the levels in brain were compared with the uptake of the same amino acids by brain slices. Uptake and brain levels were fairly parallel in a number of amino acids in that amino acids at high levels in the living brain were also taken up by brain slices to high degree, while most amino acids at low level in the living brain were taken up by brain slices to lower extent (Table VII). However, a number of exceptions were found, in particular, that the sequence of uptake in slices was not closely parallel with the sequence of levels in the living brain. References pp. 215-216

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When changes in slice uptake during development were compared with changes in the in vivo level of such amino acids during development, again, similar parallel behavior was found, with important exceptions. Most amino acids that increased during development also showed an increased uptake by slices during development ; however, amino acids that decreased during development did not show a regular pattern in slice uptake. In a comparison of differences in species with differences in slice uptake, approximately parallel behavior was shown with a number ofspecies, particulary mice, rats, and hen, while no parallelism was shown between rats and frogs, since many but not all amino acids at higher level in one species were taken up by brain slices also to higher degree. The parallel behavior of brain slice transport with in vivo brain levels in a number of cases clearly emphasizes the importance transport processes have in determining the level of cerebral metabolites. The fact that there are a number of discrepancies between in vitro accumulation and in vivo levels shows that those factors that can be observed in slices are not the only ones that contribute to the determination of cerebral amino acid levels in vivo. The reasons for this may be in the obvious differences between brain slices and living brain, such as the circulation, hormonal controls, etc., but it is also possible that the carriers behave somewhat differently in brain slices as compared to the living brain, in that they are made more available, or are unmasked, in the slice, or that the substrate affinity to the carriers changes upon preparation of the slices. The possible role of transport processes in determining the levels ofcerebral metabolites is further emphasized by the evidence (Table XII) that changes in cerebral amino acid pool and in cerebral amino acid fluxes in vivo are parallel, at least in the case of alterations caused by protein-free diet. Further research is needed to elucidate the mechanisms of cerebral transport processes and the mechanisms by which these processes may influence cerebral metabolite levels, but perhaps we should already consider the brain barrier system as not only a mechanism protecting cerebral homeostasis, but, beyond that, a mechanism that by controlling cerebral metabolite levels in a dynamic manner has an important influence on cerebral metabolism. ACKNOWLEDGEMENTS

This investigation was supported in part by Public Health Service Research Grants No. NB-03226 and NB-04360 from the National Institute of Neurological Diseases and Blindness. The work presented is the result of cooperation with several of my colleagues, in particular Drs. G . Levi, A. Cherayil, S . Lahiri, J. Kandera, R. Blasberg, and J. Toth, A. Mazeika, Z. Ronay.

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REFERENCES ABADOM, P. N. AND SCHOLEFIELD, P. G . (1962a) Amino acid transport in brain cortex slices. I - The relation between energy production and the glucose-dependent transport of glycine. Canad. J. Biocheni. Physiol., 40, 1575-1 590. - (1962b) Amino acid transport in brain cortex slices - 11. Competition between amino acids. Canad. J. Biochenr. Physiol., 40, 1591-1602. - (1962~)Amino acid transport in brain cortex slices - Ill. The utilization of energy for transport. Cutrad. J. Biochenr. Pliysiol., 40, 1603-1618. AGRAWAL, H. C., DAVIS, J. M.AND HIMWICH, W. A. (1966) Postnatal changes in free amino acid pool of rat brain. J. Neitrochem., 13, 607-615. BLASBERG, R. AND LAJTHA, A. (1965) Substrate specificity of steady-state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. - (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Res., 1, 86-104. CHERAYIL, A,, KANDERA, J. AND LAJTHA,A. (1966) Cerebral amino acid transport in vitro - 1V. The effects of inhibitors on exit from brain slices. J. Neurochem., 13, in press. P. A N D UDENFRIEND, S. (1960) Uptake of tyrosine by rat brain in vivo. CHIRIGOS, M.A., GREENGARD, J. Biol. Chem., 235, 2075-2079. CHRISTENSEN, H. N. (1962) Biological Transport. New York, W. A. Benjamin, Inc. - ( I 964) A transport system serving for mono- and diamino acids. Proc. Nut. Acad. Sci., 51,337-344. ELLIOTT,K. A. C. A N D VAN GELDER, N. M. (1958) Occlusion and metabolism of y-aminobutyric acid by brain tissue. J. Neitrochem., 3, 28-40. LAHIRI,S. AND LAJTHA, A. (1964) Cerebral amino acid transport in vitro - I. Some requirements and properties of uptake. J. Neurochem., 11, 77-86. LAJTHA, A. (1959) Amino acid and protein metabolism of the brain -1. Turnover of leucine in mouse tissues. J. Neitrochem., 3, 358-365. - (1962) The brain barrier system. Neurochemistry, K. A. C. Elliott, I. H. Page, and H. J. Quastel (Eds.), Springfield, C. C. Thomas (p. 399). - (1964) The uptake of amino acids by the brain in various species. Comparative Neurochemistry. Proc. 5th Intern. Neitrochem. Symp., Oxford, Pergamon Press (p. 193). - (1967) Energy requirements of cerebral amino acid transport. Problems of biochemistry of brain. Vol. 3. Armenian Academy of Sciences, Yerevan. LAJTHA,A., BLASBERG, R. AND LEVI, G. (1966) Control of cerebral amino acid concentrations. Significance of Changes in Plasma Amino Acid Patterns. New Brunswick. Rutgers University Press. LAJTHA,A,, LAHIRI,S. AND TOTH,J. (1963) The brain barrier system - IV. Cerebral amino acid uptake in different classes. J. Neurochem., 10, 765-713. LAJTHA, A. AND TOTH,J. (1961) The brain barrier system - 11. Uptake and transport of amino acid by the brain. J . Neurochern., 8, 216-225. - (1962) The brain barrier system - 111. The efflux of intracerebrally administered amino acids from the brain. J. Neurocheni., 9, 199-212. - (1963) The brain barrier system - V. Stereospecificity of amino acid uptake, exchange and efflux. J . Neurochem., 10, 909-920. - (1965) The effects of drugs on uptake and exit of cerebral amino acids, Biochem. Pharmacol., 14, 729-738. - (1967) Control of cerebral metabolite levels - 111. Relationship of alterations in the free amino acid pool and in amino acid transport. J . Neurochem., 14, in press. LEVI,G., BLASBERG, R. A N D LAJTHA, A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biochem. Biophys., 114, 339-351. LEVI,G., CHERAYIL, A. AND LAJTHA,A. (1965) Cerebral amino acid transport in vitro - 111. Heterogeneity of exit. J. Neiirochem., 12, 757-770. LEVI.G., KANDERA, J. AND LAJTHA, A. (1967) Control of cerebral metabolite levels - I. Amino acid uptake and levels in various species. Arch. Biochem. Biophys., 116, in press. LEVI,G . AND LAJTHA,A. (1965) Cerebral amino acid transport in vitro - 11. Regional differences in amino acid uptake by slices from the central nervous system of the rat. J. Neurochem., 12,639-648. NEAME, K. D. (1961a) Phenylalanine as inhibitor of transport of amino acids in brain. Nuture, 192, 173-174. (1961b) Uptake of amino acids by mouse brain slices. J. Neurochem., 6, 358-366.

-

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- (1964) Effect of amino acids on uptake of L-histidine by rat brain slices. J. Neurochem., 11,67-76. OXENDER, D. L. AND CHRISTENSEN, H. N. (1963) Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Eiol. Chem., 238, 3686-3699. ROBERTS, E. AND SIMONSEN, D. G. (1962) Free amino acids in animal tissue, Amino AcidPuols, J. T. Holden (Ed.). Elsevier Publishing Company, Amsterdam, pp. 284-349. ROBERTS, S. AND ZOMZELY, C. E. (1965) Regulation of protein synthesis in the brain. Protides o f t h e Biological Fluids, 13, H. P e t e r s (Ed.). Elsevier Publishing Company, Amsterdam, pp. 91-102. TSUKADA, Y., NAGATA, Y.,HIRANO,S.A N D MATSUTANI, T. (1963) Active transport of amino acid into cerebral cortex slices. J. Neurochem., 10, 241-256.

DISCUSSION

H. MCILWAIN:I would like t o comment on effects of the membrane potential: that it conditions the distribution of substances between intracellular and extracellular locations. This I don’t think has been fully discussed here. In deciding what is concentrative uptake, and what is simply a passive distribution, several factors operate on a metabolite. The cell membrane is charged so that any positively or negatively charged metabolite will not distribute itself according to concentration gradient only, but also according to the potential gradient. In other words: it is an electrochemical system, and the passive distribution for positively charged substances will be very much in favor of the intracellular environment. Now 1 think some of the amino acids were actually distributed in that sense in the data quoted: you have factors of 2,4, and 8 for arginine, lysine, and histidine in some instances, while with other amino acids the gradient was reversed. This consideration emphasizes the unusual position of glutamic acid, which was one of those distributed most in favor of the intracellular phase. Here one has a situation in which not only has the concentration gradient to be explained, but the electrochemical gradient also. For this reason I am not quite sure that the description of concentrative uptake is adequate. Electrochemical considerations might operate against the concentration gradient, causing the observed gradient to differ by a factor of 100 t o 200 from that what would exist in an unchanged system. A. LAJTHA:In the brain slice, which is (one has to emphasize this point) an artificial system, but a system which we probably can approach experimentally and begin t o understand, the concentrative uptake as we define it is the uptake above the medium, and this is hard to imagine as being mostly passive, or due to charge gradients, etc. Most metabolic inhibitors abolish or decrease uptake, but they may affect charge distribution. Of course, as an alternative we may consider that the metabolic inhibitors alter (which they may) the membrane itself and increase its leakiness. H. MCILWAIN: The membrane potential exists in vivo at the same level, so that these considerations are equally applicable in vivo.

K. A. C. ELLIOTT: I would like to make a comment: I think that we cannot always be speaking of “transport”. This is “concentration” we have been talking about today, mostly in your talk, Dr. Lajtha. And concentration: just what does it mean? I am quite sure that those amino acids that are present in brain in large amounts (glutamic acid, glutamine, alanine, GABA, and aspartic acid) are not in free solution. You cannot just say that they are taken up against a concentration gradient. N o doubt they are, but that is not by any means the whole thing. All of those amino acids, as shown by Dr. Van Gelder and by Bilodeau and Paula Strasberg in my lab., are in large part bound, if I may use the term (not quite knowing what I mean) in certain subcellular particles. They are not in free solution, they are not simply transported across a membrane but are transported probably across more than one membrane or transported and then adsorbed on a membrane. So we can scarcely think in terms of simple constants for ingress and egress. A. LAJTHA:Of course the brain is not a homogeneous solution, and the amino acids are inhomogeneously distributed, a significant amount of them in particulates. Whether the distribution is really explained by binding I am not sure. I find carrier-mediated transport just as good an explanation. if not better, than “binding”. Very few people approached the problem experimentally, but recently Udenfriend reported evidence that the intracellular amino acids are indeed in free and not in bound state.

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K. A. C. ELLIOTT:We have situations where you d o not get exchange between the medium and the particle.

R. V. COXON:I wonder if there was any correlation between the amounts of these various amino acids in the brain and their relative abundance in any known protein in the nervous system. A. LAJTHA:No. Amino acids that are taken up into the free amino acid pool to a high degree are not particularly high in brain proteins. The ratio of free to protein-bound amino acid is different from compound to compound, from I : 10 about a 1 : 500 ratio (the protein-bound form being always the higher) per unit weight of tissue. The composition of the free pool seems to be very different from the protein-bound one. T. Z. CSAKY:I would like to sound a word of caution against denying the explicit role of ATP in active processes, based on the iodoacetate experiments. I think we should keep in mind that Passov recently showed that in the red cell iodoacetate does two things: it inhibits metabolism, thereby the creation of new ATP, but, what is more important, as an -SH inhibitor it renders the membrane extremely leaky. So here probably the efflux increased very much through a passive process. I think this is probably where the big discrepancy comes in in the case of iodo-acetate. I still would believe that ATP is probably the primary source of energy. A . LAJTHA:There are a few more exceptions in addition to iodo-acetate, but certainly not enough to deny definitely the significant role of ATP. ATP, I agree with you, would be the first logical choice. Low sodium seems to have, with the same ATP content, a quite different inhibitory effect on glutamate than on aspartate uptake, for example.

D. B. TOWER:I would like to comment in regard to what Dr. Elliott mentioned in this sense, that if we have to make a distinction between (if you will allow me the term) “endogenous” glutamate for example, and what is added from the outside, then when glutamate is added to brain slices (as Dr. Elliott, I think, was the first to show) there is also a tremendous amount of water uptake at thesame time. If this material were bound, I am not sure that you would get as much (if any) water increase in slices. I think the water uptake can be related to the extra negative charges introduced when the glutamate is concentrated in the cell, and the degree of concentration is about what you showed in your experiments, that is something of the order of 10- to 20-fold. So that this is a rather complicated question, depending on what the source of the glutamate is: whether it is added from the outside, or whether it is generated metabolically through the Krebs’ cycle via glucose oxidation. A. LAJTHA:Water uptake is fairly specific for glutamate, or at least the swelling of the slices is similar with most other amino acids, but it is greatly increased with glutamate - perhaps because this amino acid also increases intracellular sodium or potassium.

P. MANDEL: In relation to what Dr. Elliott thought: I think that we have to keep in mind another aspect of the concentration problem. That is that many of the free amino acids are bound to transferRNA. So that we have to find out what is the situation in virro, if the transfer-RNA binds the amino acids? A decrease in ATP may also decrease the binding possibility of the free amino acids to the transfer-RNA. This might be a factor which should be controlled also. A. LAJTHA: As far as exchangeability is concerned, we have measured only two or three amino acids until now, and we are measuring some now. Those we did measure were all exchangeable as far as we could see. We got a complete isotopic equilibrium between the exogenous and endogenous pool using tracer amounts. So that the endogenous amino acids are not irreversibly bound as far as we can see, but they are all exchangeable, under physiological conditions.

J, FOLCH-PI:Can I ask you a very specific question? I noticed that histidine seems to be off the general rules in three different aspects: It was the one that a low concentration showed at tremendous differential, the one that showed a tremendous increase in starvation, and finally the one that seemed to be competing with alanine instead of competing with a n amino acid of its own size. Do you care to make a comment on that?

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A. LAJTHA:It is interesting that a few amino acids like histidine are exceptions. It would make me think that they must therefore be in a special “transport class”.

K. A. C. ELLIOTT:I must answer this suggestion that these amino acids are not bound by describing an experiment. If you take ordinary brain tissue and homogenize it in sucrose solution and put in some tracer, like radioactive GABA, and then centrifuge it, you will find none of that tracer in the sediment though there will be plenty of GABA and other amino acids in the sediment. But if you add a little sodium with the tracer, you will then find the label and the amino acid in the sediment. There is bound amino acid, which depends for its binding and exchange on the presence of sodium. A. LAJTHA:Exchange in vitro is considerably damaged. In slices its rate seems t o be less than under living conditions.

K. A. C. ELLIOTT:Yes, but in vivo you have sodium there all the time. But there is the possibility of not-exchanging . . . A. LAJTHA:You may be right that sodium may make some difference between the in vivo and in vitro experiments. We did very few experiments on slices as far as the exchangeability of the endogenous pool is concerned, but if you add tracer amounts in the plasma in vivo the equilibrium is complete between the brain and plasma pool. P. G . SCHOLEFIELD: I would like to go back, if I may, to the discussion between Dr. Lajtha and Dr. Csaky about the directness of the role of ATP. I think there is a fair amount of evidence that ATP may not be directly involved. 1 would like to quote an experiment that Dr. Johnstone and I performed a few years ago, where we were measuring glycine uptake by ascites cells. If we let the glycine reach a steady state level and then added dinitrophenol, immediately the incorporation in the protein ceased, but we had to wait nearly 10 minutes before we got any loss of amino acids from the cell. Yet, if we took the same cell, pre-loaded it and put it into amino acid free medium, the amino acid was lost immediately. If in similar experiments we added dinitrophenol after 5-10 minutes when the glycine had reached approximately 50% of its steady state level then again the incorporation into the protein ceased immediately but amino acid kept going into the cell and did not start to come out for anothzr 10 minutes. This is an experiment that would suggest that maybe ATP is not directly involved. Of course, if there is something like a phosphoprotein formation, or confirmational changes, one could see why ATP was not being directly involved, even though ATP has a function. A. LAJTHA:I agree that ATP has an important function and we have to define what we mean by direct function. J. FOLCH-PI:Well, you would, in the very circumstances that you are describing for ATP, have to d o more than measure one compound only at one time point.

C. F. BAXTER: I would like to expand upon Dr. Elliott’s comments about the effect of sodium ion concentration on amino acid uptake. Several papers in the literature suggest an interrelation between sodium ion concentration and amino acid movement. For example, Dr. Scholefield showed some years ago that glycine uptake into brain slices was greatly influenced by the sodium chloride concentration of the incubating medium. In view of this and other findings (like those discussed by Dr. Elliott), it seems reasonable to ask whether the quantitative interrelationships of amino acid uptake in Dr. Lajtha’s slice experiments would not be altered significantly if an incubation medium with a different sodium ion concentration were used. A. LAJTHA:The sodium in the medium we used for the slice experiments is far above the optimal requirements. I would be surprised if slight variations in these levels (120 mM) would have effects on any amino acid movement. Where you start getting decreases is a t about 50 mM Na, and where you see strong inhibition and large differences it is more like 5-10 mM.

219

Regional Differences in Cerebral Amino Acid Transport G I U L I O LEV1 Istiticto Superiore d i Sanita, Viale Regina Elena 299, Rome (Italy)

INTRODUCTION

The anatomical, physiological and biochemical heterogeneity of various areas of the brain has been object of a number of studies (for a recent symposium see: Kety and Elkes, 1961). In particular, the physiological level of most free amino acids was found to be different in various regions of the central nervous system (CNS), the regional distribution pattern varying from one compound to the other (Berl and Waelsch, 1958; Okumura et al., 1959; Lajtha and Mela, 1961; Bart et al., 1962; Singh and Malhotra, 1964; Shaw and Heine, 1965; Ramirez de Guglielmone and Gomez, 1966). It has been suggested that the heterogeneous distribution of cerebral amino acids may be determined by regional differences in the transport of these compounds (Lajtha and Mela, 1961) but very few workers investigated this problem (Lajtha, 1961 ; Nakamura and Nagayama, 1966). The work to be presented here summarizes experiments in vitro and in vivo on regional differences in cerebral amino acid transport. It is known that amino acid transport in brain slices occurs through an active, carrier-mediated process (Stern et al., 1949; Guroff et a/., 1962; Neame, 1962; Abadom and Scholefield, 1962; Tsukada et al., 1963; Lahiri and Lajtha, 1964; Blasberg and Lajtha, 1965 and 1966; Levi et al., 1966); we have shown that amino acid transport processes of brain slices are in many ways similar to those operating in the living brain, except for the marked restriction of the passage of amino acids from the blood to the CNS in vivo (Lajtha et al., 1966), which is generally attributed to the presence of a complex “brain barrier system”. Brain slices appear to be a useful tool to study one aspect of this complex system: the permeability properties of nervous tissue cells, in particular their unrestricted transport capacity. For this reason the problem of regional amino acid transport was approached both in brain slices and in the living brain, and an attempt was made to compare and correlate the results obtained. The aim of these studies was not only to try to clarify some of the mechanisms by which amino acids are transported into the nervous tissue, but also to further investigate the role that transport processes may have in the determination and in the maintenance of the physiological amino acid levels in different areas of the CNS. References pp. 227-228

G. L E V 1

220

RESULTS

Table I shows the degree of steady state accumulation of five amino acids in slices from various areas of the CNS. The amino acids chosen belong to different classes and have different affinities for the membrane carriers (Blasberg and Lajtha, 1966). More areas than those presented were analysed, but only the most representative are shown in this and in the following tables. TABLE I S T E A D Y S T A T E A C C U M U L A T I O N O F A M I N O A C I D BY S L I C E S FROM D I F F E R E N T A R E A S O F T H E RAT

CNS

Tissue to medium ratio

Area

a-Aminoisobutyrate*

Cortex White matter Midbrain Cerebellum Pons-medulla

8.8 4.4 11

1.7 9.1

Leucine

Lysine *

2.1 1.7 2.4 2.8 2.2

4.0 2.6 3.3 2.8 2.5

Taurine

20 18 22 15 12

9.6 4.1 27 3.6 12

Slices were incubated for 70 min in a Krebs-Ringer medium containing initially 2 mM 14C amino acid. pmoles/ml tissue water after incubation. pmoles/ml medium From Levi and Lajtha, 1965.

Tissue to medium ratio

*

=

It can be seen that the degree of accumulation varies with each of the amino acids tested. Among them D-glutamate is concentrated to the highest degree, leucine to the lowest. The regional pattern of accumulation also varies with each amino acid. None of the areas studied has in every case the highest accumulation, and none has in every case the lowest. Some area may have the highest accumulation with one amino acid, and the lowest with an other, such as cerebellum with leucine and taurine respectively. The degree of regional variation of accumulation also varies with the amino acid tested, and is relatively small with leucine and great with taurine. The difference between lowest and highest accumulation is of the order of two-fold with a-aminoisobutyric acid (AIB), leucine, lysine and D-glutamate, but is almost eight-fold with taurine. In previous experiments (Levi et al., 1966) we have shown that the degree of amino acid accumulation at steady state is determined by the relative extent of the following processes: ( I ) Accumulation rate (influx); (2) Exit rate (efflux); (3) Homoexchange into and out of thecells. With brain slices it is often sufficient to know the extent of influx and efflux in order to be able to predict with some approximation the degree of accumulation at steady

REGIONAL DIFFERENCES I N CEREBRAL AMINO A C I D T R A N S P O R T

221

state. In order to investigate whether the regional differences of amino acid steady state accumulation could be explained on the basis of regional differences of influx or efflux, these fluxes were studied separately, with three amino acids (AIB, D-ghtamate, and lysine). With AIB it was found that regional differences of influx were very similar to those of steady state accumulation (Levi and Lajtha, 1965), making it likely that regional differences in A1B exit would not be great. Table I 1 shows that indeed the exit of AIB is very similar in all the areas except cerebellum, where it is slightly higher. This finding confirms that with AIB the heterogeneity of steady state accumulation is mostly due to regional differences of the accumulation rates and not to differences of the exit rates. T A B L E 11 E X I T O F A M I N O A C I D S FROM S L I C E S F R O M D I F F E R E N T A R E A S O F T H E R A T

CNS*

Per ceirt ititracellular loss Area

Cortex White matter Midbrain Cerebel I urn Pons-medulla

u-Anrinoisobittyrate

D-Gliitamale

Lysitie

41 18 28 25 28

57 49 61 66 64

31 33 30 39 29

Slices were preincubated for 30 min in amino acid containing medium, then transferred to amino acid free medium for 30 min. Average intracellular level after preincubation: AIB I3 ,umoles/ml

*

D-GIu 33 pnoles/rnl Lys 5.5 pmoles/ml From Levi, Cherayil and Lajtha, 1965. T A B L E 111 C O M P A R l S O N OF L-LYSINE F L U X E S IN D I F F E R E N T AREAS O F T H E R A T

CNS

Relative initial accittnulat ion*

Relative steady state accumulation*

Relative exit * *

Cortex

101

83 104 I02 98

127 87 106 91 83

94

White matter Midbrain Cerebellum Pons-medulla

Area

Relative values are calculated taking the average of total areas as 100. * Recalculated from Lcvi and Lajtha, 1965. +* Recalculated from Levi, Cherayil and Lajtha, 1965. Rtfiwnces pp. 227-2211

80 100 108 106

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G . LEV1

With D-glutamate the regional heterogeneity of influx did not parallel exactly that of steady state (Levi and Lajtha, 1965), making it likely that in this case regional differences in both influx and efflux play a role in determining the degree of accumulation at steady state. The second column of Table I1 shows in fact that D-glutamate exit varied from area to area, being highest in cortex, about average in midbrain, cerebellum, and pons-medulla, and lowest in white matter. Also with lysine the regional pattern of influx did not parallel that of steady state accumulation; in agreement with this lysine exit was not homogeneous in the various areas, as shown in the third column of Table 11. Table 111 shows that with lysine the heterogeneity of steady state accumulation is mostly due to regional differences in exit, since the influx of this amino acid is essentially the same in all the areas except white matter. It can be seen that the degree of amino acid accumulation at steady state is lower in those areas where exit is higher, and vice-versa. In white matter all influx, efflux, and steady stateare lower than average. Finding regional differences in the exit of D-glutamate and lysine, but not of AIB, is in agreement with other experiments in which the exit of these amino acids was studied with slices from whole brain (Levi et al., 1965; Levi et a/., 1966). In these studies it was shown that the exit rates of D-glutamate and lysine depend not only on the intracellular concentration of the amino acid, but also on the experimental time at which exit is measured. In contrast, no time-dependence of AIB exit could be demonstrated. To explain these findings it was suggested that exit of D-glutamate and that of lysine occur from different intracellular pools having different exit rates. The regional heterogeneity of D-glutamate and lysine exit shown in Table I1 could partly reflect this compartmentation. TABLE IV COMPARISON O F AMINO ACID ACCUMULATION

in vitro T O A M I N O A C I D L E V E L in vivo I N

DIFFERENT AREAS O F T H E R A T

Leucine Area

Hemisphere Caudate nucleus Midbrain Cerebellum Pons-medulla Total areas analysed

CNS

Lysine

Taurine

Relative Relative Relative Relative Relative Relative accumulation distribution accumulation * distribution* * accumulation distribution

79

115

78

135

83

129 143

266 35 121

66 109 35

100 (4.6)

100 (0.28)

100 (13)

100 (5.0)

86

101

107

75

106

100

106

104

125 100

100 96

91

100 (3.3)

100 (0.10)

In parenthesis values for total areas analysed are expressed as pmoles/g. Relative values are calculated taking the average of total areas as 100. * Recalculated from Levi and Lajtha, 1965. ** From Lajtha, Blasberg and Levi, 1966.

REGIONAL DIFFERENCES I N CEREBRAL A M I N O ACID TRANSPORT

223

While the picture of regional amino acid transport in vitro is relatively clear, the significance that this may have in the living brain is not quite clear. If accumulation in vitro is an indirect measure of the availability of carriers, the question arises whether or not carrier availability is the limiting factor in determining the amino acid levels in the living brain. In previous experiments we noted that amino acids that are accumulated to a high level in vitro have a high concentration in the living brain, whereas amino acids that are accumulated to lower levels have lower in vivo concentrations. We concluded that the transport capacitl- for a particular amino acid as measured by brain slices may be one of the factors determining its concentration in the living brain (Lajtha e t a / . , 1965). It seemed therefore logical to compare the relative accumulation of amino acids by different brain areas in vitro to their regional distribution in vivo. Table IV shows this comparison for leucine, lysine, and taurine. It is apparent that no parallelism exists between relative accumulation and relative distribution of any of the three amino acids presented. Indeed, with lysine and taurine, areas having higher levels in vivo tend to accumulate less in vitro, and areas that accumulate more in vitro tend to have lower levels in vivo. Although with other amino acids (such as alanine and glycine) there seems to be a better parallelism (Levi et a/., in preparation), it has to be concluded that factors other than the transport capacity as measured with brain slices are operative to regulate and control the regional distribution of a particular amino acid in the living brain. In order to investigate this problem further, the uptake and the exchange of some amino acids were studied in experiments in vivo. Table V shows the results obtained with lysine. It can be seen that the regional TABLE V RELATIVE D I S T R I B U T I O N O F L Y S I N E I N D I F F E R E N T A R E A S O F T H E R A T

Area

Hemispheres Midbrain Cerebellum Pons-medulla Total areas analysed

Control distribution'

Distribution after loading2

Distribution of tracer3

CNS t+4

75 104 129 143

74 95 136 157

88 97 119 123

86 108 111 117

I00 (0.28 / t M / g )

100

(0.34 pM/g)

100 (750 cts/g)

(34 min)

loo

I . Plasma concentration: 0.44 /rmoles/g. 2. Plasma concentration: 3.4 pmoles/g. The animals had an intraperitoneal injection of lysine (90 mg in 1 ml) and were killed 10 min later. 3. Plasma activity: 1 I 800 cts/g. The animals had an intraperitoneal injection of [14C]lysine(3 p C in 1 ml) and were killed 5 min later. 4. Calculated according to the following: t+

=

Tissue concentration x In2

The first two columns are taken from Lajtha et al., 1966. Rrferenres pp. 227-228

flux

224

C . LEV1

distribution of lysine is not significantly changed when the brain level of lysine is increased after a large intraperitoneal injection. In other words, lysine is taken up more by areas having higher lysine concentration. The third column of the Table shows that also the distribution of tracer, 5 min after an intraperitoneal injection of [Wllysine, closely parallels the relative concentration in the various areas. Higher exchange rates of lysine in areas with higher physiological level had already been shown (Lajtha and Mela, 1961). The fourth column shows that in spite of a higher rate of exchange, the half-life time of the pool of free lysine is higher in those areas where lysine level is higher. When the same type of experiment was done with leucine, whose physiological level in the various areas is very homogeneous, no significant regional difference in either uptake or exchange was found (Levi et al., in preparation). These findings suggest that at least with lysine and leucine there may be a rather close correlation between regional transport in vivo and regional physiological levels. The same type of experiment was repeated with taurine. This amino acid was chosen in view of the large differences in its concentration in the various areas. It was thought that taurine transport as well might differ widely from one area to the other and that a clearer correlation might appear between transport and concentration. Table VI shows that when the physiological plasma level of taurine is increased over 100 times, its brain concentration does not show any significant increase. The only region in which a relatively high increase of taurine level occurs is pons-medulla; this suggests the existence of regional differences in the blood-brain barrier to this amino T A B L E VI RELATIVE DISTRIBUTION OF TAURINE IN DIFFERENT AREAS OF THE RAT

Areas

Hemisphere Caudate nucleus Midbrain Cerebellum Pons-medulla Total areas analysed

CNS

ti4

Control disisrributionl

Distribution after loading2

Distribution of tracer3

115 135 66 109 35

119 125 63 98 46

98 81 117 109

113 159 54 104 31

100

I00

(5.0 / W g )

(5.3 PM/i3)

100 (530 cts/g)

100 (74 hours)

100

1. Plasma concentration: 0.20 pmoles/g. 2. Plasma concentration : 24 ,umoles/g. The animals had two intraperitoneal injections of taurine (270 mg in 3 ml), one at time 0, and one after 10 min. They were killed 20 min after the first injection. 3. Plasma activity: 13 000 cts/g. The animals had an intraperitoneal injection of [14C]taurine(3 pC in I ml) and were killed 10 min later.

Tissue concentration x In2 flux Relative values are calculated taking the average of total areas as 100.

4. Calculated according to the following: tf

=

REGIONAL DIFFERENCES I N

CEREBRAL AMINO ACID T R A N S P O R T

225

acid. Nevertheless, the relative distribution pattern remains essentially the same. The third column shows that the exchange of taurine between plasma and brain is quite slow and rather homogeneous. In contrast with lysine, it tends to be somewhat higher in areas with lower physiological level and vice versa. The half-life time of the taurine pool, whose average value is 74 h, closely parallels the pattern of taurine concentration in the various areas. I n view of these results i10 clear correlation seems to exist between transport and regional distribution of this amino acid. DISCUSSION

The data on amino acid transport in slices from different regions of the CNS confirm previous studies with slices from whole brains (Levi et a/., 1966; Lajtha et a/., 1966) in that the steady state or equilibrium level reached by the amino acids in the tissue upon incubation is mainly determined by the relative extent of the processes of accumulation and exit. Other factors, such as homoexchange, or the metabolism of the compounds studied, do not interfere significantly (Blasberg and Lajtha, 1965). The results presented seem to rule out the possibility that the regional differences of amino acid accumulation are determined primarily by factors such as regional differences in the number of cells per unit of tissue weight, or differences in the energy sources available for transport. In fact, if these were the limiting factors of amino acid accumulation in the various areas, the regional pattern of accumulation would be expezted to be the same with all the amino acids tested. It is interesting to note that although none of the areas studied has a homogeneous cell population, the histological composition of the various areas is quite different. It seems, therefore, that several constituents of the nervous tissue are responsible for the high amino acid accumulation of brain slices, and it is likely that the affinities of different amino acids or classes of amino acids for the various cell types are not the same. This may have a functional significance, and may reflect different metabolic requirements of different types of cells. It has been recently shown that neurons and glial cells do not have the same permeability to ions (Reed and Woodbury, 1963; Pappius, 1965). The problem becomes more complex if we try to correlate regional transport in vitro with regional amino acid concentrations and transport in vivo. In the living brain the physiological concentrations of free amino acids can be considered to be in dynamic equilibrium with those of plasma and cerebrospinal fluid. If brain concentrations were determined mainly by the transport capacity of the cells, a correlation would be expected between steady state accumulation in brain slices and in vivo brain concentrations. In agreement with this we have shown that in most cases amino acids that are accumulated to high levels in vitro have high in vivo concentrations, and vice-versa. This correlation was shown with mouse whole brain (Lajtha, Blasberg and Levi, 1966) and also with whole brains of other species (Levi et al., 1967). In contrast, the present report shows no clear correlation between regional pattern of amino acid accumulation in vitro and regional distribution in vivo, sugRc/c.rmces pp. 227-2211

226

G . LEV1

gesting that in the living brain other factors, such as regional differences in metabolic rates, may be operating. Also, there might be quantitative differences of the processes of influx and efflux between in vitro and in vivo conditions, as suggested by Lajtha, Lahiri and Toth (1963). According to these authors transport processes may act in vivo mostly to exclude substances from the brain, and in vitro may act mostly to accumulate substances in the slices. In other words, the exit process from brain tissue would not be as active in vitro as it is in vivo, where amino acids can be excluded from the brain also against a concentration gradient of elevated plasma levels (Lajtha and Toth, 1962). This hypothesis would explain why amino acid net accumulation into the living brain is much restricted. Regional differences in the activity of the exit process in vivo could therefore be an important factor in determining amino acid concentrations in the various areas of the brain under physiological circumstances and after plasma loading, and could explain the lack of correlation with the in vitro accumulations. In the opinion of the writer a high activity of the exit process in the living brain could be explained on the basis of the unique anatomical connections of nervous tissue cells, and of glial cells in particular. It is widely accepted that glial cells are in close contact with the basal membrane of blood capillaries (De Robertis and Gerschenfeld, 1961), and it is also accepted by most authors that there is a discrete extracellular space in the brain (Rall el al., 1962; Reed et al., 1964; Van Harreveld et al., 1965). A number of considerations would suggest that the extracellular fluid is in equilibrium with the cerebrospinal fluid, and that no barrier exists to the passage of substances from the former to the latter (Davson and Bradbury, 1965). Glial cells could take up blood amino acids from the small space existing between the capillary endothelium and the glial end feet. This space might well be occupied by a simple cdpillary filtrate (Davson and Bradbury, 1965) or by a fluid whose chemical composition is determined by the permeability properties of the capillary endothelium. Amino acids taken up would tend to exit preferentially into the extracellular space, since the concentration gradient in that direction is more favorable. Extracellular amino acids could then diffuse to the cerebrospinal fluid and could be rapidly eliminated through the arachnoid villi or by other routes. Thus the extracellular concentration would be kept low, with a mechanism similar to that described by Davson (1963) as “sink-action” of the cerebrospinal fluid; and the facilitated exit from the intracellular compartment would prevent the intracellular accumulation of amino acids. According to this model the cerebral concentration of those amino acids that are not produced or are not rapidly metabolized in the CNS would be mainly determined by the permeability properties of capillary endothelia and glial cells. Regional differences in amino acid concentration and uptake in vivo could be at least partly determined by regional differences in the size of the extracellular space, and in the readiness with which amino acids can diffuse to the cerebrospinal fluid. Since these mechanisms cannot be present in incubated brain slices, the lack of a close parallelism between regional transport i n vitro and regional distribution and uptake in vivo is not surprising.

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227

SUMMARY

Amino acid transport in different areas of the CNS was studied both in vitro and in vivo. The transport capacity of different areas of the CNS as measured in vitro varies from one area to the other, and also varies with the amino acid tested. Regional differences in the degree of amino acid accumulation at steady state seem to depend mostly on differences of accumulation rates with AIB, of exit rates with lysine, and on regional differences of both these processes with D-glutamate. It is suggested that the regional heterogeneity of cerebral amino acid accumulation and exit may be determined by differences in the permeability properties of the various cell types of the nervous tissue. The pattern of amino acid accumulation in slices from various brain areas does not correspond to the pattern of regional uptake or exchange in the living brain. This suggests the existence of regional differences in the restriction of amino acid transport in the living brain. In most cases no apparent correlation seems to exist between regional amino acid transport in vitro and regional amino acid concentration in vivo. Jnstead, the regional in vivo transport of some amino acids, such as lysine and leucine, tends to parallel the pattern of regional concentrations. The importance of transport pro:esses in the regulation of the level of cerebral amino acids may therefore vary from one case to the other. The possible reasons for the restriction of amino acid transport in the living brain, and for the low correlation between in vitro and in vivo findings are briefly discussed. ACKNOWLEDGEMENTS

The experimental work was done at the New York State Research Institute for Neurochemistry and Drug Addiction. 1 want to thank Dr. A. Lajtha for his hospitality and his advice. The cooperation of Dr. J. Kandera, Dr. A. Cherayil, and Mr. J. Toth is also gratefully acknowledged. The investigation was supported in part by Grant No. N B 04360 from the U S . Public Health Service (to Dr. A. Lajtha).

REFERENCES P. N . AND SCHOLEFIELD, P. G. (1962) Amino acid transport in brain cortex slices. 11. AHADOM, Competition between amino acids. Canad J . Biochem., 40, 1591-1602. BART,B., LOGOTETHIS, J., AELONY, Y. A N D Bovrs, M. (1962) Quantitative profile of freeaminoacids in various areas of cerebral cortex in normal guinca pigs. Exptl. Neurol., 5, 519-524. H. (1958) Determination of glutamic acid, glutamine, glutathione and BERL,S. A N D WAELSCH, y-aminobutyric acid and their distribution in brain tissue. J . Neurochem., 3, 161-169. BLASBERG, R. A N D LAJTHA, A. (1965) Substrate specificity of steady state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. - (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Res., I, 86-104. DAVSON, H. (1963) The cerebrospinal fluid. Ergeb. Physiol. biol. Cheni. exptl. Pharmacol., 52,21-73. DAVSON, H. AND BRADBURY, M. (1965) Theextracellular space of the brain. Progressin Brairi Research, E. D. P. De Robertis and R. Carrea (Eds.). Elsevier Publishing Company. Amsterdam. Vol. 15 (p. 124-134).

228

G . LEV1

DEROBERTIS, E. D. P. AND GERSCHENFELD, H. M. (1961) Submicroscopic morphology and function of glial cell. Intern. Rev. Neurobiol., 3, 1-65. GUROFF, G., KING, w.AND UDENFRIEND, s. (1961) The uptake Of tyrosine by rat brain in vitro. J . biol. Chem., 236, 1773 -1777. KETY,S. S. AND ELKES,J., Editors (1961) Regional Newochertiisfry. Pergamon Press, Oxford. LAHiRI, S. A N D LAJTHA,A. (1964) Cerebral amino acid transport in vitro. I. Some requirements and properties of uptakc. J. Nertrochem., 11, 77-86. LAJTHA,A. (1961) Exchange rates of amino acids betwcen plasma and brain in different parts of the brain. Regional Neitrochemistry. S. S. Kety and J. Elkes, Editors. Pergamon Press, Oxford. (p. 19-24). LAJTHA,A. A N D MELA,P. (1961) The Brain-Barrier System. I. The exchange of free amino acids between plasma and brain. J. Neurochern., 7 , 210-217. LAJTHA, A. AND TOTH,J. (1962) The Brain-Barrier System. I I I.Theefflux of intracerebrally administered amino acid from the brain. J. Neurocheni., 9, 199-212. LAJTHA, A., LAHIRI, S. AND TOTH,J. (1963) The Brain-Barrier System. IV. Cerebral amino acid uptake in different classes. J. Neurocheni., 10, 765-773. LAJTHA, A.. BLASBERG, R. A N D LEVI,G. (1966) Control of cerebral amino acid concentrations. Signif icance of changes in plasma amino a c i d p a t t e r m New Brunswick, New Jersey. Rutgers University Press. In press. LtvI, G. A N D LAJTHA,A. (1965) Cerebral amino acid transport in vitro. 11. Regional differences in amino acid uptake by slices from the central nervous system of the rat. J. Neurochem., 12,639-648. LEw, G . , CHERAYIL, A. A N D LAJTHA,A. (1965) Cerebral amino acid transport in vitro. 111. Heterogeneity of exit. J. Nertrochem., 12, 757-770. LEVI,G., BLASBERG, R. AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biochein. Biophys., 114, 339-351. LEVI,G., KANDERA, J. AND LAJTHA,A. (1967) Control of cerebral metabolite levels. I. Amino acid uptake and levels in various species. Arch. Biochem. Biophys., 119,303-31 1. NAKAMURA, R. A N D NAGAYAMA, M. (1966) Amino acid transport by slices from various regions of the brain. J . Neurochem., 13, 305-31 3. NEAME, K. D. (1962) Uptake of L-histidine, L-proline, L-tyrosine and L-ornithine by brain, intcstinal mucosa, testis, kidney, splcen, liver, heart muscle, skeletal muscle and erythrocytes of the rat in vitro. J. Physiol., 162, 1-12. OKUMURA, N., OTSUKI,S. AND FUKAI, N. (1959) Amino acid concentration in different parts of the dog brain. Acta M e d . Okayama, 13, 27-30. PAPPIUS, H. M., (1965) The distribution of water in brain tissue swollen in vitro and in vivo. Progress in Brain Research. E. D. P. De Robertis and R. Carrea, Editors. Elsevier Publishing Company, Amsterdam. Vol. 15 (p. 135-154). RALL,D. P., OPPELT,W. W. A N D PATLACK, C. S. (1962) Extracellular space of brain as dctermined by diffusion of inulin from the ventricular system. f i f e Sci., 2, 4 3 4 8 . RAMlREZ DE GUGLIELMONE, A. AND GOMEZ,c. J . (1966) Free amino acids in different arcas of rat brain. Ac/a Physiol. I atinoamer., 16, 26-37. REED,D. J., WOODBURY, D. M. AND HOLTZER, R. L. (1964) Brain edema, electrolytes, and extracellular space. Arch. Neirrol., 10, 604616. SINGH,S. 1. A N D MALHOTRA, C. L. (1964) Amino acid content of monkey brain: effects of reserpine on some amino acids of certain regions of monkey brain. J. Neurocliem., 11, 865-872. SHAW,R. K., AND HEINE, J. D. (1965) Ninhydrin positive substances present in different areas of normal rat brain. J. Nertrochem., 12, 151-155. STERN, J. R., ECCLESTON, L. V.. HEMS,R. AND KREBS,H. A. (1949) Accumulation of glutamic acid in isclated brain tissue. Biochenr. J., 44, 410418. TSUKADA, Y., NAGATA, Y.,HIRANO, S. A N D MATSUTANI, T. (1963) Active transport of amino acid into cerebral cortex slices. J. Neurochem., 10, 241-256. VANHARREVELD, A., CROWELL, J. A N D MALHOTRA, S. K. (1965) A study of extracellular space in central nervous system by freeze-substitution. J. Cell Biol.,25, 117-137.

REGIONAL DIFFERENCES I N CEREBRAL AMINO A C I D T R A N S P O R T

229

DISCUSSION

D. H. FORD:How much are the regional differences due to the differences in cell population, that is to the density of the cells per unit area? If you take, e.g., the uptake of almost any amino acid by the hypothalamus, one finds that the highest accumulation of label is near the arcuate nucleus, which has almost the highest nerve cell population density in the hypothalamus. Thus, the degree of accumulation of labeled compomds appears to be, in part at least, related to the number of neurons present in a particular region. G. LEVI:I did not measure the density of the cells in the different areas.

D. H. FORD: In your preparation there are cells mixed with white matter, all mixed up together. Anatomically it would be difficult to define this as cerebral cortex. G. LEVI:The preparations that I used are not anatomically pure, in the sense that in no case are we dealing only with neurons or glia or white matter. Nevertheless the cellular composition of the various areas that I used is very different. Now, what I found is that the regional pattern of transport is not the same with all amino acids. In other words, none of the areas has the highest accumulation with all of the amino acids, and since the cell population of the various areas is different, the structures involved in the transport of amino acids must also be different. Therefore, according to the type of structure and to the type of amino acid we may have different results.

D. H. FORD:I don’t think I would want to say that the structures involved in transport would necessarily be different in an anatomical sense because we are still dealing with the problem of the capillary: glia: neuronal relationship, which probably occurs in a similar manner in most areas of the brain. The difference in accumulation in different regions may again be closely related to the population density. I t makes a tremendous difference in the amount, and perhaps the rate with which a compound would be accumulated.

G. LEVI:I would like to emphasize that the conditions are different in vivo and in vitro: in brain slices there is no longer a capillary: glia barrier and the various cell types are in direct contact with the slibstances present in the medium. A. LAJTHA: There was some indication of a greater exit of glutamic acid by slices from areas richer in neurons, but since this was not true for other amino acids, we can’t say that there is a single pattern of neurons extruding all amino acids more than glia.

D. H.FORD: The incorporation may vary from amino acid to amino acid. You are getting entirely different curves, depending on what your amino acid is.

P. MANDEL:I have two questions. The first is: have you performed any tests concerning changes occurring in slices after 70 minutes of incubation in vitro? The second question is a general one: I should like to know something about the role of taurine in the brain. Maybe somebody has a hypothesis upon this? G. LEVI:I can easily answer the first question. We did experiments just to check the condition of the slices after periods even longer than 70 minutes. Both the accumulation and the exit remained unchanged after periods of - if I remember correctly - about two hours. I don’t have an answer to the second question, unfortunately. N. M. VAN GELDER: I just wanted to ask: could you give any indication of how reproduceable these figures are; how many experiments are represented by each figure, and what was the standard deviation?

G. LEVI:Yes, with in vitro experiments there was an average of 4 to 6 experiments, with a standard deviation of less than 10%. The number of in vivo experiments was approximately four, and the standard deviation was somewhat higher, being about I5 ?{.

230

G. LEV1

K. A. C. ELLIOTT:I may have missed something in both these last papers. Did you ascertain that these were the actual amino acids that were taken up and incorporated, or was it only radio-active carbon derived from them that appeared in these various spaces? G. LEVI:In our investigation, these amino acids were in the so called free amino acid pool. The radio-activity in the PCA-soluble fractions was from 85 to 90% in the form of the original amino acid provided, as determined by paper chromatography.

D. H. FORD:We have always done parallel paper chromatographic experiments with our radioautographic and accumulation studies, and feel reasonably sure that the radioautographic localizations shown after injecting lysine is due primarily to lysine. With the glutamine study, much of the labeled material in the tissue was present as glutamic acid as well as glutamine. D. B. TOWER: I did not quite understand your distinction a t the end between the in vivo and in v i m levels, because certainly amino acids (at least in vitro) achieve the in vivo levels during incubation, even if you have depleted them temporarily by some means. I am referring particularly to glutamate, aspartate, and so on, which are generated primarily by conversion from glucose. Thus, I think you have to make a distinction as to which amino acid you are talking about in this regard, since accumulation of these amino acids may not necessarily have anything to do with their transport. G . LEVI:The in vilro accumulation of the amino acids that were used in this investigation is due to transport and not to other factors. In fact the amino acids used were either not metabolized or metabolized very slowly. For example the D-form and not the readily metabolized L-form of glutamate was used. Anyhow factors such as the conversion from glucose to glutamate or to other amino acids would be negligible as compared to the transport rates in our experimental conditions; glutamate reach-s an intracellular level of over 50 mM in about half an hour when brain slices are incubated in presence of 2 mM glutamate.

GENERAL DISCUSSION

23 1

GENERAL D I S C U S S ION P. L. IPATA: Dr. Mandel, you have shown that the nucleoside enters the nerve cell and is incorporated into RNA molecules. We must therefore assume that it is phosphorylated; for instance, adenosine must give rise to AMP inside the cell in order to be incorporated into RNA. We have shown that the dephosphorylation of AMP inside the cell is strongly inhibited by ATP and other nucleoside triphosphates. This would facilitate the further phosphorylation of AMP to ADP and ATP for RNA synthesis.

P. MANDEL: We know that in several tissues the uptake of nucleosides and the synthesis of RNA from nucleosides is much faster than for purine and pyrimidine bases. In free nuclei with nucleosides incubated in vitro we have found a quite high synthesis of RNA, and a much lower synthesis of adenine, which means that the cells, or the nuclei, where the synthesis of RNA occurs, are able to produce ATP or nucleoside diphosphate from the nucleosides. P. L. IPATA:It is therefore meaningfull that the dephosphorylation of AMP is strongly inhibited inside the cell. Adenosine enters the cell, and is transformed into AMP; the dephosphorylation of AMP, however, is strongly inhibited by nucleoside triphosphates. I will show later that the inhibition is of the allosteric nature.

H. KOENIG: Pyrimidine nucleotides serve two major metabolic roles: ( I ) they are precursors of RNA and DNA; and (2) as coenzymes they participate in intermediary metabolism and biosynthetic reactions of lipids and carbohydrates. My interest in pyrimidine nucleotides in the central nervous system was stimulated by observations that neural tissues actively incorporate [I4C]orotic acid into neuronal and glial RNA (Koenig. H., 1958, Proc. Soc. Exper. Biol. Med., 97, 255; J . Biophys. Biochem. Cyrol., 4, 241 ; 1959, I n “Prog. in Neurobiol.” S. Korey. Ed., 4, 241, Hoeber, New York). Later studies revealed that the pyrimidine analogs 5-fluoroorotic acid and 5-fluorouridine (Koenig, H., 1960, AMA Arch. Neurol., 2: 463; 1962, 111 “Response of the Nervous System to Ionizing Radiation”, Haley, T., and Snider. R. (Eds.), p. 109, Academic Press, New York), and 6-azauracil and 6-azauridine (1961, Koenig, H., Young, 1. J., Wells, W., and Gaines, D., Trans. Am. Neurol. Ass., p. 219; 1963, Wells, W., Gaines, D., and Koenig, H., J . Nerrrochenz., 10: 709) are neurotoxic when administered intrathecally. These studies, which have recently been reviewed (1967, Koenig, H., International Review of Neurobiology, 10, Academic Press, New York), prompted an investigation of the entry and uptake of a number of natural and unnatural pyrimidines into brain and liver in rat. The experimental details and results are summarized in Table 1. A number of interesting findings are apparent. [14C]Orotic acid and [’4C])fluorooroticacid, both highly ionized, traversed the bloodbrain barrier to a limited extent, whereas [14CC]azauraciland [14CC]uridinewere 3 to 4 fold more effective in this regard, as judged by the specific activity of the acid soluble fraction. The intracerebral injection route gave much greater uptake of pyrimidines into the acid soluble fraction and RNA than the intravenous route of injection. Despite this, incorporation into brain RNA, as measured by the relative specific activity, was comparable for both routes of administration. [I4C]uridine was most actively taken up into RNA. [lJC]Oroticacid, 5-fluoroorotic acid and 5-fluorouracil were also well utilized. [I4C]Uracilwas incorporated to a lesser degree, while [14C]azauracilwas incorporated minimally into RNA. Liver differed significantly from brain in a number of respects. [*4C]Oroticacid and [14C]5-fluor~ orotic acid were extensively incorporated into the acid soluble fraction of liver, even when these wer: administered intracerebrally. The other pyrimidines, [14C]uracil, [14C]azauracil, [~~C]5-fluorouracil and [‘JCIuridine, appeared in the acid soluble fraction of liver to a limited degree. Without exception these pyrimidines, as measured by the R.S.A., were incorporated into brain R N A to a greater extent than into liver RNA. From these data it seems that RNA turnover in brain is considerably more active than RNA turnover in liver. The extent to which brain can utilize preformed pyrimidines conveyed by the blood-stream naturally depends upon the facility with which these traverse the bloodbrain barrier. Of the pyrimidines tested, uridine was most actively transported into brain and was also most extensively incorporated into brain RNA.

232

G E N E R A L DISCUSSION

TABLE 1 U P T A K E OF N A T U R A L A N D U N N A T U R A L P Y R I M I D I N E S I N T O R A T B R A I N A N D L I V E R

[I4C]Orotic acid Intravenous Intracerebral [W2]5-Fluoroorotic acid Intravenous lntracerebral [ W ] Uracil Intravenous Intracerebral [14C]6-Azauracil Intravenous Intracerebral [14C]5-Fluorouracil Intravenous Intracerebral [14C]Uridine Intravenous Intracerebral

** +

S.A.* of A.S.F.

BRAIN S.A.** of RNA

R.S.A.+

S.A.* of A.S.F.

LIVER S.A.** of RNA

355 67,500

I .2 378.0

3.4 x 10-3 5.7 x 10-3

199,000 34,800

190.0 42.0

1.0 x 10 1.2 x 10-3

428 80,000

1.7 349.0

4.1 x 10-3 4.3 x 10-3

132,000 34,800

203.0 42.0

1 . 6 x 10-3 1.2 x 10-3

198 27.500

0.5 66.0

2.3 x 10-3 2.5 x 1 0 - 3

566 869

0.3 0.2

4.8 x 10 1.6 x 10

1,339 10,622

0.4 2.3

3.0 x 1 0 - 4 2.1 x 1 0 - 4

1,567 568

0.2 0.2

1.5

-

0.1

3.2 x

0.1

0.9 x 10-3 1 . 4 x 10-3

-

-

34,400

193.0

5.6 x 10-3

253

947 43,000

6.7 359.0

6.5 x 10-3 8.3 x 10-3

111.3 64.7

0.I

R.S.A.+

X

10

1.9 x 10-4

Specific activity (S.A.) of acid-soluble fraction (A.S.F.) = counts per minute (C.P.M.)/g fresh weight. S.A. of RNA = C.P.M. per optical density unit at 260 mp (ODZEO). Relative specific activity (RSA)

=

S.A. of R N A S.A. of A.S.F.

Experinienral Derails: All isotopes (S.A. = 2.4 mC/mMole) were injected intravenously into rat in a dose of 1 pC/100 g, or intracerebrally in a dose of 0.5 pC/100 g. After a 4 hour exchange period, heads were plunged into liquid nitrogen, frozen brains removed with a chisel and homogenized in ice cold 1076 trichloracetic acid to yield an acid soluble fraction. After water and lipid extraction, the protein residue was hydrolyzed in 0.3 M NaOH at 37’C for 1 h., neutralized with HCI, and DNA and protein precipitated by adding cold TCA to a concentration of 10%. RNA was measured in the acid-soluble supernatant by ultraviolet spectrophotometry. Radioactivity was assayed in an automatic gas flow Geiger counter.

P. MANDEL:I think that we cannot assume that you have a higher incorporation in liver than in brain, since you relate the relative specific activity to the whole RNA, and d o not know the specific activity of RNA in the various types of cells. In the neurons you have about the same concentration of RNA as in the liver, but the apparent synthesis is lower in the liver. But I am not sure that this is so since, for example, in measuring the RNA polymerase activity, you find it either in a similar, or in a lower quantity in the brain than in the liver. H. KOENIG:RNA polymerase activity gives only the potential for RNA synthesis, as RNA synthesis depends on the amount of available template, and the availability of precursors, etc.. In brain, of course, the various cell types differ greatly in the rate of R N A synthesis. Large cells show the greatest RNA synthesis. These have the same amount of template DNA as glia. However, their chromatin is largely in a diffuse state, and is therefore metabolically active in the RNA polymerase reaction. The difference between a big nerve cell and a small nerve cell with respect to RNA synthesis is great, and still greater for glia. So this is a picture of the “average” nerve cell,

GENERAL DISCUSSION

233

P. MANDEL: We measured template independent RNA polymerase.

D. H. FORD:I t is very interesting to hear that your uptakes of adenine in the liver and brain were so comparable. We have just completed an in vivo study in rats where we have injected [3H]adenine and studied the uptake per unit wcight of ventral horn neuron, using a modified Hyden procedure to isolate the neurons. Our data indicate that nerve cells accumulate slightly more than liver cells, which emphasizes the tremendous affinity which the neuron has to have for various substrates to maintain its high metabolic needs. What the biologic nature is of the labeled material in the neurons from these preparations can only be inferred from what can be determined for blocks of grey matter from the same animals analyzed chromatographically. Such data suggest that most of the labeled material present in the neurons is probably associated with the high energy phosphates. P. MANDEL:Here again the problem is whether you do short-time experiments or longer-time experiments. If you d o longer-time experiments this is quite different. We mainly did very short-time experiments to look at the diffusion in the brain or in the liver.

D. H. FORD:Ours were for a half hour to 24 hours. Ours were from 2 minutes. P. MANDEL: P. L. IPATA: Can you answer the question if, in your experimental conditions, adenosine is deaminated to inosine in the brain? And furthermore; what is the ratio of the radioactivity among the four bases in the RNA? P. MANDEL:I cannot say if it is deaminated or not. I did not control this. But the deamination of adenosine in brain is more artifact, because if you kill the animal in good conditions you have a very low amount of inosine, and if you kill it in bad conditions you have a high amount of inosine. R . KATZMAN: One would assume from your data that polynucleotides would not enter the brain. Do you have direct evidence on this? P. MANDEL:Polynucleotides are something else. Polynucleotides might enter into the brain by pinocytosis. This may be low. I t is possible that by pinocytosis macromolecules can enter another site, but I have no evidence.

P. G . SCHOLEFIELD: Have you any evidence on how the ribosides are converted to the nucleotides? Do they go down to the base? I don't know. If you incubate the nucleotides the radioactivity is not decreased by the P. MANDEL: addition of thc adenine. And if you had to pass through the adenine step you should have a decrease. P. G. SCHOLEFIELD: If you use ascites cells under similar conditions you can get an almost equivalent quantity of lactic acid from ribosides. P. MANDEL:Yes, but that is a peculiar phenomenon, I think, for ascites cells, and perhaps also for red blood cells, which use for energy metabolism the ribose of nucleosides.

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235

Influence of Elevated Circulating Levels of Amino Acids on Cerebral Concentrations and Utilization of Amino Acids S I D N E Y ROBERTS Departnien t of Biological Chemistry, and the Urain Research Institule, UCLA Center for the Health Sciences, I 0 s Angeles, California 90024 ( U S A )

INTRODUCTION

The relationship between circulating and tissue levels of various amino acids does not follow any consistent pattern, partly as a consequence of competitive interactions in membrane transport, variations in intracellular metabolism, and differential effects of other regulatory factors (see Roberts, 1963). In the central nervous system, this relationship is further complicated by the existence of specialized barrier mechanisms which selectively restrict net uptake of certain amino acids more than others (Lajtha, 1964a). However, even where the restriction is great, parenteral administration of large quantities of an amino acid results in elevated concentrations of this substance in cerebral tissues (see, for example, Schwerin et al., 1950; Kamin and Handler, 1951; Tigerman and MacVicar, 1951 ; Lajtha, 1958; Dingman and Sporn, 1959; Chirigos et al., 1960; Lajtha and Toth, 1961; Guroff and Udenfriend, 1962). Under these circumstances, cerebral concentrations of certain other amino acids may be acutely depressed (Roberts and Morelos, 1965). Data in the literature suggested that two principal mechanisms might be responsible for the latter finding, i.e., competition among amino acids for brain transport systems (Chirigos et al., 1960; Lajtha 1964a; Neame, 1964) and enhanced intracerebral utilization of amino acids in general (see Hanking and Roberts, 1965). The investigations summarized in this report provide evidence that both of these processes were responsible for the acute lowering of cerebral levels of several amino acids in the presence of elevated circulating concentrations of another amino acid. METHODS

The methods employed have been described in detail elsewhere (Roberts and Morelos, 1965). Male rats of an inbred Sprague-Dawley strain were maintained on Purina laboratory chow until they were about 6 weeks old and weighed 160-170 g. They were then placed in individual cages and either fed the same diet ad libitum or fasted overnight (16-20 h). Animals scheduled to be killed within 5 min after administration of amino acids were lightly anesthetized by subcutaneous injection of sodium pentoRrfcrencrs pp, 242-243

236

S. R O B E R T S

barbital (Nembutal) in 0.9% saline (5 nig per 100 g body wt). Otherwise, they were injected with amino acid solution while under light ether anesthesia, allowed to recover, and given Nembutal 10 min before autopsy. Non-labeled and “Clabeled amino acids were dissolved in saline, neutralized, and administered via the saphenous vein in a volume equivalent to 0.5 ml per 100 g body wt. At intervals of from 5-30 min, the rats were exsanguinated from the dorsal aorta with a heparinized syringe over a 15-sec collection period. Free amino acids were extracted from plasma and cerebral cortical tissue with 5 % TCA as described previously (Roberts, 1963). The extracts were then passed through Dowex-2 resin columns to remove the TCA (Lajtha, Furst, Gerstein and Waelsch, 1957; Roberts, 1963). Amino acid content was measured with the Beckman Model 120 Amino Acid Analyzer (Moore, et al., 1958). Just prior to analysis, ~~-[l-~~C]b-2-thienylalanine was added to each sample (5 mpC per 5 mpmoles per ml), to be used as an internal standard for calculations of concentrations (Siege1 and Roach, 1961) and radioactivities of the separated amino acids. Radioactivity in individual amino acids was determined directly on the effluent from the column of the Amino Acid Analyzer by means of a Packard Scintillation flow detector equipped with an anthracene cartridge. RESULTS

Cerebral Free Amino Acids after Amino Acid Administration An acute lowering of cerebral concentrations of certain amino acids in the presence of elevated levels of one amino acid was first noted after intravenous injection of Lleucine to fed rats (Roberts and Morelos, 1965). Within 5 min after the administration of 50 pmoles L-leucine per 100 g body wt, total ninhydrin-reacting substances were depressed approximately I5 %. Several amino acids participated in this decline, notably proline, alanine, valine, phenylalanine and arginine. Concomitantly, plasma concentrations of these and other amino acids appeared to be slightly enhanced. Both cerebral and plasma free amino acids tended to return to normal or supranormal levels within 15-30 min after the injection of leucine. The degree of response and the exact nature of the amino acids participating in this response varied from experiment to experiment, probably as a result of uncontrollable variations in the feeding patterns of the animals. For this reason, similar studies were undertaken with rats which had been deprived of food for 16-20 h. More consistent and more profound effects were obtained in these fasted animals. Thus, intravenous administration of 100 pmoles L-leucine per 100 g body wt resulted in an average 22 % lowering of cerebral amino acids in 10 min under L-leucine). Significant declines were these circumstances (Table 1, fasted vs. fasted observed for most amino acids in the presence of a 2-3 fold increase in the cerebral concentration of leucine. The short period of fasting per se resulted in a lowering of certain cerebral amino acids (Table 1, fed vs. fasted). Longer periods of fasting (e.g., 48-72 h) had the opposite effect on cerebral concentrations of aminoacids and did not appear to result in a sensitization to alterations in circulating levels of these metabolites. Concomitant with the depression in cerebral concentrations of amino acids, plasma

+

237

CIRCULATION A N D CEREBRAL TISSUE AMINO ACID LEVELS

TABLE 1 I N F L U E N C E O F F A S T I N G A N D E L E V A T E D L E V E L S O F L - L E U C I N E O N C O N C E N T R A T I O N S OF

FREE AMINO ACIDS I N RAT CEREBRAL ~~

~

~

Fed ~~

Taurine Urea Aspartic acid Threonine Serine Asparagine I Glutarnine Proline Glutamic acid Glycine Alanine ci-Amino-ti-butyric acid Valine Cyst i t hionine Met hionine lsoleucine Leucine Tyrosine Phenylalanine fi-Alanine y-Aminobutyric acid Ornithine Lysine Histidine Arginine Total NRS 8 No. of analyses

CORTEX*

~~

Cerebral free atvitro acids (pmoleslg tissue) Fasted Fasted i L-leucine

~~

~~

5.40 5 0.38 4.00 I t 0.37 3.36 f 0.18 0.639 rt 0.072 1.33 f 0.070

5.43 f 0.060 3.66 5 0.220 3.63 5 0.040 0.607 -+ 0.033 1.39 0.020

4.33 f 0.06 2.77 & 0.07 # 2.76 0.08 # 0.488 f 0.033* 1.12 f 0.04 *

2.88 & 0.34 0.107 f 0.012 13.62 -L 0.96 0.650 f 0.037 0.700 0.024 0.018 f 0.007 0.109 f 0.004 0.021 5 0.003 0.061 0.009 0.037 IO.001 0.074 i0.004 0.084 i 0.008 0.043 5 0.004 0.045 f 0.006 1.95 f 0.04 0.014 f 0.001 0.215 -+ 0.01 0.080 -40.003 0.106 I 0.004 43.105 4

4.74 + 0.06+ 0.068 L 0.002+ 11.50 1 0.24 0.699 t 0.011 0.625 f 0.009+ 0.034 f 0.003 0.105 5 0.003 0.016 1 0.001 0.048 0.002 0.052 t 0.001+ 0.084 i0.002 0.076 f 0.004 0.053 0.002 0.034 0.001 1.58 f 0.05 0.013 i0.001 0.235 f 0.008 0.079 f 0.003 0.092 _t 0.004t 41.674 6

3.79 f 0.11 0.054 f 0.003+ 8.97 f 0.22 0.543 f 0.017+ 0.461 f 0.01 If 0.029 f 0.003 0.064 fi 0.003+ 0.012 0.00lt 0.033 f 0.00If 0.024 f 0.002+ 0.208 f 0.009# 0.041 f 0.00If 0.023 & 0.001+ 0.034 f 0.004 1.31 f 0.080 0.01 1 0.187 5 0.009+ 0.048 $I 0.004" 0.061 & 0.003+ 32.750 6

+

+

+

+

+

f

+

+

* All animils wzr: f:d Purim laboratory chow adlibititrii prior to the experiment. The fasted groups were d e p r i c d of foxl for 16-20 h. All animals were killed 10 rnin after intravenous injection of 0.9% NaCI or L-leucins (100 pmoles par 100 g bDdy wt). The results given are means i S.E.M. t p ;. 0.05 for difference b e t w z x this value and the corresponding value in the column t o the left. ' p 0.01 to 0.02 for difference bctwesn this value and the corresponding value in the column to the left. 0 Ninhydrin-reacting substances. 2

levels tended to rise after intravenous administration of L-leucine to fed rats (Roberts and Morelos, 1965) or fasted rats (Table 2). However, significant increases were noted only in a few plasma amino acids (serine, a-amino-n-butyric acid, lysine, and arginine) as a consequence of a 9 fold enhancement in the circulating level of leucine in fasted animals. Fasting for 16-20 h per se tended to decrease plasma levels of many amino acids in a manner analogous to the effects of overnight fasting on cerebral concentrations of these metabolites. Enhanced secretion of adrenal cortical steroids was not responsible for the observed responses to intravenously administered leucine or overRefirrncrs p p . 242-243

238

S. R O B E R T S

TABLE 2 I N F L U E N C E OF F A S T I N G A N D E L E V A T E D L E V E L S O F L - L E U C I N E O N C O N C E N T R A T I O N S O F FREE A M I N O ACIDS I N RAT PLASMA*

Fed

Taurine Urea Hydroxyproline Aspartic acid Threonine Serine Asparagine Glutamine Proline Glutamic acid Citrulline Glycine Alanine u-Amino-n-butyric acid Valine Half cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Total NRS 8 No. of analyses

+

Plasma free amino acids (,umoles/ml) Fasted Fasted

+ L-leucine

0.069 f 0.010 4.74 f 0.21 0.051 f 0.005 0.015 f 0.001 0.222 f 0.015 0.267 f 0.018

0.049 f 0.003 3.86 f 0.32 t 0.037 f 0.006 0.01 3 f 0.002 0.226 f 0.013 0.277 f 0.007

0.049 -+- 0.003 4.00 f 0.34 0.046 f 0.006 0.01 1 f 0.002 0.271 f 0.026 0.334 & 0.013#

0.567 f 0.036 0.220 f 0.014 0.170 f 0.019 0.091 f 0.006 0.400 f 0.006 0.493 f 0.016 0.01 I f 0.002 0.152 f 0.005 0.022 f 0.003 0.049 f 0.001 0.075 f 0.002 0.1 18 f 0.005 0.078 f 0.002 0.050 f 0.001 0.072 f 0.008 0.412 f 0.015 0.065 f 0.001 0.139 f 0.005 9.024

0.556 f 0.038 0.150 f 0.006# 0.252 f 0.028t 0.069 f 0.006t 0.366 f 0.004* 0.359 f 0.0102 0.015 -f 0 0.157 f 0.012 0.016 f 0.003 0.041 f 0.002# 0.085 f 0.007 0.122 f 0.012 0.069 5 0.010 0.065 f 0.001# 0.057 f 0.005 0.393 f 0.024 0.064 f 0.005 0.1 14 f 0.010 8.010 3

0.087 & 0.056 0.145 f 0.003 0.263 f 0.058 0.084 & 0.007 0.400 0.024 0.380 f 0.016 0.020 f 0.001+ 0. I76 f 0.01 3 0.0 I3 0.003 0.045 f 0.003 0.090 f 0.007 1.098 f 0.057 0.061 0.001 0.067 0.003 0.056 0.001 0.493 5 0.005# 0.075 rt 0.003 0.147 0.003+ 9.682 3

I

+

+

* All animals were fed Purina laboratory chow adlibitum prior to the experiment. The fasted groups were deprived of food for 1 6 2 0 h. All animals were killed 10 min after intravenous injection of 0.9% NaCl or L-leucine (100 pmoles per 100 g body wt). The results given are means f S.E.M. t p < 0.05 for difference between this value and the corresponding value in the column to the left. p = 0.01 to 0.02 for difference between this value and the corresponding value in the column to the left. 8 Ninhydrin-reacting substances. +

night fasting since these responses also occurred in adrenalectomized animals and could not be duplicated by the injection of cortisol. Acute alterations in cerebral amino acids were also noted after intravenous administration of L-phenylalanine, L-proline or L-lysine to fasted rats. The amino acidlowering effect was not restricted to those amino acids for which common carriers are thought to exist (see Blasberg and Lajtha, 1966). Thus, after injection of L-phenylalanine (100 pmoles per 100 g body wt), significant declines were noted in cerebral histidine as well as leucine and valine (Table 3). In fact, depressed cerebral concentrations of histidine (and leucine) were observed in 5 min, whereas valine was decreased only

C I R C U L A T I O N A N D C E R E B R A L TISSUE A M I N O A C I D L E V E L S

239

TABLE 3 I N F L U E N C E OF E L E V A T E D L E V E L S O F L - P H E N Y L A L A N I N E O N C O N C E N T R A T I O N S OF F R E E A M I N O ACIDS I N RAT CEREBRAL

Minutes ajier injec!ion

0.054 0.002 0.182 i.0.009+ 0.205 0.003+ 0.168 0.009+

15

30

Cerebral free aniitio acids (pmoleslg tissue) Tyrosine Leucine Valine

Plienylalarrine

+

Control 5

CORTEX*

0.063 f 0.002 0.060 f 0.007 0.079 f 0.008 0.183 f 0.007#

0.082 f 0.003 0.061 f 0.005# 0.061 f 0.006# 0.062 & 0.003#

0.1 1 1 f 0.005 0.105 f 0.006 0.105 f 0.007 0.083 f 0.005+

Histicline

0.075 f 0.002 0.063 f 0.005t 0.063 0.001# 0.083 f 0.008

+

* All animals were fed Purina laboratory chow ad lihituni prior t o the experiment and were then deprived of food for 16-20 h. Control animals were injected intravenously with 0.9 % NaCl(0.5 ml per 100 g body wt) and killed at intervals of 5-30 min; these procedures were without effect on cerebral amino acid levels. All other animals were injected intravenously with L-phenylalanine (100 /moles per 100 g body wt). The results given are means f S.E.M. for 9 animals in the control group and 3 animals in each of the other groups. t p 0.05 for difference between this value and the corresponding value in the control group. # p = 0.01 for difference bztween this value and the correspmding value in the control group. :<

TABLE 4 I N F L U E N C E O F E L E V A T E D L E V E L S OF L - P H E N Y L A L A N I N E O N U P T A K E A N D U T I L I Z A T I O N O F

L-[14C]VALlNEI N

RAT CEREBRAL

CORTEX*

Minutes after injec!ion

Cerebral aciti-saluble ra .lioac!ivitv cpmlg tissue

5 I5 30

8,930 8,380 6,550

7,230 6,200 3,210

5

4.260 5.300 5,890

Phenylalanine-injected 2,900 27,400 3,500 33,700 2,180 26,050

15

30

Radioactivity in cerebral free valine cpmlg tissue cpmlpmole 76 0.f total

Saline-injected 69,300 52,350 28,400

81 rt 1 74 f 2 49 f 2 68 f 3# 66 f 2t 37 & 3+

* All animals were fed Purine laboratory chow adlibifurn prior t o the experiment and were then deprived of food for 16-20 h. L-phenylalanine was injected in an amount equivalent t o 100 pmoles per 100 g bodywt. Uniformly-labeled L-[l1C]valine (26 m C per mmole) was injected in a n amount equivalent t o 3.75 ,uC per 100 g body wt. The results given are means of values obtained from 3 animals in each instance. In addition, S.E.M. are shown for the % of total radioactivity in the column effluent from the Amino Acid Analyzer which remained in the valine peak. t p I. 0.05 for difference between this value and the corresponding value in the saline-injected group. p .z: 0.02 for difference between this value and the corresponding value in the saline-injected group. f

Rcjwiwcrs pp. 242-243

240

S. R O B E R T S

after 30 min. By 30 min, histidine levels were back to normal. Since cerebral phenylalanine exhibited a 3 fold elevation within 5 min and tyrosine levels were not enhanced until much later, the results observed may be presumed due principally or entirely to the administered amino acid. Subsequent experiments substantiated these data and, i n addition, revealed significant declines in cerebral concentrations of isoleucine and arginine 15 minafterphenyl alanineadministration. Decreases in cerebral levels ofthese or other amino acids have also been noted after intraperitoneal administration of very large amounts of phenylalanine (Carver, 1965; Boggs, D. E. and McKean, C. M., unpublished observations). Intravenous administration of L-proline or L-lysine to fasted rats (100 pmoles per 100 g body wt) caused only slight alterations in cerebral levels of certain amino acids within 15 min. However, under these circumstances, cerebral concentrations of proline and lysine were each elevated only about 40%. It may be anticipated that more profound increases in the cerebral levels of these amino acids would result in more extensive changes. Cerebral Uptake and Utilization of Amino Acids after Amino Acid Administration The existence of competitive interactions among amino acids for transport into cells has been recognized since the pioneering work of Christensen and his colleagues. Interactions of this nature were considered to be responsible for the apparent displacement of accumulated amino acids from tissues following administration of another acid in vivo (Christensen et al., 1948) or in vitro (Riggs e t a / . , 1954). In the present investigations, the lowering of cerebral levels and the elevation of plasma levels of certain amino acids after intravenous injection of L-leucine or L-phenylalanine were associated with inhibition of the cerebral influx of these amino acids. This phenomenon is clearly illustrated in Table 4. Cerebral accumulation of uniformly labeled ~-[14C]valine, administered intravenously in tracer quantities, was depressed by the concurrent injection of L-phenylalanine (100 pmoles per 100 g body wt). This depression was most striking during the first 5-1 5 min. Acid-soluble radioactivity in plasma was approximately one-third higher in the phenylalanine-injected rats than in the saline controls over this period and was localized principally (85-95%) in the valine peak in the effluent from the Amino Acid Analyzer. Intravenous injection of an equimolar amount of L-leucine instead of L-phenylalanine produced entirely comparable results. Lproline, which did not result in a significant lowering of cerebral valine in 15 min under similar experimental conditions, nevertheless produced a n inhibition (about 20 %) in the apparent uptake of this amino acid. L-lysine altered neither the cerebral level nor the uptake of valine. Earlier investigations had revealed that intravenous injection of large doses of L-leucine not only enhanced cerebral uptake and exchange of this amino acid and depressed cerebral concentrations of other amino acids but also strikingly stimulated the intracerebral utilization of leucine (Roberts and Morelos, 1965). Suggestive evidence was obtained that the intracerebral metabolism of other amino acids was also increased under these conditions. The data in Table 4 provide definitive support for this conclusion. The percentage of total acid-soluble radioactivity in the cerebral cortex

CIRCULATION A N D CEREBRAL TISSUE A M I N O ACID LEVELS

24 1

remaining in valine was significantly reduced by prior injection of phenylalanine, indicating that the utilization of valine was enhanced by this procedure. Similar results were obtained when leucine was injected in large doses instead of phenylalanine and when the radioactive amino acid was phenylalanine or leucine respectively, instead of valine. Major identifiable radioactive products in the column effluent from the Amino Acid Analyzer included the dicarboxylic acids and their amides in all instances, plus tyrosine when ~-[“T]phenyalaninewas administered. DISCUSSION

The dependence of normal brain activity and mental performance on the proper functioning of amino acid metabolism has been well documented in recent years (see Waisman and Gerritsen, 1964; Nyhan and Tocci, 1966). Abnormalities involving the central nervous system in man have been demonstrated in the presence of defects in metabolism of phenylalanine, brached-chain amino acids, tryptophan, histidine, cystathionine, homocystine, p-alanine, etc. In several instances, the metabolic error appears to be associated with an enzymic defect in the brain. However, the primary abnormality in amino acid metabolism may occur elsewhere in the body. Under the latter circumstances, elevated circulating levels of the amino acid or its metabolites are presumed responsible for the neural and mental aberrations. The mechanisms involved in the deleterious actions of these substances are unknown, but the explanations most frequently advanced involve effects on membrane transport, enzyme action, and protein synthesis in the brain (Udenfriend, 1963; Lajtha, 1964b; Roberts and Morelos, I965 ; Roberts and Zomzely, 1966). The present investigations clearly demonstrate that elevated circulating levels of one amino acid can result acutely in the lowering of cerebral concentrations of other amino acids. The nature of the amino acids involved and the extent of the decline depended upon the amino acid injected, its concentration, and the time after administration. However, the depression in cerebral concentrations of amino acids was not limited to amino acids within a given transport group. This finding could be explained by the fact that the lowering effect was due not only to competitive interactions among amino acids for entry into the brain, but also to increased utilization of cerebral amino acids in general. The mechanisms underlying these metabolic alterations have not yet been elucidated, but appear to occur under similar circumstances in other tissues as well (Hanking and Roberts, 1965). A relationship between these observations and the naturally-occurring derangements in cerebral function which accompany the aminoacidurias remains to be demonstrated. In the latter instances, chronic rather than acute alterations in amino acid metabolism appear to be responsible for the pathologic manifestations (see Waisman and Harlow, 1965). However, the basic biochemical mechanisms underlying these defects would appear to have common characteristics in both acute and chronic situations and may be elucidated more readily in acute experiments where the complications of secondary, delayed alterations in metabolism are more readily avoided. Rt.lr.rmrrs pp.

242-243

242

S. R O B E R T S

SUMMARY

Intravenous administration of large amounts of one amino acid (SO-I00 pmoles per 100 g body wt) resulted in the acute depression of cerebral cortical concentrations of a number of other amino acids. The amino acid-lowering effect was not limited to amino acids within the same cellular transport group. The decline was more pronounced in rats fasted overnight (16-20 h) than in fed rats. L-leucine and L-phenylalanine were more effective in eliciting this response than L-proline or L-valine, apparently because the former were more readily concentrated by the cerebral cortex. Within 5 min after the injection of L-phenylalanine, cerebral uptake of ~-[“C]valine was decreased. Concomitantly, cerebral utilization of this radioactive amino acid was enhanced. These results indicated that elevated cerebral levels of one amino acid acutely lowered cerebral concentrations of other amino acids not only by inhibiting their uptake, but also by stimulating their utilization in cerebral tissue. ACKNOWLEDGEMENT

This work was aided by grants from the United Cerebral Palsy Research and Educational Foundation and from the National Institutes of Health, U.S. Public Health Service (NB-07869). Mrs. Beatrice S. Morelos provided expert technical assistance. REFERENCES BLASBERG, R. AND LAJTHA,A. (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Bruin Res., 1, 86-104. CARVER, M. J. (1965) Influence of phenylalanine administration on the free amino acids of brain and liver in the rat. J. Neurochenr., 12, 45-50. P. AND UDENFRIEND, S. (1960) Uptake of tyrosine by rat brain in vivo. CHIRIGOS,M. A., GREENGARD, J. Biol. Chem., 235, 2075-2059. CHRISTENSEN, H. N., STREICHER, J. A. AND ELBINGER, R. L. (1948) Effects of feeding individual amino acids upon the distribution of other amino acids between cells and extracellular fluid. J. Biol. Chem., 172, 5 15-524. DINGMAN, W. A N D SPORN,M. B. (1959) The penetration of proline and proline derivatives into brain. J. Neurochem., 4, 148-153. GUROFF,G. AND UDENFRIEND, S. (1962) Studies on aromatic amino acid uptake by rat brain in 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. HANKING, B. M. AND ROBERTS, S . (1965) Stimulation of protein synthesis in vitro by elevated levels of amino acids. Biochini. Biophys. Actu, 104, 427-438. KAMIN,H. AND HANDLER, P. (1951) The metabolism of parenterally administered amino acids. 11. Urea synthesis. J. Biol. Chem., 188, 193-205. 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 a t different ages. J. Neurochem., 2, 209-215. -, (1964a) Protein metabolism of the nervous system. Intern. Rev. Neurobiol., 6, 1-98. -, (1964b) Alteration and pathology of cerebral protein metabolism, Intern. Rev. Neurobiol., 7,l-40. LAJTHA,A,, FURST,S., GERSTEIN, A. AND WAELSCH, H. (1957) Amino acid and protein metabolism of the brain - I. Turnover of free and protein bound lysine in brain and other organs. J. Neurochem., 1, 289-300. LAJTHA,A. AND TOTH,J. (1961) The brain barrier system - 11. Uptake and transport of amino acids by the brain. J. Neurochem., 8, 216-225. MOORE,S.. SPACKMAN, D. H. AND STEIN,W. H. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Fed. Proc., 17, 1107-1115.

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NEAME, K. D. (1964) Effect of amino acids on uptake of L-histidine by rat brain slices. J. Neurochem., 11, 67-76. NYHAN, W. L. A N D TOCCI,P. (1966) Aminoaciduria. Ann. Rev. Med., 17, 133-160. RIGGS, T. R., COYNE,B. A. AND CHRISTENSEN, H. N. (1954) Amino acid concentration by a free cell neoplasm. Structural influences. J. Biol. Chenr., 209, 395-41 1. ROBERTS, S. (1963) Regulation of cerebral metabolism of amino acids - 11. Influence of phenylalanine deficiency on free and protein-bound amino acids in rat cerebral cortex: relationship to plasma levels. J . Neurocheni., 10, 931-940. S. A N D MORELOS, B. S. (1965) Regulation of cerebral metabolism of amino acids - IV. ROBERTS, Influence of amino acid levels on leucine uptake, utilization and incorporation into protein in vivo. J. Nerrrocheni., 12, 373-387. ROBERTS, S. A N D ZOMZELY, C. E. (1966) Regulation of protein synthesis in the brain. Protides ofthe Biological Fluidv, 1965. H. Peeters (Ed.). Amsterdam, Elsevier (91-102). SCHWERIN, P., BESSMAN, S. P. AND WAELSCH, H.(1950) The uptake of ghtamic acid and ghtamine by brain and other tissue of the rat and mouse. J. Biol. Cheni., 184, 3 7 4 . SIEGEL,F. L. A N D ROACH,M. K. (1961) &?-ThienyI-o~-alanine, internal standard for automatic determination of amino acids. Anal. Chem., 33, 1628. TIGERMAN, H. A N D MACVICAR, R. (1951) Glutamine, glutamic acid, ammonia administration, and tissue glutamine. J . Biol. Chenr., 189, 793-799. UDENFRIEND, S. (1963) Factors in amino acid metabolism which can influence the central nervous system. Attier. J. Clin. Nutr., 12, 287-290. WAISMAN, H. A. AND GERRITSEN, T. (1964) Biochemical and clinical correlations. in: Mental Retardution. A review of research. H. A. Steevens, and R. Heber (Eds.). Chicago, The University of Chicago Press (307-347). WAISMAN, H. A. A N D HARLOW,H. F. (1965) Experimental phenylketonuria in infant monkeys. Science, 147, 685-695.

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245

Specificity of Cerebral Amino Acid Transport: A Kinetic Analysis R O N A L D G. B L A S B E R G New York State Research Itutitute for Neurochetiiistry and Drug Addiction, Wards’ Island N . Y. (U.S.A.)

Metabolite flux between brain cells and the surrounding fluid compartments (blood and cerebrospinal fluid) is one aspect of the “brain barrier systems” that has recently become the focus of considerable investigation. The distinctive composition of the free amino acid pool of brain in comparison to that of plasma and other organs (Tallan, 1962) was at first thought to be due to a barrier that limits the passage of these metabolites between brain and plasma. More recently, however, it was found that a rapid exchange exists between plasma and brain amino acids (Lajtha and Mela, 1961; Lajtha and Toth, 1961; Lajtha, 1961, 1962). It has also been shown in in vitro studies with brain slices that amino acids are actively transported from a medium into the cells against an electrochemical gradient (Stern et a/., 1949; Elliott and van Gelder, 1958; Guroff, et a/. 1961 ; Neame, 1962; Abadom and Scholefield, 1962a, c; Tsukada et al., 1963; Lahiri and Lajtha, 1964). A comparison of amino acid transport in vivo and in vitro as discussed in this symposium and elsewhere demonstrates many qualitative similarities which suggest that cellular transport mechanisms are an important constituent of the brain barrier systems (Lajtha el a/., 1966). Amino acid transport across brain cell membranes, both in vivo and in vifro, is specific for amino acids and is selective among different amino acids (Guroff et a/., 1961; Guroff and Udenfriend, 1962; Abadom and Scholefield, 1962b; Neame, 1964; Blasberg and Lajtha, 1965, 1966). Since all a-amino acids have the same basic structure (a-carboxyl group and a-amino group), differences in their physical and chemical properties are determined by differences in the structure, charge, and configuration of the side chain. As suggested by Christensen the selectivity of amino acid transport would also be expected to be determined by differences in the amino acid side chain (Christensen, 1962; Oxender and Christensen, 1963). This appears to be the case in brain, since the transport of an amino acid is usually inhibited by a structurally related analog, whereas those amino acids whose side chains are structurally dissimilar usually have little or no effect. The specificity of amino acid transport as discussed in this symposium and elsewhere indicates that a number of membrane transport systems exist (Blasberg and Lajtha, 1965, 1966). Since it appears unlikely that a specific transport system exists for each amino acid, a particular transport system is thought to mediate the entry of a particular group or class of amino acids whose side chains are Refcrenrrs p. 256

246

R.

C. B L A S B E R C

similar in structure and charge. The transport system itself is partially defined by the characteristics of the particular class of amino acids it mediates into the cell. However, some inhibition of transport between amino acids within different classes was found to exist. This indicates that the specificity patterns of transport are not absolutely defined and suggests that a particular amino acid may be transported into the cell by more than one transport system. The significance of the interaction between certain amino acids in different classes with respect to the specificity patterns and the number of transport systems mediating amino acid passage across the brain cell membranes can be studied in greater detail by a kinetic analysis of amino acid transport and amino acid inhibition of transport. The present report is a comparative kinetic analysis of the inhibition of transport of four representative amino acids (a-aminoisobutyric acid, L-phenylalanine, L-arginine, and L-aspartic acid respectively) by various amino acids of different classes (small neutral, large neutral, large basic, and acidic amino acids). M E T H 0D S

The experimental procedure has been described previously (Blasberg and Lajtha, 1965, 1966); briefly, slices 0.42 mm thick were prepared from cerebral hemispheres of young adult Swiss mice. The slices were equilibrated for 30 min at 37" in a KrebsRinger Tris medium; 14C-labeledamino acid (or W-labeled amino acid plus 2 mM [Wlamino acid inhibitor) was added and the slices incubated for an additional 2-4 min. Initial uptake was measured from five different concentrations of l4C-labeled amino acid in the medium : 0. I , 0.15,0.25, 0.5, and 2.0 mM respectively (except in the case of L-aspartate, in which only the first four concentrations were studied). The rate of amino acid uptake (influx) into the cells was calculated from the 14C-labeled amino acid concentration in the intracellular water space. The intracellular water space was estimated from an inulin space of 52.7 per cent per gm tissue wet weight and a dry weight of 20 per cent (unpublished observations). Short incubation periods were necessary to avoid significant return of labeled amino acid from the cells to the medium. The metabolism of the 14C-labeledamino acid during the incubation period was negligible (Blasberg and Lajtha, 1965). It must be emphasized that the initial rate of intracellular amino acid penetration (influx) must be determined if meaningful kinetic constants for transport are to be calculated for brain slices. In other cells, however, kinetic constants for metabolite transport have been calculated from steady state uptake experiments (Heinz, 1954; Wilbrandt and Rosenberg, 1961). With brain slices, kinetic constants for amino acid transport cannot be determined from steady state uptake experiments, since it has been shown that amino acid efflux from brain cells shows a tendency towards saturation and that efflux is altered by the presence of other amino acids in the intracellular phase as well as extracellular phase (Levi et al., 1966). The kinetic constants for amino acid transport reported here were determined from initial uptake experiments which essentially measure initial unidirectional flux. The initial flux of many amino acids into brain cells can be described phenomenol-

S P E C I F I C I T Y O F C E R E B R A L A M I N O A C I D TRANSPORT: A K I N E T I C A N A L Y S I S

247

ogically by Michaelis-Menten kinetics: v, = (V,xA)/(A x K,) where V a is the observed velocity of amino acid entry and A is the concentrationofamino acid in the medium. The transport constants K, and V, are formally equivalent to the MichaelisMenten constants Km and Vma,. The contribution of physical diffusion (Kd x A) to the observed flux (v,) was negligible under the experimental conditions since A < Ka and the diffusion constant ( K d ) was relatively small (unpublished observations). The transport constants were determined from a Lineweaver-Burk plot of reciprocal initial intracellular entry rates (influx) vs. reciprocal medium concentrations (Lineweaver and Burk, 1934) as shown in Fig. 1 for n-aminoisobutyric acid (AIB). Va was 15-

10

x L-LEU 2 m M

,

I

I

0.5

2

4

I

6.7

J

10

7/5

Fig. 1 . Graphical analysis according to Lineweaver and Burk of a-aminoisobutyric acid (AIB) influx, alone and in the presence of an amino acid inhibitor (L-alanine and L-leucine). Each point represents an average o f at least four experiments and a standard error not greater than 20 per cent.

determined from the reciprocal ordinate intercept by averaging four independent estimates and has an average error of approximately 20 per cent. Ka was calculated from the slope of the Lineweaver-Burk plot and V,. In the presence of a second amino acid (B), the inhibition of amino acid (A) transport may be considered to be of the competitive type if Vmax remains unchanged, i.e., V, + b 2 Va. It can be seen from Table 1 that V, + b is equal to V, within the limits of experimental error for all the amino acid combinations studied, indicating that the inhibition of transport is a competitive inhibition. The transport constant in the presence of the inhibitor (K, + b) wascalculated from the slope of the Lineweaver-Burk plot and V,; V, rather than the individual V, + b value was used to avoid carrying the variation, which was within experimental error, through the calculation of K, + b. The inhibitor constant (i;) was calculated from the relationship: i; = (Ka x B)/(Ka + b - K,) as given in Fig. 2. K h and v h are phenomenological Michaelis-Menten constants for the initial transport into the cell of the amino acid inhibitor (B) itself; the Kb for various amino acids is presented in Table 3 and the VI, for the same amino acids in Table 1. R&encrs

p. 256

248

R. G . B L A S B E R G TABLE 1

M A X I M A L RATE OF A M I N O ACID T R A N S P O R T : A L O N E A N D I N THE P R E S E N C E OF A N A M I N O

ACID INHIBITOR

Amino Acid Studied ( A ) inhibitor ( B ) (2 m M )

vb

*

AIB'

va+b+*

Glycine L-alanine D-alanine Cycloleucine L-proline L-histidine L-glu tamine L-met hionhe L-leucine L-lysine L-glutamate D-glutamate

3.3 3.3 4.0 3.3 4.0 2.0 3.3 1.3 1.3 0.67 4.0

2.4 2.1 1.9 2.3 2.6 2.7

L-Phe2

L-Arg3

L-A@

Va+b

Va t b

va

1 .o

1 .o

4.1

I .2 1.3 0.87

0.67

+b

1.1

1.2 0.77 0.85 0.95

1.3 1.4

2. I 2.4

5.0 4.2

10

a-aminoisobutyric acid (AIB): Va* = 2.5 pnoles/ml I.W./min. L-phenylalanine: Va = 1 . 1 pmoles/ml I.W./min. L-arginine: Va = 0.77 pmoles/ml I.W./min. L-aspartate: Va = 4.4 pnoles/ml I.W. min. * Vs and Vb are phenomenological Michaelis-Menten constants formally equivalent to Vmaxfor the transport of amino acid A and B respectively, into the cell and are expressed as pmoles/ml intracellular water/minute. Va and Vb were determined from the reciprocal ordinate intercept on their respective Lineweaver-Burk plots: four independent estimates were obtained and the average is presented. The average variation was approximately 20 per cent. * * Va + b is the maximum velocity of transport for amino acid A in the presence of amino acid inhibitor B. Va + b was determined in the same manner as Va and Vb and is expressed similarly: pmoles/ml intraacellular water/minute. The average variation was between 15 and 20 per cent.

A0

+

+

carrier

gAm

A1

carrier

Bo

+

carrier

0m K, =

k 2 +kl k3

,where k 2 s J k 3

Kb=

!k!-bk4

,Where k5,u,k6

Fig. 2. A is the substrate whose initial rate of transport is measured: B is a competitive inhibitor of substrate transport. Ka and Kb are phenomenological Michaelis-Menten constants (Km)for the transport of A and B respectively across the cell membrane. KI is the inhibitor constant, i. e., the transport constant of B for the same carrier system(s) mediating the transport of A. K a + b is the transport constant for A in the presence of B a t a given medium concentration.

SPECIFICITYOF C E R E B R A L A M I N O A C I D TRANSPORT: A K I N E T I C ANALYSIS

249

In the experiments to be discussed, the specificity of four amino acids (a-aminoisobutyric acid, L-phenylalanine, L-arginine, and L-aspartic acid) was studied and each amino acid is referred to as substrate or amino acid (A). The transport inhibitors in these experiments were other amino acids in different transport classes and are referred to as inhibitor or amino acid (B). RESULTS A N D DISCUSSION

A kinetic model for amino acid transport across cell membranes is presented in Fig. 2. The model is constructed so the transport process or reaction can be described phenomenologically by Michaelis-Menten kinetics. The transport constants Ka and V, are therefore formally equivalent to the Michaelis-Menten constants K m and Vm,, for the transport of amino acid A into the cell. The affinity of the amino acid for the carrier at the transport site on the external surface of the cell membrane will be determined by the ratio of the rate constants kl and kz. The individual rate constants for the as yet unknown sequence of reactions which occur during passage of the substratecarrier complex a.;.ross the membrane and the dissociation of the complex at the inner surface of the membrane are summated into a single rate constant k3. If the rate constants k3 are similar for all the amino acids transported by the same carrier system, the transport constants will reflect the relative affinities of the amino acids for the carrier. The fact that amino acids within the same transport class have similar maximum transport rates (Vmax) may be significant in this respect (Table I). The small neutral amino acids have a Vmay. between 3 and 4 ,umoles/ml intracellular water/min; the large neutral, large basic, and acidic amino acids have a Vma, between I .O and 1.5,0.5 and I .O, and 4 and 4.5 ,umoles/ml I.W./min respectively (except for D-glutamate in the acidic group of amino acids). The difference in the maximum rate of transport between amino acids in the various transport classes is greater than can be explained by experimental error, which suggests that different carrier systems are involved in the transport of the different classes of amino acids. The transport model in Fig. 2 describes only the unidirectional passage of amino acid across the cell membrane; the subsequent fate of the substrate within the intracellular space is essentially of no consequence to the kinetics of the transport reaction. Whether the amino acid remains in a “free” intracellular pool, is accumulated within subcellular particulates, or is bound to intracellular protein is relatively unimportant in experiments designed to measure the initial rate of uptake. Such factors, however, may have to be considered in experiments which do not measure the initial uptake process (Blasberg and Lajtha, 1965; Levi et a]., 1966). The initial rate of amino acid (A) entry into the cell may be inhibited by the presence of another amino acid (B) in the medium. If this inhibition of amino acid transport is due to a reversible affinity of the inhibitor (B) for the same carrier mediating the transport of the substrate (A), the inhibition will be of the competitive type and the maximum rate of transport will remain unchanged, i.e., Va + b 2 V, (Table I ) . The transport constant in the presence of the inhibitor (K, + b) can be determined as previously described and the inhibitor constant Kj can be calculated knowing K,, Re/c.rmces p. 256

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R. G . B L A S B E R G

Ka + b, and B as shown in Fig. 2. If the predominant mode of entry for the inhibitor (B) is by the same transport system utilized by the substrate being measured (A), the value of Ki should approximate the value of the transport constant Kb obtained when the kinetics of the transport of B is studied independently of A. In other words, Ki and Kb should be numerically equal if A and B are transported by the same transport system. If A and B do not enter the cell by the same transport system they would not be expected to interact during transport and KI will be infinitely large. A situation may exist, however, in which A and B show some degree of competitive interaction during transport, but predominantly enter the cell by different transport systems. That is, a particular amino acid may have an affinity to two or more transport sites and may enter the cell by more than one carrier system. The observed flux or entry velocity (va) of the amino acid from a given medium concentration (A) would then be determined by the sum of the fluxes at the individual transport sites. If the individual transport reactions can be described phenomenologically by MichaelisMenten kinetics, the following equation can be written : VIa x A

Va =

V2a x A Vna x A + +...+ A + Kla A + K2a A S Kna

The contribution of diffusion (Kd x A) to the observed flux in these experiments was negligible as discussed in the previous section. The transport of an amino acid across cell membranes by more than one carrier system is analogous to an enzymatic reaction in which two or more different enzymes independently catalyze the same overall reaction (Dixon and Webb, 1964). It can be shown that a number of conditions exist when it is virtually impossible to differentiate more than one transport reaction (or enzymatic reaction) by graphical analyses of the kinetic data, when in fact two independent transport reactions (or enzymatic reactions) are taking place. For example, the Lineweaver-Burk plot will remain essentially linear when Kl, z K2,; K, z (Kla K2a)/2 when V1, z Vza. Only when there is a large discrepancy between Kl, and Kz, (i.e., greater than 10 fold) will the Lineweaver-Burk plot show significant deviation from linearity. Furthermore, the deviation from linearity may be seen only within a particular range of substrate concentration. Preliminary results for many amino acids (acidic amino acids in particular) indicate that the kinetic data obtained at concentrations higher than those studied for this report (2 mM to 50 mM) does not fit a straight line on a Lineweaver-Burk graphical analysis as is shown in Fig. 3 for AIB. The break in linearity of the graphical analysis seen at higher amino acid concentrations does not appear to be totally accounted for by the increasing significance of diffusion relative to mediated flux (unpublished observations). It may be significant that the Kt values for various amino acids are within the same concentration range as the deviation from linearity seen in the respective Lineweaver-Burk plots (Tables 2 and 3, Fig. 3 and unpublished observations). Whether or not this preliminary kinetic data at higher amino acid concentrations reflects a true break in the graphical analysis which would suggests that another carrier system(s), or the same carrier system(s) with different kinetic properties, functions at high amino acid levels, cannot be answered at present. Suffice it to say that graphical analysis of the kinetic data over the particular

+

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251

TABLE 2 TRANSPORT CONSTANTS FOR A M I N O A C I D I N H I B I T I O NO F

Kn I, tnM

Inhibitor ( B )

2

ti1

1

M

Glycine L-alanine o-alanine Cycloleucine L-methionine L-proline L-histidine L-leucine 1

Ki

Kh

IllM

t71M

AIB

INFLUXI

KiIKh 171 M

2.7 3.5 1.7 2.4

1.9 1.2 7.2 2.5

2.4 1.6

1.7 I .2 3.0 1.6

1.9

4.4 18 9.9 8.9

0.69 2.4 1.2 0.54

6.4 7.5 8.3 16

I .5 1.6 1.6

u-aminoisobutyric acid (AIB): Kn

-

1.1 1 .o

1.3 mM.

TABLE 3 KINETIC EVALUATION O F AMINO A C I D INHIBITION OF T R A N S P O R T

Itihihitor ( B )

Kb

AIB'

L-Phes

L-Arg3

( 2 niM)

tnM

KilKh

KiIKh

KiIKh

Glycine L-alanine D-alanine Cycloleucine L-proline L-histidine L-glutamine L-methionine L-leucine L-l ysine L-glutamate D-glutamate

1

2

4

1.1

I .o

2.4 I .6 2.4 1.2 2.5 0.69 0.54 1 .o

1 .7 1.2 3.0 1.6 7.5 8.3

7.5 3.2 5.4 1.9

6.4 16

2.0 2.0

0.48 3.0

a-arninoisobutyric acid (AIB): K:, L-phenylalanine: K , 0.51 m M . L-arginine: Kn 1.0 rnM. L-aspartate: K , 0.49 m M .

21 23

L-Asp.'

KIIKII 11

1.8

21 13 9.3 1.4

2.0 1.8

-

1.3 niM.

~

concentration range studied in this report can be consistent with a single transport reaction when in fact two or more independent transport systems are contribution to the observed flux. The mediated passage of amino acids by more than one carrier system could explain the interaction between amino acids in different transport classes (i.e., small neutral w. large neutral amino acids) which are presently thought to enter the cell by different carrier systems. One method which will demonstrate the presence of at least Rcjrrcnci~rp. 256

252

R . G. B L A S B E R G

Fig. 3. See legend to Fig. 1. The kinetic data is uncorrected; the contribution of physical diffusion to the observed flux has not been substracted.

two carrier systems mediating amino acid flux would be to selectively inhibit one carrier system. This approach is difficult to implement with reasonable confidence, given our is present knowledge of the transport process. Another method would be to study the interaction between amino acids in different transport classes by a kinetic analysis of amino acid (B) inhibition of amino acid (A) flux and then compare the transport constants Ki and Kb as discussed previously. Since the inhibitor constant (KI) is a measure of the inhibitor’s (B’s) transport at the same site(s) mediating the transport of the substrate (A), the numerical relationship of the inhibitor constant (KI) and the transport constant of the inhibitor (Kb) will depend on the extent of interaction between A and B during transport. The value of Kb would not be expected to be identical to K I if the inhibitor primarily enters the cell by a transport system(s) not predominantly utilized by the substrate; in fact KI will be greater than Kb when the values of Ka and Kb are similar. Therefore, a comparison of Ki and Kb will indicate whether compounds A and B are transported into the cell at the same transport site(s) or whether they are predominantly transported at different transport sites but show some degree of competitive interaction during entry. A gra,>hicalanalysis according to the method of Lineweaver and Burk of the initial inflsx 0; a-aminoisobutyric acid (AIB), alone and in the presence of an amino acid inhibitor (L-alanine and L-leucine), is presented in Fig. 1. The line drawn between the (Va) of individual points for control AIB influx is linear and extrapolates to a V,, 2.5 ,umole/ml intracellular water/minute; the transport constant Ka calculated from the slope of the line and Va is 1.3 mM. In the presence of an amino acid inhibitor (Lalanine or L-leucine), the individual points also fit along a straight line and extrapolate

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to a V,,, similar to the control, i.e., V, + b z V, (Table I). This indicates that the inhibition of AlB flux by these amino acids is a competitive inhibition. The transport constant in the presence of inhibitor ( K , + b) was determined and the inhibitor constant Ki calculated as previously discussed; the value of Ki for L-alanine and L-leucine inhibition of AIB influx was I .9 mM and 8.9 mM respectively. The inhibitor constants Ki for other amino acids were determined similarly and are given in Table 2. In each case the inhibition appeared to be a competitive inhibition (Table I). The transport constants for the inhibitors themselves ( K b ) were determined by a similar graphical analysis as previously described and are also presented in Table 2. I t can be seen that four amino acid inhibitors (glycine, L-alanine, malanine, cycloleucine) all within the small neutral group of amino acids have similar Ki and Kb values. The values of Ki and K b for the other four amino acids (L-methionine, Lproline, L-histidine, and L-leucine) in contrast are quite dissimilar. In othe words the ratio Kj/Kb is close to unity for the first four amino acids but not for the latter four amino acids. This would indicate from the previous discussion that the first four amino acids are predominatly transported by the same carrier system(s) mediating the transport of AIB over the concentration range studied. Since AlB is classified as a small neutral amino acid and the first four amino acids (glycine, L and D-alanine, and cycloleucine) are also recognized to be within the small neutral classification, their transport into the cell would be expected to be mediated by the same carrier system(s) (Blasberg and Lajtha, 1966). The fact that the K i / K b ratio is somewhat greater than unity for all of these amino acids could be due to experimental error; however, it may suggest some as yet unresolved differences in the transport of neutral amino acids with short side chains. Since the KI/Kb ratio for the latter four amino acids (L-methionine, L-proline, Lhistidine, and L-leucine) is at least 6, it is apparent that these amino acids are not predominantly transported by the same carrier system(s) mediating AIB influx. This group of amino acids does interact with AIB competitively for transport, however, which indicates that these amino acids have some affinity to the carrier(s) mediating AIB influx or that AIB is transported into the cell to some extent by the carrier system(s) primarily responsible for the transport of the amino acid inhibitor, or possibly that both conditions apply. A number of models for substrate-inhibitor interaction or carrier-inhibitor interaction could be constructed to explain the observed inhibition in terms of a multi-carrier transport reaction. Whatever the model, it is apparent that AIB flux is representative of small neutral amino acid transport. Although AIB influx is inhibited competitively by neutral amino acids with long side chains (methionine, leucine, and possibly histidine) as well, the transport of AIB and the latter amino acids are predominantly mediated by different carrier systems over the coiicentration range studied. The specificity of L-phenylalanine, L-arginine, and L-aspartate transport is summarized in Table 3. These amino acids are representatives of thelarge neutra1,large basic, and acidic amino acid transport classes, respectively (Basberg and Lajtha, 1966). The transport constants K a and V, were determined similarly to AIB as were the inhibitor constants Ki; in each case the inhibition appeared to be a competitive inhibition R i p k r m w s p. 256

254

R. G . B L A S B E R G

(Table I). The specificity of phenylalanine transport is essentially the mirror image of that for AIB with respect to the neutral amino acid inhibitors. The KJKb ratio for the first three inhibitors (glycine and L- and D-alanine) is significantly greater than that for the latter four inhibitors (cycloleucine, histidine, methionine, and leucine). The Ki/Kb ratio indicates that the small neutral amino acids (glycine and L- and D-alanine) are not predominantly transported by the same carrier system(s) mediating phenylalanine. Those amino acids with long side chains (cycloleucine, methionine, leucine, and possibly histidine), however, would be expected to be transported by the same carrier system(s) mediating phenylalanine influx; this is indicated by the Kt/Kb ratio for these amino acid inhibitors. Cycloleucine has been included in both the small neutral and large neutral amino acid transport classes because the Kr/Kh ratio was close to unity for the inhibition of both AIB flux and phenylalanine flux. This condition would exist if cycloleucine were transported to a similar extent into the cell by the carrier systems predominantly mediating AIB and phenylalanine at the concentration studied. The specificity of arginine transport demonstrates that only lysine of the amino acid inhibitors studied has a Ki/Kb ratio close to unity. This would be expected if the large basic amino acids were primarily transported by a carrier system which is relatively specific for amino acids with a long cationic side chain. Neutral amino acids inhibited arginine influx to a limited extent, but the Ki/Kb ratio indicates that neutral amino acids are predominanlty transported by another carrier system(s). In previous studies it has been shown that large basic amino acids (lysine and arginine) have little or no effect on neutral amino acid influx (Blasberg and Lajtha, 1966). This would suggest that large basic amino acids have little or no affinity to the carrier systems mediating neutral amino acid transport. The inhibition of aspartate by other acidic amino acids (L- and D-glutamate) demonstrates that the C4 and Cg acidic amino acids are transported by the same carrier system(s). Although glycine inhibits aspartate transport to some extent, the Ki/Kb ratio clearly indicates that glycine is primarily transported by a carrier system other than that which predominantly mediates aspartate flux. Other amino acids with neutral or long basic side chains have been shown to have essentially no effect on acidic amino acid influx; similarly, acidic amino acids have been shown to have little or no effect on neutral or basic amino acid influx (Blasberg and Lajtha, 1966). This suggests that C4 and C5 acidic amino acids are primarily transported by a carrier system that is relatively specific for amino acids with an anionic side chain. CONCLUSIONS

( I ) The mediated passage of amino acids into brain cells can be described phenomenologically by Michaelis-Menten kinetics; the kinetic constants for the transport reaction are formally equivalent to Michaelis-Menten constants K, and Vmax. (2) Transport constants for amino acid influx in brain slices must be determined from initial uptake experiments, which essentially measure the initial unidirectional flux; transport constants for influx cannot be determined from steady state uptake

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255

experiments, since amino acid exodus from brain cells is a mediated process and demonstrates saturation kinetics. (3) Amino acids within the same transport class have similar maximum transport rates ( V d . (4) Amino acid inhibition of amino acid transport into brain cells appears to be a competitive inhibition. ( 5 ) A comparison of the Ki/Kt, ratio for the transport inhibition of four amino acids (u-aminoisobutyric acid, L-phenylalanine, L-arginine, and L-aspartate), as representatives of different amino acid transport classes (small neutral, large neutral, large basic, and acidic amino acids, respectively), demonstrates that at least four transport systems mediate amino acid passage across brain cell membranes and indicates that an amino acid may enter the cell by more than one transport system. (a) The small neutral amino acids are predominantly transported by the same carrier system(s) mediating AIB influx; large neutral amino acids inhibit AIB flux competitively but are not primarily transported by the same carrier system(s) at the concentration studied . (b) Large neutral amino acids are predominantly transported by the same carrier system(s) mediating phenylalanine influx; small neutral amino acids inhibit phenylalanine flux competitively but are not primarily transported by the same carrier system(s) at the concentration studied. (c) Cycloleucine appears to be transported by two carrier systems (AIB’s carrier system and phenylalanine’s carrier system) to a similar extent at the concentration studied. (d) Lysine and arginine are transported by the same carrier system(s), which is relatively specific for amino acids with long cationic side chains. (e) Aspartate and glutamate are transported by the same carrier system(s), which is relatively specific for c 4 - C ~amino acids with an anionic side chain. I n summary, amino acid transport in brain slices involves a number of carrier systems, which can be partially characterized by the particular group of amino acids it predominantly mediates across the cell membrane. These transport systems apparently do not possess absolute specificity, since a number of amino acids appear to have some capacity for transport by carrier systems other than the one which primarily mediates their passage into the cell. A kinetic analysis of amino acid transport and amino acid inhibition of transport provides one method to evaluate the number and characteristics of the carrier systems in greater detail. ACKNOWLEDGEMENTS

The experimental work was done at the New York State Research Institute for Neurochemistry and Drug Addiction. I want to thank Dr. A. Lajthafor his hospitality and his advice, Dr. S. R. Cohen for his criticisms of the manuscript, and Mr. A. Mazeika and Z. Ronay for expert technical assistance. The investigation was supported in part by Grant No. NB04360 form the U.S. Public Health Service (to Dr. A. Lajtha.) RrJerenres p.

256

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REFERENCES P. G. (1962a) Amino acid transport in brain cortex slices. 1. ABADOM, P. N. A N D SCHOLEFIELD, Relation bEtween energy production and the glucose-dependent transport of glycine. Canad. J. Biochem., 40, 1575-1 590.

-, (1962b) Amino acid transport in brain cortex slices. 11. Competition between amino acids. Canud. J. Biochem., 40, 1591-1602.

-, (1962~)Amino acid transport in brain cortex slices. 111. Utilization of energy for transport. Canud. J. Biochern., 40, 1603-1618. BLASBERG, R. AND LAJTHA,A. (1965) Substrate specificity of steady state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. -, (1966) Heterogeneity of mediated transport systems of amino acid uptake in brain. Brain Res., 1, 86-104.

CHRISTENSEN, H. N. (1962) Biological Transport. W. A. Benjamin, New York (p. 54-65). DIXON, M. A N D WEBB,E. (1964) Enzymes. Academic Press, New York (p. 87-90). ELLIOTT, K. A. c. A N D VAN GELDER, N. M.(1958) Occlusion and metabolism of y-aminobutyric acid by brain. J. Neurocheni., 3, 28-5 I . 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. Chem., 237, 803-806.

HEINZ,E. (1954) Kinetic studies on the “influx” of glycine I-CYinto the Ehrlich mouse ascites carcinoma cell. J. Biol. Chern., 211, 781. LAtiIRI, s. A ND LAJTHA,A. (1964) Cerebral amino acid transport in vitro. I. Some requirements and properties of uptake. J. Neurochem., 11, 77-86. LAJTHA, A. (1961) Exchange rates of amino acids between plasma and brain in different parts of the brain. Regional Neurochemistry. S. S. Kety and J. Elkes (Eds.). Pergamon Press, Oxford (p. 19-24). -, (1962) The brain barrier system. Neurochemistry. K. A. C. Elliott, I. H. Page, and J. H. Quastel (Eds.). Charles C. Thomas, Springfield (p. 399-430). LAJTHA,A. AND MELA,P. (1961) The brain barrier system. 1. The exchange of free amino acids between plasma and brain. J. Neurochem., 7, 210-217. LAJTHA, A. AND TOTH,J. (1961) The brain barrier system..II. Uptake and transport of amino acids by the brain. J. Neurochem., 8, 216-225. LAJTHA,A., BLASBERG, R. AND LEVI,G. (1966) Control of cerebral amino acid concentrations. Significance of Changes in Plasma Amino Acid Patterns, Rutgers University Press, New Brunswick, New Jersey, In press. LEVI,G., BLASBERG, R. AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biochem. Biophys., 114, 339-351.

LINEWEAVER, H. AND BURK,D. (1934) The determination of enzyme dissociation constants. J. Amer. Chern. SOC.,56, 658.

NEAME, K. D. (1962) Uptake of L-histidine, L-proline, L-tyrosine, and L-ornithine by brain, intestinal mucosa, testis, kidney, spleen, liver heart muscle, skeletal muscle, and erythrocytes of the rat in vitro. J. Physiol., 162, 1-12. -, (1964) Effect of amino acids on uptake of L-histidine by rat brain slices. J. Neurochem., 11,67-76. OXENDER, D. L. AND CHRISTENSEN, H. N. (1963) Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem., 238, 3686-3699. STERN,J. A., EGGLESTON, L. V., HEMS,R. AND KREBS,H. A. (1949) Accumulation of glutamic acid in isolated brain tissue. Biochem. J . , 44, 410-418. TALLAN, H. H. (1962) A survey of the amino acids and related compounds in the nervuus tissue. Amino Acid Pools. J. T. Holden (Ed.). Elsevier, Amsterdam (p. 471-485). TSUKADA, Y.,NAGATA,Y., HIRANO,S. AND MATSUTANI, T. (1963) Active transport of amino acids into cerebral cortex slices. J. Neurochem., 10, 241-256. T. (1961) The concept of carrier transport and its corollaries in WILBRANDT, W. AND ROSENBERG, pharmacology. Pharmacol. Rev., 13, 109-183.

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DISCUSSION P. G. SCHOLEHELD: We faced the choice of measuring steady state levels or initial velocities for the determination of Km-values several years ago. I think there are many factors that have to be considered, and the reason that we rejected initial velocities was very simple; it was that initially a tissue, whether it is brain slice, or pancreas, or ascites cells, always contains amino acids inside the cell, and these can always exchange with the amino acid that is added to the medium. So when initial velocities are measured there is always a danger. When we measure steady state values all the amino acids inside the cell have been swept out or have exchanged as much as they possibly can and this would be a relatively sniall amount compared with the amount that one puts in the medium. We tried to substantiate this directly by preincubating cells or tissues to get rid of endogenous amino acids, and then we found somewhat different Km values. So that this, I think, is a note of warning, if 1may.

R. BLASBERG: To answer your point: our initial velocities were calculated from 3-minute intracellular uptake values, and for most of the amino acids, intracellular uptake was approximately linear between 2 and 5 minutes. Undoubtedly there is reflux and physical diffusion of amino acid between the intracellular and extracellular phases during this period, but we believe this to be relatively sniall. Secondly, our tissue is pre-incubated for 30 minutes in amino acid-free medium, during which time the endogenous amino acids will tend to equilibrate with the medium. Whether exchange processes significantly alter the calculated unidirectional flux, I cannot really answer, except to say that the Lineweaver-Burk plots are linear below 2 mM for most amino acids. This concentration is considerably higher than the endogenous tissue level of most amino acids. In any event, Dr. Scholefield’s point is a good one and must be kept in mind in any kinetic study of transport processes. A. LAJTHA:May I ask you, Dr. Scholefield, if you have any evidence of moles of ATP used per mole amino acid transported in your system at least approximately? No, we have never really calculated this. It is rather difficult to assay ATPP. G. SCHOLEFIELD: utilization in ascites., We did some years ago attempt to study 32Pturnover, to get after this but it is difficult because the 3?P turnover is determined mainly by the rate at which 32Penters the ascites cells or the brain slice. I f brain slices or ascites cells are incubated with 3zP it will take about a n hour to get a steady state label in ATP, whereas, assuming a P/O ratio of three and a normal rate of oxygen uptake, all the ATP should be labelled within, I think, 45 to 60 seconds. Study of 3eP turnover is therefore of no use in efforts to answer this question from studies in which an intact cell preparation is used. Perhaps I could make one point to Dr. Mandel on this general topic. In studies of brain slices we tried to elevate the level of ATP in the slice, and we found adenine of little use since its presence could lead to perhaps 5 or 10% increase. Addition of adenosine led to an increase of the total ATP level of the slice so our in vitro experiments would lend some support to your in vivo experiments.

K. D. NEAME:I would just like to comment on the relationship between the review slides I gave on the various transport systems and Dr. Blasberg’s presentation. The systems that I gave were based, as I indicated, on a multitudinous amount of work by many people, but essentially it rested on the interpretations of net uptake, that is, uptake expressed as influx plus the effect of efflux over a certain time, or, as Dr. Lajtha indicated, the steady state situation where there is a balance. Whereas Dr. Blasberg’s presentation was on influx only, and some of Dr. Scholefield’s comments were on intracellular amino acids coming out of the tissue, Dr. Blasberg’s comments seem to correlate very well with the steady state situation as regards the different transport systems. They seem to agree very well indeed. D. B. TOWER: But is it not true that if you pre-incubate brain slices for 30 minutes under optimal conditions you reach a steady state? You are simply putting in a tracer to see what is going on. Is that not correct? A. LAJTHA: No, it is not tracer amounts that were added in Dr. Blasberg’s experiment at 30 minutes. We wanted to measure uptake rather than exchange. It is interesting that endogenous amino acid levels in brain slices d o not disappear if you incubate them for 30 minutes. You get an ATP level that is about half of the in vivo levels, but the amino acid levels seem to be about the same in the incubated

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R. G . B L A S B E R G

slice as they were in the living brain. This high endogenous pool was the reason for our using 2 mM amino acids in the incubation medium. The level reached in the slice with 2 mM medium concentrations is way above the endogenous levels, even after 2 minutes. Therefore 1 would not think that the exchange with the small amount of endogenous amino acid would be very significant. When there is a lower concentration in the medium - perhaps below 0.1 mM - then exchange with endogenous amino acids may contribute to initial uptake rates. Our measurements therefore, were mostly of net uptake. At present we are measuring exchange which shows somewhat different behavior. T. Z. CSAKY:I would like to ask Dr. Scholefield: what is the other source of energy if ATP is rejected? If there are other supplies of energy, how does digitalis act in inhibiting the active transport? P. G. SCHOLEFIELD: I think the key word in what I said is direct. I have been trying to find out whether ATP may not be the direct material involved. If, for example, phosphoprotein is involved, the ATP may cause a conformational change, and the conformational change of the phosphoprotein may be what is directly involved in the transport.

T. Z. CSAKY:And where does the ouabain enter into this whole picture?

P. G. SCHOLEFIELD: In the phosphorlation-of the phosphoprotein. P. MANDEL: But this is quite rapid. The phosphorylation of phosphoprotein is a very rapid phenomenon. P. G. SCHOLEFIELD: While we are discussing amino acids and cerebral systems I think the phenomena of the excitatory amino acids should be mentioned. There are extremely rapid interactions between amino acids and cerebral systems, whereby amino acids (including some which are not metabolized at all) can cause cell-firing within seconds or milliseconds of their application to the cerebral cortex in vitro; corresponding phenomena occur in vivo. 111 vitro this was shown to depend on a relationship between glutamic acid and sodium ions. The metabolic relationship between glutamic acid and cerebral systems in vivo and in vitro is already complex. The mechanism for this immediate provoking of cell-firing appears to be that glutamic acid increases permeability to sodium, and this also occurs within milliseconds. The acid allows the entry of sodium, and this is through the initiation of part of the normal mechanism that causes cell firing. The membrane potential is lowered through entry of positive ions, and this can cause cell-firing in vivo. In vitro there occurs a diminution in membrane potential, and this sets in motion processes which normally include the entry of sodium. The system is normally restored by sodium extrusion: the sodium pump comes into operation. This accounts for the loss of creatine phosphate that takes place, and as Dr. Elliott mentioned some time ago, the entry of potassium and the entry of water afterwards are a secondary phenomenon to the action of the sodium pump, which is removing the sodium and bringing in potassium at the same time.

A. LAJTHA:I just wanted to say that, from what we know about the reactions in which ATP participates, and about ATP turnover, one would expect that the energy rich compounds always equilibrate fairly rapidly with each other. So probably the ATP levels fluctuate quite rapidly and in parallel fashion with an intermediate that, in turn, is, as Dr. Scholefield said, more directly utilized.

T. Z. CSAKY:I think it is very important, physiologically, that the ATP we talk about is not identical to the chemists’ interpretation. ATP is a system, an equilibrium between the adenosine compounds and creatine phosphate, and it is fairly rapidly turned over. So, physiologically, when we talk about ATP, what we talk about is really higher energy phosphates.

259

A Possible Enzyme Barrier for r-Aminobutyric Acid in the

Central Nervous System N l C O M. V A N G E L D E R Departirient of Physiolog??. Fuatlty of’ Medicitre Uiriversity of’ Moiiireal, Montreal, @(e. (Canada)

In 1958 van Gelder and Elliot demonstrated that high concentrations of y-aminobutyric acid (GABA) in blood did not result in an elevation of brain GABA levels. Their work indicated that, as is the case for many compounds, a barrier exists which restricts the passage of GABA from blood to nervous tissue. The nature of the barrier is unknown, but recent histochemical studies on the distribution of GABA-a-ketoglutarate transaminase in the mammalian central nervous system (van Gelder, 1965a, b) suggested that the barrier may be in part a reflection of high GABA-transaminase activity in the walls of cerebral blood vessels and in the cells lining the cerebrospinal fluid spaces (see below). In order to explore this suggestion further, a study was made of the distribution of GABA-transaminase in kidney and liver. GABA appears to pass freely from blood to kidney, since as much as fifty per cent of the total GABA injected can be recovered from urine (van Gelder and Elliott, 1958). Similarly, the concentration of GABA in liver after injection parallels that of the blood. In view of these findings, an enzyme barrier similar to the one observed in the central nervous system should not exist in these organs. The present results indeed suggest that no such barrier prevents GABA from being filtered by the kidney from blood since very little GABA-transaminaseappears to be present i n either the blood vessels of the kidney or the capsule surrounding this organ. In liver also, GABA-transaminase does not appear to be concentrated particularly around blood vessels, and it may even be present in below normal concentrations whenever the vessels are surrounded by hepatic connective tissue. METHODS

The histochemical procedures for the localization of GABA-transaminase have been described previously (van Gelder, I965a). Frozen sections of brain are incubated in a 0.5 % agar-saline medium (pH 7.4) having the following composition: Nitro BT, 2.0 mg/ml; NAD, 2 mg/ml; (1-ketoglutarate, 5 mg/ml; GABA, 5 mg/ml. Incubation is carried out at 40” for 10 min i n a moist chamber. The method is based on the fact that, in the course of the metabolism of GABA to succinic acid, NADH is formed, which in turn reduces a tetrazolium salt (Nitro BT) RcWenic’s p. 268

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to its formazan form. Formazan is insoluble and will precipitate out at sites in the tissue where the combined presence of GABA-u-ketoglutarate transaminase and succinic semialdehyde dehydrogenase have caused the conversion of GABA to succinic acid. For further details of the procedure as well as determination of the specificity of the reaction for GABA metabolism, see van Gelder, 1965a and 1966. RESULTS

When sections of mammalian brain are incubated with a transaminase medium (see Methods), strong formazan precipitation occurs at sites where GABA-transaminase and succinic semialdehyde dehydrogenase are present in combination (van Gelder, 1965a). Although the presence of both enzymes is a prerequisite for the reaction, recent studies (van Gelder, 1966) suggested that the transamination step is ratelimiting with respect to the histochemical demonstration of GABA metabolism. The ependymal cell layer which lines the cerebrospinal fluid spaces is among the sites in the central nervous system which exhibit strong GABA-transaminase activity. This is demonstrated, for example, in Fig. I , which shows the central canal in the

Fig. 1. GABA-u-ketoglutarate transarninase in central canal of mouse. A heavy zone of formazan precipitation, indicative of strong transaminase activity, is present in ependyrnal cells which line the cerebrospinal fluid space. Activity is directed towards the lumen of the canal. These results apply to all cerebrospinal fluid spaces and are also obtained with brain tissue of monkey or rabbit. Section (10 p ) through cervical region ( x 400 magnification).

cervical region. The heavy layer of formazan in the ependymal cells which is directed towards the lumen of the canal indicates that GABA can be rapidly metabolized in this region. Similar results are obtained in other areas of the cerebrospinal fluid

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Fig. 2. GABA-transaminase activity in mouse cerebral blood vessels. 2a = Control: section incubated in a medium lacking GABA. 2b = Experimental: consecutive section incubated in the same medium after GABA had been added. Enzyme activity, visualized by formazan precipitation, surrounds all blood vessels and is also present in the pia-arachnoid. a = artery; bl = blood vessels; pa = pia-arachnoid. Section (10 p ) through mouse brain stem ( x 400 magnification).

spaces, where the choroid plexus is also found to show a pronounced histochemical reaction. With the possible exception of very large blood vessels, a layer of GABAtransaminase activity appears to surround cerebral blood vessels as well. Fig. 2 shows two consecutive brain sections, one of which was incubated with a control medium lacking GABA while the other was incubated in the same medium after Rcfiwnces p. 268

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GABA had been added. As illustrated here, the production of formazan is dependent on the presence of GABA (van Gelder, 1965a), but once GABA metabolism has been initiated by the addition of GABA to the medium, the resulting tetrazolium reduction is heavily concentrated around cerebral blood vessels of various sizes, as well as the pia-arachnoid (pa). In sections incubated in this manner, most capillaries appear to be outlined by a formazan precipitate. Results such as these suggest that diffusion of GABA from the cerebrospinal fluid spaces or from the blood must occur through a zone of strong GABA-transaminase activity. It would appear that these zones act as a barrier against the penetration of exogenous GABA into the brain parenchyma. In order to investigate the distribution of GABA-transaminase in kidney and liver, which do not appear to possess a barrier for GABA, frozen sections of these organs were incubated with the same medium as that used for the brain sections. Figs. 3a and

Fig. 3. GABA transaminase activity in mouse kidney. A relatively large artery (a) is shown, partially surrounded by proximal tubules (p). Control section (3a) exhibits a certain degree of non-specific tetrazolium reduction, but enhancement of staining in proximal tubules of experimental section (3b) indicates the presence of GABA-transaminase. Little increase in formazan production has occurred in artery of experimental section, as compared to that of control section. Section (10 p); ( x 400 magnification).

4a illustrate that, unlike brain tissue, kidney sections will reduce tetrazolium salts to some extent in the absence of GABA. Such reduction, therefore, cannot be ascribed to the presence of GABA-transaminase. However, after incubation of sections in the medium to which GABA had been added, a strong enhancement of formazan production was observed in certain areas. This enhancement was especially noted in the proximal tubules (p), which are therefore believed to possess considerable GABA-

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Fig. 4. Glomerulus (9) in mouse kidney surrounded by proximal tubules (p). The glomerular parenchyma is stained about equally in control (4a) and experimental (4b) section, indicating low GABA-transaminase activity at this location. Compare to surrounding proximal tubules.

Fig. 5 . Section through mouse kidney showing cortex (c) and outer medulla (m). Only proximal tubules in the outer medulla exhibit appreciable GABA-transaminase activity. Portions of the tubules in the cortex or inner medulla as well as kidney blood vessels (bl) are devoid of enzyme activity. Connective tissue capsule (ca) surrounding the kidney is also not stained. Section (10 p ) ; ( x 100 magnification). Rrfercnces p . 268

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transaminase activity. In contrast, little enhancement in formazan production was seen in blood vessels. This is shown in Fig. 3b and Fig. 4b, which respectively represent a relatively large artery (a) and the glomerular tuft (g) surrounded by proximal tubules (p). In both photographs, no striking difference in formazan precipitation is seen in the blood vessels of the experimental sections as compared with those of the control sections. It is especially noteworthy that little GABA-transaminase activity appears to be present in the capillary tuft of the glomerulus, since it is at this site that GABA must pass into the kidney tubules. Fig. 5 represents a kidney section incubated in the transaminase medium. It shows that neither the connective tissue capsule surrounding the kidney (ca), nor the kidney cortex (c) are stained for GABA-transaminase activity. Only the outer medulla (m), which is composed primarily of the distal portion of the proximal tubules and the descending and ascending loops of Henle, shows appreciable enzyme activity. Close examination of this area revealed that the loop of Henle exhibits no tetrazolium reduction, beyond that found in control sections. The inner medulla is also not stained by GABA-transaminase activity, nor are the walls of the blood vessels (bl). Finally, to determine the specificity of the histochemical reaction for GABAtransaminase in kidney, hydrazinopropionic acid (1 5 mg/kg) was injected into a mouse and its kidney was sectioned two hours later. Such sections, when incubated in the transaminase medium, were identical in appearance to normal sections which had been incubated in a control medium lacking GABA. Hydrazinopropionic acid, which was recently synthesized in this laboratory, is a very strong and probably quite specific in vivo and in vitro inhibitor of GABA-transaminase (to be published). This compound is a close structural analog of GABA. The results obtained with liver sections are to a large extent similar to those obtained with kidney sections. In this organ too, a certain degree of tetrazolium reduction occurs which is not dependent on GABA-transaminase activity (Fig. 6a). The formazan precipitate which is formed in these control sections has a pin-point appearance (Fig. 6b), which is not found in the kidney. The addition of GABA to the medium will result in a considerable enhancement of formazan formation as shown in Fig. 6c. The exact localization of this tetrazolium reduction, due to GABA-transaminase activity, is difficult to determine because of the background of non-specific formazan precipitation. However, Figs. 7a and 7b clearly show that transaminase activity in the connective tissue cells (ct) surrounding hepatic blood vessels is low as compared with hepatic parenchyma, while the bile ducts (bi) appear to have a high activity. The low transaminase activity in hepatic connective tissue is in sharp contrast to the high enzyme activity which surrounds cerebral blood vessels. DISCUSSION

Both Dobbing (1961) and Lajtha (1962), in reviewing studies on the blood-brain barrier, concluded that in addition to anatomical barriers, metabolic processes had to play an important role in preventing a variety of different compounds from penetrating into the central nervous system. Similarly, Barrnett and co-workers in a long

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Fig. 6. GABA-transaminase in mouse liver. As in the kidney, non-specific tetrazolium reduction occurs in control section (6a) which has a characteristic pin-point appearance (6b; x 400). After addition of GABA to the medium (6c) sections exhibit enhanced formazan production. Connective tissue surrounding blood vessels (bl) has low transaminase activity (see Fig. 7). Section (10 p ) ; ( x 73 magnification).

series of histochemical studies (e.g., Rostgaard and Barrnett, 1964; Marchesi and Barrnett, 1965) suggested that enzymes located at membrane surfaces aided in the transport of substances across such membranes. This suggestion presumably implies that, in the course of being transported, compounds are altered in some manner (Rostgaard and Barrnett, 1964). Hrfcwnci.s p. 268

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Fig. 7. Two hepatic veins in an experimental section. Connective tissue (ct) surrounding veins is noticeably lower in GABA-transaminase activity than liver parenchyma. High enzyme activity is seen in bile ducts (bi). Non-specific tetrazolium reduction (Fig. 6b) partially obscures localization of GABA-transaminase in parenchyma. Section (10 p ) : ( x 400 magnification).

The present histochemical data is in agreement with the above observations, since they suggest that in the process of diffusion across cerebral blood vessels or across the cerebrospinal fluid spaces, GABA can be rapidly converted to succinic acid. Little information is available regarding rates of diffusion of amino acids at these sites, but the results of Kuttner, Sims and Gordon (1961) indicate that this process may be slow. Using a-aminoisobutyric acid, which is metabolically inert, these

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authors reported that equilibration of the concentrations between plasma and brain required up to 10 h. This slow diffusion process would presumably allow ample opportunity for the destruction of GABA in passage from blood or cerebrospinal fluid to brain (see also Levin, Garcia Argiz and Nogueira, 1966). The situation i n kidney appears somewhat different, since very little destruction of G ABA seems possible during passage through the glomeruli (Figs. 3 and 4). The rate of removal of GABA from the blood by the kidney would therefore depend simply on the glomerular filtration rate, which varies from approximately 6.5 ml/min/kg in rat to about 120 ml/min/1.73 m2 in man. The results suggest that once GABA has entered the proximal tubule, a certain percentage will be destroyed by GABAtransaminase present in this part of the tubule. The amount destroyed should depend on both the concentration of GABA in the filtrate and the flow rate through the tubule. Once past the descending loop of Henle, the subsequent redistribution of water in the more distal portion of the kidney tubule would tend to concentrate the remaining G ABA. This would account for the high concentrations of GABA which are found in kidney and urine after intraperitoneal or intravenous injections of GABA into animals (van Gelder and Elliott, 1958). The distribution of GABA-transaminase in liver indicates that here, too, no particular barrier exists for the penetration of GABA into this organ. On the contrary, the concentration of the enzyme in connective tissue cells surrounding the blood vessels appears to be below normal when compared to the liver parenchyma or the bile ducts. While the histochemical data as well as pharmacological studies (van Gelder, 1966) tend to support the concept of a metabolic barrier for GABA, it must be pointed out that biochemical evidence so far islacking. Attempts to demonstrate entrance of GABA into the central nervous system of mice (van Gelder, 1966) and rats (Fisher, Hagen and Colvin, 1965) by in viva inhibition of GABA-transaminase with aminooxyacetic acid have been unsuccessful even after injection of as much as 1500 mg/kg of GABA (unpublished data). Possibly, indirect biochemical evidence may have been provided by several studies in recent years which have shown that injections of radioactive amino acids result in labeling of brain constituents without a concomitant net increase in their brain levels (e.g., Bed, Lajtha and Waelsch, 1961 ; Gaitonde, 1965). Contrary to prediction, such data indicated that the specific activities of expected metabolites from these amino acids were often higher in brain than the specific activity of the original amino acid injected, This led to the suggestion that several independent metabolic compartments for the same amino acid exist in brain. If the concept of a metabolic barrier is extended to other amino acids, the simplest explanation of this discrepancy would indeed be that such amino acids are metabolized while passing from blood or cerebrospinal fluid to nervous parenchyma. Bed, Lajtha and Waelsch (1961) have in fact suggested that the compartment for amidation of administered glutamate is located in the cerebral surface membranes or the ependymal lining. More recently, Levin, Garcia Argiz and Nogueira (1966) reported that ventriculo-cisternal perfusion of labeled GABA (or glutamate) in cats resulted in the appearance of radioactivity in the periventricular tissue (3-4 mm deep). At the same time no net increase in the concentration of this amino acid could be demonstrated R~~/r.rcncrs p.1268

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at these sites. These findings are also in accordance with the histochemical demonstration of GABA-transaminase in the ependymal lining of the cerebrospinal fluid spaces. In conclusion, the available data, when taken together, seem to support the suggestion that various enzymes, localized in cells bordering blood and cereborspinal fluid spaces, form a barrier towards the entrance of exogenous amino acids into the central nervous system. It is clear, however, that more direct biochemical evidence must be forthcoming before such an important concept can be accepted. ACKNOWLEDGEMENTS

This work was supported by a grant from the National Multiple Sclerosis Society. I wish to thank Miss Ann Larratt for her valuable assistance. REFERENCES BERL,S., LAJTHA,A. AND WAELSCH, H.(1961) Amino acid and protein metabolism - VI. Cerebral compartments of glutamic acid metabolism. J. Neurochem., 1, 186-197. J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. DOBBING, FISHER, M. A., HAGEN,D. Q. AND COLVIN,R. B. (1966) Aminooxyacetic Acid: Interactions with Gamma-Aminobutyric Acid and the Blood-Brain Barrier. Sci., 153, 1668-1 670. GAITONDE, M. K. (1965) Rate of Utilization of Glucose and 'Compartmentation' of a-Oxoglutarate and Glutamate in Rat Brain. Biochem. J., 95, 803-810. 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, 311-317. LAJTHA,A. (1962) Neurochemistry. Illinois. Charles C. Thomas. LEVIN,E., GARCIAARGIZ,C. A. AND NOGUEIRA, G. J. (1966) Ventriculocisternal perfusion of amino acids in cat brain - 11. Incorporation of glutamic acid, glutamine and GABA into the brain parenchyma. J. Neurochem., 13, 979-988. MARCHESI, V. T. AND BARRNETT, R . J. (1964) The localization of nucleosidephosphatase activity in different types of small blood vessels. J. Ultrastruc. Res., 10, 103-1 15. ROSTGAARD, J. AND BARRNETT, R. J. (1964) Fine structure localization of nucleoside phosphatases in relation to smooth muscle cells and unmyelinated nerves in the small intestine of the rat. J. Ultrastruc. Res., 11, 193-207. VAN GELDER, N. M. (1965a) The histochemical demonstration of y-aminobutyric acid metabolism by reduction of a tetrazolium salt. J. Neurochem., 12, 231-237. -, (1965b) A comparison of y-aminobutyric acid metabolism in rabbit and mouse nervous tissue. J. Neurochem., 12, 239-244. -, (1966) The effect of aminooxyacetic acid on the metabolism of y-aminobutyric acid in brain. Biochem. Pharmacol., 15, 533-539. VAN GELDER, N. M. AND ELLIOTT,K. A. C. (1958) Disposition of y-aminobutyric acid administered to mammals. J. Neurochem., 3, 139-143.

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DISCUSSION Did you use a-ketoglutarate for the transamination reaction? H. KOENIG: N. M. VAN GELDER: The transamination reaction requires GABA plus a-ketoglutarate. One then gets succinic semi-aldehyde and glutamate.

H. KOENIG:Glutamate dehydrogenase has exactly the same histochemical distribution as your enzyme (Koenig and Barron, 1962, Acta Neurol. Scan& Suppl., p. 72). This enzyme system, strategically located at the Blood-Brain Barrier, might serve the function of protecting the brain from ammonia, because glutamic dehydrogenase brings about the reductive amination of a-ketoglutarate to form glutamate. (Koenig, 1964, in "Morphological and Biochemical Concepts of Neural Activity", Cohen, M. M. and Snider, R. S. (Eds.), p. 39, Hoeber, New York). N. M. VAN GELDER: 1 would like to point out that there are many enzymes localized in the ependymal cells: e.g., succinic dehydrogenase. H. KOENIG: Glutamate-dehydrogenase occurs not only in the perivascular glial cells but also in the neuropil. Did you look at grey matter for this enzyme?

N. M. VAN GELDER: I did not look for glutamate-dehydrogenase.

P. MANDEL:The first problem is, I think, to demonstrate that there are enzymes which recognize glucose in the intestine; then you can be sure that glucose is going across the intestinal epithelium. You also need enzymes which are able to destroy the substance or complex because it is not the albumin that is going through.

N. M . VAN GELDER:I am not saying that albumin can. The point is how to demonstrate the glucose enzymes as such. If you look at the intestine, it appears that glucose goes through, but it is phosphorylated in the process of penetrating, and dephosphorylated before it enters the blood. P. MANDEL:But anyway, it isgoing through! N. M. VAN GELDER: Yes, but I am suggesting that if glucose arrives in the blood as, e.g., succinic acid, it is just as good a substrate.

P. MANDEL: The second remark is that if you purify GABA-transaminase 100-fold, you also have glutamate-transaminase activity because you cannot separate one from the other. Even with several methods of electrophoresis you always have the two enzymatic activities together. VAN GELDER: But what you are measuring here is not glutamate-transaminase. I have tried these same brain sections with glutamate, and one does not get the tetrazolium reduction with glutamate as substrate, at least not under the experimental conditions employed.

N. M.

P. MANDEL:Perhaps it is because it is altering the cell. Anyway, even if the enzyme is purified, the two activities remain together. A. LAJTHA:For the removal of GABA in the reaction sequence you discuss, you would need an equivalent amount of ketoglutarate to transaminate with. I wonder if there is enough ketoglutarate present in the brain, or whether enough can be generated, especially in experiments in which you administered significant amounts of GABA. As you know, ketoglutarate levels in the brain are rather low; and although enough enzyme may be present at the barrier site you propose, one of the substrates may not be there in sufficient amount to allow the reaction to go to completion.

N. M. VAN GELDER: I agree with you that there has to be a direct biochemical proof before one can really accept this type of blood-brain barrier. I found it attractive, and it struck me just from the distribution of GABA transaminase that an enzymic barrier may exist.

D. B. TOWER:I have two questions. One is in regard to the other tissues where, if memory serves

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me correctly, Eugene Roberts found a GABA-transaminase in large amounts. How do your histochemical qualitative observations coincide with the quantities which he has found for enzyme-activity in the liver and kidney and elsewhere? And the second question relates to the question of the entry of GABA into the CSF, since this is one place where we measured it. We did not use radioactive GABA. GABA was given to human subjects, in doses of several millimols per kg and measured in the blood and in the CSF by Robins’ method which has some disadvantages. 1 think, however, since it was confirmed by paper chromatoaraphy it is fair to say that we were actually measuring GABA. In normal subjects it is quite true that you cannot demonstrate any GABA entering into the central nervous system, but in certain patients with epilepsy you can show this. My question is: if this barrier that you have proposed is the mechanism which normally keeps GABA out of the central nervous system following a systemic administration, what would you anticipate has happened in these seizure patients to modify the barrier? I don’t expect a specific answer to this question, but I wonder if you have thought about this problem. It is a puzzling problem because it relates to a very important fundamental question, namely the modification of the barrier systems. We are going into that tomorrow, but here is one example which is already presented to us, where in certain seizure patients (not all) there is apparently a modification of whatever the mechanism is that normally excludes GABA. N. M. VAN GELDER: Offhand I can suggest at least two possible explanations. First of all: a lower substrate level, assuming for the moment that there is enough, and I am not saying that there is, but assuming that there is enough u-ketoglutarate in normal brain tissue, then in the seizure areas there may just not be enough a-ketoglutarate available to allow for transamination. A second possibility may be that in many of the seizure patients one finds abnormal circulation which may completely alter the permeability of the blood vessels, or even the transaminase distribution in these blood vessels; but I don’t know anything about this at the moment. With respect to the second question, the chemical results in liver and kidney appear to correlate well with the biochemical data of Roberts. C. F. BAXTER: We have some experimental evidence on the movement of GABA out of the cerebral ventricles which would tend to corroborate Dr. van Gelder’s findings. In collaboration with Dr. M. Rubinstein we have found that there is also a high concentration of GABA metabolizing enzyme around the ventricular walls in the rat. The technique used was virtually identical to that described by Dr. van Gelder. In other experiments we have injected stereotaxically 1 [14C]GABA into the lateral ventricle of rats. Five to 45 minutes after the injection, the rats were frozen in liquid nitrogen and radioautographs prepared of frozen brain sections. These autographs showed that the movement of radioactivity from the ventricle to the more distal parts of the brain was surprisingly slow. At the same time 1 [“TIGABA injected intraventricularly, is rapidly metabolized (Baxter, 1963). It is quite possible, therefore, that deep penetration of radioactivity from 1 [14C]GABA into cerebral tissues is hindered by the rapid metabolism of the carboxyl group of this amino acid in areas adjacent to the ventricle. This concept agrees with Dr. van Gelder’s observations. In the mouse it is more difficult to show slow penetration from the ventricle into brain tissues except into the cerebellum. However, it is apparent that there is an effective barrier which prevents the movement of GABA out of the ventricle into the rest of the body. This barrier is compromised if the vascular bed of the brain tissue is damaged during the injection procedure. This finding is illustrated by the figure.

We injected mice intraventricularly with 2 [WIGABA using the Haley technique (1957). After various time periods each mouse was quick frozen, embedded in methyl cellulose and radioautographs made of longitudinal microtome sections. These techniques were developed in Sweden (UW berg, 1954). The top third of the figure shows a typical section. The middle third shows a radioautograph which grossly indicates the distribution of 2 [I4C]GABA injected 25 minutes earlier. It can be seen that radioactivity is almost exclusively confined to the central nervous system and parts of the spinal cord. A little activity in the urine (bladder) can also be detected. There is very little radioactivity in liver and kidney. This result should be compared with the bottom radioautograph in the figure. In this experiment the intracerebral injection of the isotope was accompanied by rupture of a small cerebral blood vessel, observable by the fact that a little blood was on the injection needle when it was withdrawn.

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Under these conditions, a substantial amount of radioactivity in liver and kidney was detectable within 5 minutes after intracerebral injection. These incidental findings clearly illustrate some of the difficulties encountered when attempting to determine the "normal" rates at which substances move in vivo within the brain and from the brain into other body tissues. REFERENCES BAXTER,C. F. (1963) Cerebral Metabolism of Some Amino Acids in v i v a Fed. Proc., 22 : 301. HALEY,T. J., AND MCCORMICK, W. C. (1957) Pharmacological Effects Produced by lntracerebral Injection of Drugs in the Conscious Mouse. British J. Pharinacol. Chetiiother., 12 : 12-1 5. U L L B ~ RS. G ,(1959) Autoradiographic Studies on the Distribution of Labeled Drugs in the Body. Progress in Nitclear Energy - Series 6,22,29-35. N. M. VAN GELDER:I find Dr. Baxter's observation very interesting. One would almost suspect that if the barrier works in one way, structures forming the barrier may just as well work in the other way in the reverse direction. This would support the fact that even when you load up GABA-levels in the brain to fantastic amounts, you don't find anything in the blood. As a matter of fact, you don't find much GABA in the CSF either. So it looks as if the barrier would indeed work both ways. All 1 am saying is that the localization of GABA transaminase seems to be compatible with an enzymic barrier but I am not suggesting that it is the only barrier. G. PAPPAS:I just wanted to ask Dr. French if he has any comments to make about the action of rhodanase, or anybody else, about rhondanase and thiocyanate?

C . M. FRENCH: It is an enzyme which breaks or converts thiocyanide, and it seems to have an equilibrium position such as the cyanide. It is present in very small concentrations, probably 0.1 mequiv./l, whereas the thiocyanate will be at something like I 0 0 rnequiv./l. This was the point I was making when asked what causes the definite ion movement; that very small quantities of thiocyanate d o get converted to cyanide. The quantity is so small that it does not affect the analysis of how much thiocyanate there is present. On the other hand, it may have a marked effect on the function of the cells. Kogan has suggested that this is the cause of the reduced arterial venous oxygen difference. He suggested that cyanide is blocking oxygen uptake. If thiocyanate i s converted to cyanide, it is an indication of course, that the thiocyanate can become intracellular, and it would support the concept that some cells are resistant to cyanide. It occurs mainly in vitro in red cells of rats. I have been able to show a conversion of thiocyanate into cyanide in vitro, but I have not been able to show this in vivo.

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Ion Movements

Ion Movements in Isolated Preparations from the Mammalian Brain HENRY Mcl LWAIN Department of Bioclieriiistry, Institute of Psychiatry (British Postgraduate Medical Federation. University oj’lotidon), Maudsley Hospital, Denmark Hill, London, S. E.5 (England)

INTRODUCTION

This contribution is concerned mainly with the barriers or restrictions to diffusion which operate at the cellular level in the brain. At cellular level is manifested that manipulation of barriers which is the most characteristic property of any neural system. In about a millisecond, restrictions normally imposed on the movements of Na and K are greatly altered : diminished to a fraction of their previous value and then reasserted, these changes occurring in the defined sequence which produces the nerve action potential. Demonstration and understanding of such events need co-ordinated observations by chemical and electrical means, and these are greatly furthered by separating the neural system concerned from the organism of which it forms part, and examining it in vitro. This practice has long been established in relation to amphibian peripheral nerve and brilliantly applied to the giant axons of crustacea and squids. Application to the central nervous system may have been delayed by exaggerated opinions of the damage caused in preparing the tissue samples, and for this reason the following appraisal is given (Mcllwain, 1956; Mcllwain and Joanny, 1963). A P P R A I S A L OF ISOLATED TISSUE P R E P A R A T I O N S FROM THE B R A I N

To obtain portions of the brain for biochemical work, a usual practice is to prepare sheets about 30-150 mg in weight and 0.35 mm in thickness. This allows access at their outer surfaces to materials normally exchanged with the blood stream at the cerebral capillaries. Note that this implies immediately that one category of barrier phenomena important in vivo is eliminated in using isolated tissues suspended in aqueous media; those operating between the blood capillaries and the extracellular fluids of the brain. Such simplification is a major reason for using in vitro techniques. In particular, opportunity is given in vitro for direct observation of events between extracellular and intracellular phases, which in vivo may need indirect computation. In appraising such systems it is valuable to know their area of cell surface (i) in reRrfirenrrs p. 280

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TABLE I C O M P U T E D AREAS IN CEREBRAL C O R T I C A L SPECIMENS

Data

(i) Area of external surfaces mmz (ii) Cut area, mrn2 (iii) Neuronal area (partial), mm2 (iv) Cut area, as approx. % of neuronal area (v) Capillary area, mmL

Valites, per 100-nig sample 600 300 82,000

0.1 1,000

Notes

Cut 0.35 mrn thick

-

Rabbit cervical vagus, 60,000 mm2/100mg Giant axon, 0.5

-

Most data are approximate only, and for rat, guinea pig or rabbit: for further details, including the parts referred to in line (iii) see text.

lation to the external area of tissue sample, and (ii) in relation to the area of capillaries which normally traverse the tissue sample. Data summarized in Table I involve the following considerations. (i) A tissue slice of 100 mg, prepared 0.35 mm in thickness from the surface of the brain, has an external area of about 600 mm2, of which 300 mm2 has been formed by cutting. In the cerebral cortex of the rabbit, SchadC and Baxter (1960) calculated the volume and surface area of neuronal components from microscopical measurements. The cell body, apical and basal dendrites with branches (but not including the axons leaving the cortex) gave total areas of 850 mm2/mm3 of tissue. At a specific gravity of about 1.04 (Thudichum, 1884), this corresponds to 82 600 mm2/100 mg. The measured portions of the neuronal surface thus presented an area over 270 times the total cut area. The elements which had been measured totalled only about 1 1 % of the volume of the cortex, and it is thus likely the newly-cut surface represents no more than 0.1 % of the cell-surfaces of the tissue. The cell bodies, it may be noted, formed only 4 % of the volume of neurones. Data consistent with this, but indicating the variation occurring in different cortical areas and in different species, are given by Scholl (1956), Tower (1954) and Heller and Elliott (1954). In a preponderantly non-myelinated mammalian nerve, the rabbit cervical vagus, Keynes and Ritchie’s (1965) data indicate that as used in experiments involved with ion movement, the specimens contained non-myelinated fibers, at a density of a similar order, namely one which totalled 60 000 mm2/mg. Comparison with giant axons as investigated by Hodgkin and Keynes (1955, 1956) indicates that axons 0.2-0.6 mm in diameter and 4 to 10 cm long have been used. Taking 0.5 mm diameter and 50 mm length as typical sizes, the area of the two ends of a cylinder of this magnitude is found to be 0.5 % of the area of the curved surface. (ii) The external area of tissue samples, prepared from the mammalian brain by sectioning at 0.35 mm intervals, approaches that of the blood capillaries which they carry. Estimates of capillary area can be made from the data of Cragie (1938); they are approximate only. In the rat brain the average capillary diameter was 2.9 p, and the average length of capillaries in the cerebral cortex was 1 100 mm/mm3. This gives

ION MOVEMENTS IN MAMMALIAN BRAIN

275

the value of c 10 mm2/mm3 for capillary area per unit volume, which is included in Table I. For variation in different parts of the brain and other circumstances see Cragie (1938) and Horstmann (1960). Thus the external surfaces which must form the route of supply to tissue sections of the type described, are quite similar in area to that of the capillary walls which form the route of supply in vivo. This is understandable, for diffusion factors must condition the biological structure and are also involved in the calculations which lead to choice of the dimensions of the tissue samples. E L E C T R I C A L E VI D E N C E

In most neural systems the observations which required the supposition of rapidly adjustable ion-barriers, were first made electrically. Electrical observations with isolated cerebral tissues may therefore be described first, though they are of relatively recent development and come largely from our own laboratory. The phenomena observable depend greatly on the part of the brain which is sampled and maintained in vitro. The piriform lobe is the most versatile of these so far examined; its position in the rat or guinea pig brain is described by Allison (1953), Cajal (191 I , 1955) and Valverde, 1965). These data indicate that a sample taken superficially from the surface of the lobe, as a sheet 0.3 mm or more in thickness, can be expected to contain the fibres ofthe lateral olfactory tract, and also much ofthe neurons on which these fibres terminate. For in vitro experiments, we(Yamamoto and McIlwain 1966; Mcllwain 1966) have cut sheets 0.35 mm thick, for these can still be adequately oxygenated, and sufficiently large to include most of the surface of a piriform lobe of the guinea pig: that is, the portion medial to the rhinal fissure. These are about 7 x 14 mm, and 35 mg in weight. They have been immersed in bicarbonate-buffered balanced salt mixtures, with glucose and equilibrated with 0 2 - 5 %COz, in the slice chamber of Gibson and McIlwain (1965). After 20-30 min preincubation, incubating fluid has been withdrawn so that the tissue rested on a nylon grid, now at the surface of the liquid and with its upper surface in the moist gas phase. On this upper surface, formerly part of the ventral surface of the brain, the lateral olfactory tract could be seen and two stimulating electrodes were placed at its anterior end. Effects of applying to these electrodes brief, isolated pulses of rectangular time-voltage relationships were then examined by placing recording electrodes on the tissue. With extracellular recording electrodes on the lateral olfactory tract itself at distances of I to 10 mm from the stimulating electrodes, the first brief response to effective stimuli was biphasic and travelled along the tract at 12 m/sec (Yamamoto and Mcllwain, 1966). This was concluded to represent conduction along the myelinated fibres of the tract itself. A response of different characteristics, however, preponderated when the recording electrodes were moved away from the tract and placed on the surface of the pre-piriform cortex. This was a negative wave of much longer duration (10-20 msec), which was of maximum amplitude about 1.5 mm from the tract. The conducted response was lost at distances more than about 0.7 mm from the tract. Response to paired and repetitive stimuli, and also the effects of some inhibitory agents, suggested that the negative wave represented a post-synaptic potential. Ri~erc~ PJnp. ~ 280

276

H. M C I L W A I N

In several characteristics the responses now observed in vitro resembled those seen in the piriform cortex in vivo on stimulation of the lateral olfactory tract (MacLean et a/., 1957). The magnitude of respiratory responses suggested that stimulation at the lateral olfactory tract affected an appreciable proportion of tissue beyond the tract itself (McIlwain, 1966). Electrical activities such as those described normally imply appreciable resting potentials at the excitable cell membrane, detectable by intracellular electrodes. Ample evidence for this has been obtained in samples isolated from the mammalian brain. Observations made with micropipette electrodes showed (Li and McIlwain, 1957; Hillman and McIlwain, 1961; Gibson and Mcllwain, 1965) resting membrane potentials of about -60 mV in mammalian neocortical samples, relative to extracellular fluids. Moreover, the ionic basis for these potentials was adumbrated by observing their requirement for sodium and diminution on increasing the extracelM a r potassium concentration. Spike potentials were observed in vitro in such tissues, which involved a transitorily positive potential at the peak of the spike. It therefore appears likely that the sequence of changes in Na and K permeability, established in other neural systems, operates in the cerebral samples in vitro. Because of the opportunity which such systems afford for obtaining fundamental data, they have been explored by chemical methods now to be described. T A B L E I1 ION C O N C E N T R A T I O N A N D D I S T R I B U T I O N I N I N C U B A T E D C ER EBR A L N EOC OR TEX

Values based on: Constituent

Potassium: [Klr, mM Ratio [Kl{/[Klp Sodium: [Nali, m M Ratio [Na]i/[Na]p Chloride: [Clli, mM Ratio [Cl]i/[Cl]c

( a ) Passive distribution of CI-

( b ) lnirlin deterniitiation

136 22 41 0.3 1 14.5 0.108

131 21 53 0.35 33 0.25

Data from Gibson and Mcllwain (1965), for guinea pig cerebral cortex after incubation in media which contained (mM): NaCI, 124; KCI, 5; KHzPO.1, 1.24; MgSOJ, 1.3; CaCIz, 2.6; NaHCOz, 26 and glucose, 10 in equilibrium with 5 % COz in 0 2 . Suffix i, intracellular; e, extracellular. Values ( a ) based o n observed membrane potential, at which passive distribution of CI- gives Cle/ CIi of 9.3. Values (b) assume that all the extracellular space and none of the intracellular space is accessible to inulin. CHEMICAL ANALYSIS A N D ISOTOPIC MEASUREMENTS

Direct chemical analysis of samples of cerebral tissues which have been maintained satisfactorily in vitro, shows that among the major diffusible ions the tissues are enriched in K and are poorer in Na and CI than the fluids in which they are incubated.

ION MOVEMENTS I N MAMMALIAN B R A I N

277

This gives an immediate demonstration of some of the most important barriers in cerebral tissues and also qualitative basis for some of the electrical phenomena just described. Quantitative statements need knowledge also of the amount of extracellular space in the tissue. In tissues as complex structurally as are those of the brain, calculation of the extracellular space involves simplifying assumptions, a matter discussed more fully in other sessions of this meeting. Ion gradients and resting potential Two different assumptions are used in alternative calculations of the data of Table I1 and these give consistent accounts of the distribution of Na and K ; that of C1 is less satisfactory. Among these ions the effects of K must preponderate in normal, unstimulated cerebral tissues, for the resting membrane potential is immediately affected by change in Ki and to a smaller extent and more slowly by change in Na or CI. However, the equilibrium potential corresponding to a K gradient of 21 or 22 (Table 11) is -83mV, markedly more negative than the observed-60mV. Presumably, therefore, the Na gradient is normally partly effective. With the relationship (i) derived from constant-field equations : (i)

v=-

RT

F

In

+ b “la, + c [CIle [Kli + b “ l a i f c [Clli

[Kle

(see Hodgkin and Katz, 1949) and for the present ignoring the C1 gradient, the K and Na values of Table I can be used to calculate a value for b. This factor gives the permeability of the tissue to Na as a ratio of its permeability to K+, and the value obtained is 0.06. Thus the resting tissue may be concluded to present a greater barrier to Na than to K . Values obtained for the squid axon, normal or perfused but also unstimulated were of 0.01-0.08 (Baker et al., 1962; Hodgkin, 1965), but on excitation the value can rise momentarily to 20, corresponding to the positive overshoot at the peak of the action potential. Following addition of KCI, the time-course of the diminution of membrane potentials in cerebral tissue was measured sufficiently accurately to merit comparison with calculated values (Gibson and McIlwain, 1965). Calculation was based on a diffusion equation and on equation (i). Quite close agreement was found when b was taken as 0.06, c as 0.5 and allowance made for diffusion of KCI in the available extracellular space. Net movements on stimulation Alterations in many metabolic properties of isolated cerebral tissues occur on electrical stimulation in vitro (see McIlwain, 1966; McIlwain and Rodnight, 1962). Included are increase in tissue Na and decrease in K. These changes which are in the direction of the concentration gradient for each ion show some simple relationships to characteristics of the applied electrical pulses (McIlwain and Joanny, 1963). The stimuli, ofcharacteristics similar to those which in vivo produce motor or other responRcyerences p. 280

278

H. M C l L W A l N

T A B L E 111 ION MOVEMENTS I N I N C U B A T E D C E R E B R A L N E O C O R T I C A L S A M P L E S

Movement

Tissue not stimulated 42K influx and efflux 24Nainflux and efflux Tissue maximally stimulated Net loss of K 42Kefflux Net entry of Na “Na influx

Rate

-~

( i ) Observed, pequiv./glh

( i i ) Computed pequiv.lcmz/h

330-400 175-21 5

45 21

240480 600-150 460 1050-1 I80

44 82 56 136

~-

Tissues were preincubated underconditionssimilar to thoseofTable I1 and reached stable composition before electrical stimuli were applied or isotopically-labelled salts were added; data from Cummins and McIlwain (1961), Keesey and Wallgren (1965) and Keesey et a / . (1965). A range of values in column (i) implies different conditions of measurement, and mean values have been used in calculating the data of column (ii). Column (ii) in addition uses the value of 82000 mm2 of neuronal surface1 100 mg of tissue, derived as explained in the text.

ses from the cerebral cortex, may be applied to electrodes which surround the tissue samples while they are immersed in media such as that quoted in Table 11. With stimuli of rectangular time-voltage relationship, the Na and K movements increased with duration of pulse between 0.03 and 0.4 msec; threshold corresponded to potential gradients of about l V/mm. Applied at frequencies between 2 and 30/sec, the stimuli resulted in initial changes of Na and K which were approximately equal and of between 5 and 6 mpequiv./g tissue/pulse (Table 111). A similar rate of K movement per pulse was subsequently observed (Keynes and Ritchie, 1965) for a mammalian non-myelinated peripheral nerve : the rabbit vagus. Movements of Na and K shown isotopically The applicability of equation (i) to data from isolated cerebral tissues implies that the tissues are permeable to the ions concerned, but does not indicate the magnitude of such permeability. Measurements using salts of 24Na or 42K show that an appreciable proportion of the tissue content of Na and K can undergo exchange each minute (Table 111). Entry of 24Na into the cellular compartments of incubated cerebral tissues was found readily separable from its movements into the extracellular phase (Keesey and Wallgren, 1965; Keesey et al., 1965). The greater part of the intracellular Na exchanged rapidly, at some 250 pequiv./g tissue/h; only in small proportion exchanged less freely. The electrical stimulation which altered the tissues’ net content of Na also altered greatly its rate of movement. The rate of turnover increased 4-6

ION MOVEMENTS IN M A M M A L I A N B R A I N

279

fold and became about 1100 pequiv./g tissue/h, implying an exchange of about one third of the intracellular Na each minute. I t will be noted that stimulation caused an immediate increase in Na efflux, even though the net movement of the ion was inwards; emphasizing that stimulation did indeed increase the permeability of the tissue to the ion, rather than prompting a unidirectional transfer. Comparable phenomena were found also in relation to potassium ions (Table 111; Cummings and McIlwain, 1961). In neocortical samples from the guinea pig, the normal flux of K+ was greater than that of Na+ and was increased by electrical stimulation. Again, stimulation increased both influx and exflux of 42K although the net result was loss of K. Applied pulses caused increased efflux of K from tissues maintained under a variety of conditions, including some conditions under which maximal gradients in K were not maintained. Thus in absence of added glucose, when stimuli did not increase tissue respiration or glycolysis, they nevertheless diminished tissue K. This gives support to the view that increase in ion permeability is a primary result of stimulation rather than its resulting from the major metabolic changes which usually accompany it. COMMENT

Electrical and ionic events of tissues from the mammalian brain are thus similar in several respects to events in simpler neural systems. Much can be ascribed to permeability barriers which show specificity to simple ions, and which can be caused to undergo equally specific modification by chemical and electrical means. The chemical nature of the cell membrane at the points at which it is penetrated by Na and K requires specification in cerebral as in other excitable tissues. In the more thoroughly-investigated systems, characterization of the groups interacting with Na and K still does not go far: the probable involvement of multiplycharged sites at some of which Ca may play a regulating role (Hodgkin, 1965). Certain actions of Ca in peripheral neural systems are reproduced in the brain and an examination of Ca in relation to excitation of cerebral tissues showed that it conditioned tissue Na and K content (Lolley, 1963; Lolley and McIlwain, 1964). Polybasic and polyacidic peptides and lipids also interact in conditioning tissue excitability and ion content (McIlwain et a/., 1961). A proposal relating ion passage to membrane potential postulated pores lined by molecules which carried acidic groupings on chains a few carbon atoms in length (Mcllwain, 1963). Others have related the sequence of Na and K movements to ion displacement at acidic groupings at the outer and inner cell surfaces (Blank, 1965); further chemical characterization may come from the susceptibility of cerebral ion movements to protoveratrine, to local anaesthetics and to tetrodotoxin (Wollenberger, 1955; McIlwain and Joanny, 1963; Hillman el al., 1963 and McIlwain, 1967). The manner in which the Na and K gradients normal to the brain have beenestablished, is deliberately not approached in the present account. The energy-consuming, pump mechanisms merit discussion at a meeting comparable to the present one for the utilization of energy-rich compounds which is necessary to active Na and K Rrfirences p . 280

280

H. M C I L W A I N

movements is giving valuable insight into the processes involved. I t is to be noted, however, that in the maintenance of many differential concentrations in neural systems the role of energy-assisted movements may preponderate over that played by barrier phenomena. REFERENCES ALLISON, A. C. (1953) Biol. Rev. (Cambridge), 28, 195. BAKER, P. F., HODGKIN, A. L. AND SHAW,T. I. (1962) J. Physiol., 164, 355. BLANK,M. (1965) J. Coll. Sci., 20, 933. CAJAL,S. R. Y (1911, 1955) Studies on the Cerebral Cortex, translated Kraft, L. M. Lloyd-Luke, London. E. H. (1938) Proc. Ass. Res. Nerv. Mental Dis., 18, 3. CRAGIE, CUMMINS, J. T. AND MCILWAIN, H. (1961) Biochem. J., 79, 330. GIBSON,I. M. A N D MCILWAIN, H. (1965) J . Physiol., 176, 261. HELLER, I. H. AND ELLIOTT,K. A. C. (1954) Canad. J . Biochem. Physiol., 32, 584. HILLMAN, H. H., CAMPBELL, W. J. AND MCILWAIN,H. (1963) J. Neurocheni., 10, 325. HILLMAN, H. H. AND MCILWAIN, H. (1961) J. Physiol., 157, 263. HODGKIN, A. L. (1965) The conduction of the nervous impulse. University Press, Liverpool. HODGKIN, A. L. AND KATZ,B. (1949) J . Physiol., 108, 37. HODGKIN, A. L. AND KEYNES, R. D. (1955) J . Physiol., 128,28. - (1956) J. Physiol., 131, 592. HORSTMANN, E. (1960) in: Structure and function of the cerebral cortex. Tower and Schadk (Eds.). Elsevier, Amsterdam (p. 59). KEESEY, 5. C. AND WALLGREN, H. (1965) Biochem. J., 95, 301. KEESEY, J. C., WALLGREN, H. AND MCTLWAIN, H. (1965) Biocheni. J., 95, 289. KEYNES, R. D. AND RITCHIE, J. M. (1965) J . Physiol., 179, 333. LI, C. L. A N D MCILWAIN, H. (1957) J. Physiol., 139, 178. LOLLEY, R. N. (1963) J. Neurochem., 10, 665. LOLLEY, R. N. AND MCILWAIN, H. (1964) Biochem. J., 93, 12P. MCILWAIN, H. (1956) Physiol. Rev., 36, 355. - (1966) J. Physiol., 185, 65P. - (1966) Biochemistry and the central nervous system. 3rd ed. Churchill, London. - (1967), J. Physiol., 190, 39 P . MCILWAIN, H. AND RODNIGHT,R. (I962) Practical Neurochemistry, Churchill, London. MCILWAIN, H. AND JOANNY, P. (1963) J. Neurochem., 10, 313. R. J. AND CUMMINS, J. T. (1961) Biochem. J., 81, 79. MCILWAIN, H., WOODMAN, MACLEAN, P. D., ROSNER, B. S. AND ROBINSON, F. (1957) Amer. J. Physiol., 189, 395. SCHADE,J. P. AND BAXTER, C. F. (1960) Inhibition of the Nervous System and y-Aniinohutyric Acid, E. Roberts et a/. (Eds.). Pergamon, New York. SHOLL,D. A. (1956) Progress in Neurobiology, 1, 324. J. A. Kappers, Editor. Elsevier, Amsterdam. THUDICHUM, J. L. W. (1884) A Treatise in the chemical constitution o j t h e Brain. Bailliere, Tindall & Cox, London. TOWER,D. B. (1954) J. Comp. Neurol., 101, 19. VALVERDE, F. (1965) Studies 011 the Piriform Lobe. Harvard University Press, Mass. YAMAMOTO, C. AND MCILWAIN, H. (1966) Nature, 120, 1055. WOLLENBERGER, A. (1955) Biochem. J . 61 68.

I O N M O V E M E N T IN M A M M A L I A N B R A I N

28 1

DISCUSSION D. B. TOWER:I think all of us owe Dr. Mcllwain a debt of gratitude. What you saw today represents many years of work, and much thought in devising these very ingenious experiments. For those of us who work with incubated slices it is gratifying to see how close one really comes in vitro to the situation in vivo. I t is of tremendous value to be able to do a number of observations in v i m which one could not do otherwise with confidence. Obviously the best preparation is the living brain in situ, but this presents many technical problems in studying the questions which we are considering hcre. I f you can work at them in isolation, and vary one parameter at a time as Dr. Mcllwain has indicated, you can really gain a great deal of understanding. With that little complement I would like to ask one question now, and that is about the chloride: We have done some calculations similar to yours about the potentials that one would expect, based on passive distribution of chloride in comparison to caiculations with the Hodgkin-Huxley type of equation. There are some peculiar abnormalities with chloride as you suggested in one slide, especially when one deals with a medium in which you alter some of the ionic constituents. There seems to be a disparity between the resting potentials which one would expect for a distribution of chloride on a passive electrochemical basis. 1 wonder if you have thought about this enough to give us some ideas, because frankly, I am puzzled as to how to explain some of the apparent behavior that chloride shows in these in vitro preparations. H. MCILWAIN: We hope to make a more detailed study of chloride, but we have not yet done so. The problems posed by chloride are being examined in several systems. Those who are studying muscle, for example, have shown somewhat similar features in potassium and chloride distribution. It is still debated whether there is an active distribution of chloride or a passive one. Because of tte cellular complexity of cerebral tissues, I hesitate to draw a conclusion from our data. K. A. C. ELLIOTT: I really wanted to say more or less the same thing as Dr. Tower. I am very impressed with this work. and the stage in this work which Dr. Mcllwain has reached. I feed that over the years the electrophysiologists have tended to dismiss Dr. Mcllwain’s approach, perhaps because it was done by a biochemist, or perhaps because it was done on a tissue slice. I think that the sort of work he is doing now is going to bring us to the highly desirable stage of marrying electrophysiology to biochemistry. We will reach the stage where the writing of the recording pen will not just be. telling us about an electrical phenomenon which means nothing; it will be an index of a chemical event, which is what we want. Dr. Mcllwain has already led to this with a lot of hisearlier studies. R. KATZMAN: One of the problems that has arisen in understanding the electrolytes in the central nervous system has been the very large amounts of sodium in brain, as compared to other tissues. I t has been postulated that this has been extracellular or in the glia, but your evidence would indicate that this is intraneuronal. If the sodium is this high within the neurons, this would indicate that the action potential ought to have a very small overshoot. Have you in fact measured the action potential, and does it have a small overshoot? H. MC~LWAIN: The overshoots were small in our preparations. Our preparations contain a higher concentration of sodium ions than in vivo, even though we have looked very carefully a t the medium constituents and so forth, in order to analyze this. It is fairly certain t o be due in part to sectioning of the tissue, but it is often overlooked that the brain in vivo has some 20-25% of extracellular space. R. KATZMAN: In other words; you feel that this may not necessarily reflect what is going on in vivo in terms of the sodium concentration. May this be in part due to the incubation situation then? H. M C ~ L W A IINthink : we do get a high sodium content, and this is due not so much to the particular fluid that we useirr vitro, but is contributed by the obvious fact that the tissue has been sectioned, and we actually gain sodium beyond that found in normal preparations in vivo.

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283

Cation Exchange in Blood, Brain and CSF ROBERT KATZMAN, L E O N A R D G R A Z I A N I

AND

STANLEY GINSBURG

Saul R . Korey Departnierit of Neiirology, Albert Einstein College of Medicine, Bronx, N . Y. (U.S.A.)

In the presence of marked changes in serum and body electrolytes the constancy of brain and CSF cations, K, Ca, and Mg, has been repeatedly confirmed (Bradbury et al., 1963; Katzman, 1966; Kemeny et al., 1961; Leiderman and Katzman, 1953; MacIntyre and Davidson, 1958; Oppelt et al., 1963a,b; Schain, 1964; Wallach et al., 1964). In the case of these cations, one factor in maintenance of the brain level may be the slowness of exchange across the blood-brain barrier. Thus, injected 42K, which exchanges with most body tissues within minutes, was shown by Katzman and Leiderman (1953) to exchange very slowly with brain K. Twenty-four to 36 h were required for half of the brain K to exchange, the rate of 42K influx being about 3 mequiv./kg/h in a brain containing 100 mequiv./kg of total K (Table I). Adrenalectomy which alters body and muscle electrolytes and causes a rise in serum electrolytes of over 50% did not alter the influx rate or total K. However, it is well established that CSF K is maintained independently of serum K. The value for CSF K of 3 mM is remarkably constant among species of mammals and some invertebrates extending back to sharks (Rall and Cserr, unpublished observations), even though the serum concentrations of the varying species are quite different. Such constancy of CSF K would certainly have survival value, if this represented the extracellular fluid K, for then cell membranes would have a rather constant environment biasing their membrane potentials. In searching for the mechanisms underlying this constancy of CSF K, early experiments were carried out in which the specific activity of the CSF was compared to that of plasma after intravenous and intracisternal injections (Table 11). It is apparent that equilibrium between CSF and plasma is reached much more rapidly than between brain and plasma. Moreover, much of the intracisternal injection is carried rapidly from the CSF, appearing both in plasma and in adjacent brain, particularly the medulla. With the development of ventriculocisternal steady state perfusions the movement of isotopes between plasma, CSF, and brain can be more accurately analyzed and the effects upon such movement of various factors such as serum concentration, inhibitors, etc. studied. Although ventriculocisternal perfusions have been utilized for many years particularly by Leusen (1950) and by Bhattacharya and Feldberg (l958), the application of this method to the measurement of ion flux has been developed largely by Pappenheimer and his associates (1961, 1962), by Heisey (1962), and by Davson ReJiwncrs pp. 293-294

284

R. K A T Z M A N et

al.

and Pollay (1963). Pappenheimer el al. (1961) introduced the use of inulin dilution as a measure of the rate of formation of CSF occurring during the ventriculocisternal perfusion. During a steady state ventriculocisternal perfusion at the inflow rate, Vt, ml/min, assuming that inulin does not diffuse across ependyma, glia, or arachnoid, then Vf, the newly formed CSF can be measured by inulin dilution

v,

=

vs

(Ct - CO)

ml/min

/.(

L O

(Heisey et al., 1962) where Ct is inflow concentration of inulin, and Cois outflow concentration of inulin. The rates of formation of CSF measured by this method varies from 0.0096 in the rabbit (Davson and Pollay, 1963) to 0.37 in man (Rubin et al., 1966). Recently, TABLE I* EFFECT O F ADRENALECTOMY O N BRAIN

K

AND

K - F L U XI N

Kbr

No.

Normal Adrenalectomy

* From Leiderman and Katzman,

Influx

mequiv./L

mequiv./kg we, weigh, nieqctiv./kg/h

5.48 f 0.1 7.60 f 0.2

100.9 f 0.9 98.7 f 1.2

Kpl

30 18

RATS

2.89 2.97

1953.

Ames et al. (1965a) have demonstrated that the rate of CSF produced is related to the pCO2 of the blood. We have, indeed, found in a series of cats perfused during nembutal anesthesia without artificial respirations, that the Vf of 0.022 was almost 20% greater than the Vf of 0.016 observed in anesthetized animals of the same size in whom artificial respiration had been carried out and in whom hyperventilation had probably occurred (Graziani, in preparation). The second use of inulin data has been the determination of inulin clearance as a TABLE 11* SPECIFIC ACTIVITY O F

CSF

A N D BRAIN AFTER I N T R A C I S T E R N A L A N D I N T R A V E N O U S I N J E C T I O N SO F

Intravenous Intracisternal

2h 10 min 60 rnin 100 rnin

42K I N

RATS

S A CSFISA plasma

SA brainlSA plasma

0.63 30,000 85

0.085

* From Katzman and Leiderman, 1953.

S A medullalSA C S F

0.03 0.97

C A T I O N E X C H A N G E IN B L O O D , B R A I N A N D

CSF

285

measure of perfusate absorbed or diverted into the subarachnoid space, Va. During a ventriculocisternal perfusion, some perfusate may not be recovered through the outflow catheter but instead may be diverted into the subarachnoid spaces and later absorbed at the arachnoid villi. Va must be known in order to carry out further calculations; it may be determined easily, since it is identical to inulin clearance. Assuming, inulin is removed from cerebrospinal fluid spaces as part of bulk absorption of CSF at Va ml/min, and steady state perfusion at inflow Vt ml/min, outflow Vo ml/min then

v,

vr Cr - v o co =

ml/min

CO (Heisey et al., 1962) With the measurement of Va, it now becomes possible to study ion flux by adding isotope either to the ventricular perfusate or giving the isotope intravenously. In the succeeding paragraphs, the equations used in the study of this ion flux are explicitly presented. Although several of the equations may appear to be trivial, their inclusion has become necessary in order to show step by step how coefficients derived from isotopic data can be used to measure ion flux per se. This is important in view of the criticisms of isotopic data recently expressed by authors such as Nims (1966).

Eflux coeficient With the addition of an isotope to the simulated spinal fluid, the efflux of this isotope into brain and plasma can be expressed as a clearance coefficient as shown by Cserr (1964, 1965) and Katzman et a/. (1965). Thus, for an isotope C* the efflux equation becomes k e f f l u x C* = (kvp kVBr) C* = Vi Ci* - Vo Co* - Va Co* (1) where kcfPlux is the ml of perfusate cleared of C*/min; is the average concentration of C* in the ventricle (taken arbitrarily as either the simple arithmetic average of Ct* and Co* or as the exponential mean); Vi is the perfusion rate in ml/min; V, is the cisternal effluent i n ml/min, and Va is the measure of the perfusate absorbed elsewhere in the system as determined by the inulin clearance. This efflux coefficient can be separated into components kvp and kvBr representing the movement into plasma and brain, respectively.

+

c*

Transport coeficient, plasma to ventricle

Provided the efflux coefficient has already been determined, the movement of an isotope from plasma to ventricle can be measured as a clearance coefficient of the plasma. During steady state ventriculocisternal perfusion with non-radioactive perfusate, if C* is perfused intravenously and assuming that brain uptake of C* from plasma is negligible then the following material balance holds R+rences

p p . 293-294

R. K A T Z M A N et al.

286

+

+

k p v C*P = Vo C*o Va C*o k e f t l u x C* (2) where k p v is the transport coefficient from plasma to ventricle, or clearance of c* from plasma in ml/min.

Isotopic j u x e s

The model we are dealing with is a 3 compartment system in which a constant flow is maintained through the middle compartment, the CSF.

In this system, we ignore the exchange that must occur between plasma and brain. At steady state the system is described by 6 unidirectional isotope fluxes, J * , J*r J*PV J*BV = J * o J*VP J*VB -(3) where J * t = Vr C*r, J*o = ( V o Va) C*o,the sum of J * V P J*VB = kefrlux c*, J*PV = kruriux C*P. An approximation which permits a separate calculation of JVP and JVB is obtained by determining the ratio of counts remaining in the brain and the counts lost to the plasma during a recovery experiment following isotopic perfusion. To determine JEW*, we use the fact that the brain is not appreciably labeled during the influx measurements. If our perfusate is initially free of both isotope and parent species, then the non-radioactive isotope which appears in the effluent can be treated as if it were a tracer coming from the brain. Here,

+

J*BV = J*PV

+

+

+

)1

+

+

(S.A.) effluent

S.A. effluent

(S.A.) plasma

S.A. plasma

(1 -

(4)

where S.A. is the specific activity in counts/min/moles, (C*/C). All of the isotopic fluxes are now calculated. Flux of parent species The unidirectional fluxes between compartments I and 2 ofparent species in moles/min may be determined simply by utilizing the general expression that J12 = J * i 2 / ( S . A . ) i

(5)

where J is the flux in moles/min, and S.A. is the specific activity in counts/min/mole. We may then rewrite equation (3) Jr JPV J B ~ V= J o JVP JVB (6) Among the simplifying assumptions in this analysis are the following: I . During the time course of the experiments inulin does not diffuse appreciably into the brain or blood vessels.

+ +

+ +

CATION EXCHANGE IN BLOOD, B R A I N A N D

CSF

287

2. That the rate of exchange of isotope between plasma and brain is negligible in the time course of the experiment. This is probably a reasonable assumption for K and Mg, less so for Ca, and not valid for Na. 3. The interfaces between CSF and brain and CSF and plasma are each treated as homogeneous entities even though it is evident they include ependymal surfaces, pia-glial surfaces, etc. We have applied this analysis to the study of the exchange of K (Katzman et a/., 1965), Mg (Ginsburg and Katzman, in preparation), and Ca (Graziani et a/., 1967) in the anesthetized cat. Similar studies for K have been carried out by Cserr (1965) and Bradbury and Davson (1965) with comparable results. In determining the fluxes, the clearance of isotope from the perfused fluid, kcflux, must be measured first, since this value is needed for the later calculations. The values obtained for clearance of 42K, ZRMg, and 45Ca during ventriculocisternal perfusion T A B L E 111 C A T I O N F L U X FROM P E R F U S A T E T O P L A S M A A N D B R A I N

No. of animals

K" Ca" MgC

7 9 4

Rate of perJision. Vi, nil/niin

0.19 0.19 0.09

coejicient

kernux

0.096

0.025 0.032

90-riiin Perfusion Counts % Counts recovered 0;

I0

in brain

21.9 4.8 8.0

kr1.

k\.Hr*

*

plasma'

15.3 8.6 15.3

0.041 0.016 0.021

0.055 0.009 0.011

* Estimated as difference between total counts perfused and counts recovered in cisternal effluent, brain, and accounted for by Va. * * kvl%ris underestimated here, since re-entry of isotope into perfusate from adjacent brain tissue has not been taken into account. a Katzman et a/., 1965. Graziani et a/., 1965. Ginsburg and Katzman, 1966. in the cat are shown in Table 111. It is evident that the clearance of 42K is much greater than that of 4SCaand 28Mg, which are similar to each other. The value for the clearance of 42K, 0.096 ml/min, is 4 to 5 times the usual rate of formation of spinal fluid in the ventriculocisternal system of the cat. At the end of a perfusion experiment a portion of the isotope cleared can be found in the brain, the rest having entered the circulation. The distribution of the cleared isotope between brain and plasma is estimated by carrying out 90-min perfusions. The animal is then sacrificed, and the total percentage of perfused material recovered in the effluent and accounted for by Va and the per cent recovered in the brain are determined and then the amount entering the plasma is estimated by difference. Here again, a different pattern for K emerges where a greater proportion enters the brain, than for Ca and Mg, where the greatest proportion is lost to the plasma (Table 111). The pattern of movement of the isotope into brain tissue is shown in Fig. 1 for 42K. Rrfercnccs pp. 293-294

288

R. K A T Z M A N et al.

Fig. I . Distribution of isotope in cat brain following 90-min ventriculocisternal perfusion with perfusate containing 42K. The numbers represent the per cent of total counts in entire perfusate received from each region of the brain.

As was expected, the highest concentration is immediately adjacent to the ventricles,

but it should be also noted that there is very considerable recovery of the material from the base of the brain stem. It would appear that the isotope can move equally well across the ependymal surface into the brain stem and across the subarachnoid, pia-glial surface. This is a useful situation ir. which to compare the movement across these two surfaces, since the brain stem is more or less cylindrical, and the surface areas of the ventricle and of the base are at least of the same order of magnitude. With the determination of the efflux coefficient, the rate of the unidirectional flux can be calculated. However, this in itself gives no information about the mechanism of transport. Information can be obtained about mechanisms by determining the unidirectional fluxes or efflux coefficients while parameters such as cation concentration are varied. One can also use such measurements to determine the effect of inhibitory agents such as ouabain. The effects of variations in K, Mg, and Ca concentrations of the perfusate upon clearance of the respective isotopes have been previously reported (Katzman et a / . , 1965; Ginsburg et al., in prep.; Graziani et al., 1965) and are summarized in Table 1V. Ineach instance, theclearancecoefficient is independent of the mean CSFconcentration of cation. Hence, the flux of the cation from the CSF is directly proportional to this concentration. This is consistent with but does not establish the existence of a simple diffusion process. Once the efflux coefficient has been determined, the influx coefficient, kpv, can be obtained by utilizing equation (2). From this value and the serum concentration, the influx JPVcan be calculated. Values of Jpv obtained for K, Ca, and Mg are shown in Table V. By determining the specific activity of the effluent and assuming little labeling of the brain during the time course of the perfusions, the influx JBV can also

C A T I O N E X C H A N G E I N BLOOD, B R A I N A N D

CSF

289

TABLE IV

K, Ca,

I N D E P E N D E N C E O F C L E A R A N C E C O E F F I C I E N T S , kernuS, A N D C O N C E N T R A T I O N S O F AND

0- I

3- 4 9-10

0.91 0.95 0.89

Mg

IN PERFUSATE

0 -0.08 1.2-1.7 1.8-2.5

0.026 0.021 0.024

0 4.04 0.08-1.2 2.1 -2.5

0.049 0.038 0.047

be calculated. Again, the flux of K is much greater than that of Ca and Mg. The flux of K from brain to ventricle is greater than from plasma to ventricle, however, the reverse is true for Ca and Mg. The effect of alterations in plasma concentrations of the cations upon kpv and upon the flux from plasma to ventricle is very different from that of the effect of alterations of CSF concentrations upon keeflux and flux from ventricle to plasma and brain. In studies of Mg exchange, our initial data indicate that the flux from plasma to ventricle is independent of plasma cation concentration; under the circumstances, kpV is inversely proportional to plasma concentration. I n studies of Ca exchange, the influx coefficient has been found to be a hyperbolic function of the plasma concentration (Graziani et al., 1966). Flux from plasma to TABLE V U N I D I R E C T I O N A L F L U X OF C A T I O N I N P E R F U S E D C A T

JPV pM/niin J I N pM/rnin Jv I’ J v 15

0.121 0.178 0.039 _K 0.057 K

0.026 0.013 0.021 Mg 0.01I M g

0.026 0.015 0.016 0.009 Ca

The units of the coefficients are nil/min. K, etc. average concentration of K in ventricle in pM/ml.

ventricle could be resolved into a major component that was concentration independent and presumably represented movement via a carrier-mediated or active transport and another component which was proportional to serum concentration and represented a concentration-dependent, presumably diffusional, component. The equation found was: 0.0037 Cap1 JVP = k v p Cap1 = 0.0156 with 0.0156 pMlmin representing the carrier-mediated; 0.0037 (ml/min) Cap1 (,uM/ml) representing the diffusional transport. We have not carried out similar experiments with K, but the data of Ames et al. (1965b) on K content of the choroid plexus fluid as a reflection of seru I K concen-

+

Rtfi.ri*nc.cspp. 293-294

-

R. K A T Z M A N et al.

290

tration and the data of Cserr as reported in her thesis (1964) both indicate that the movement of K from plasma to ventricle is similar to Ca; largely a concentration independent flux with the addition of a small diffusional component. Thus, the evidence from these studies indicates an asymmetrical type of transport system in which the movement out of CSF acts as if it were diffusional, whereas the movement into CSF appears to be largely carrier-mediated. The interpretation of these findings in the case of K is made more difficult because of the known effect of K on the electrical gradient. It has been shown by Held et a/. (1964) that alterations of K concentration within the CSF alters the electrical potential between the ventricle and venous system. If this is so, the efflux of K should not depend solely upon the internal concentration of K, as it does, but should also be modified by the changing electrical gradient. The asymmetry of the exchange process cannot be understood in terms of a single membrane between blood and ventricle. It may be interpreted to indicate at least two membranes in series with different functional capacities. Thus, the efflux from CSF is across a membrane which acts as a diffusional barrier and is not the site of the electrical potential difference. However, the movement from plasma into ventricle is via some specialized nondiffusional process such as carrier mediation or active transport. The effects of inhibitors upon the system are in the process of being studied. When added in usual concentrations to the CSF some of the ordinary inhibitors such as dinitrophenol produce little effect upon ion transport, but perhaps this may be due to their inability to penetrate into essential regions. So far no attempts have been made to correlate simultaneously the effects of inhibitors upon ion transport and upon other functional parameters such as oxygen utilization by the ependymal or other cells. Most striking effects are produced by the introduction of ouabain in concentrations of 10-4 to lo-’ M. Here, there is a marked alteration in K exchange. The K concentration in the cisternal effluent becomes elevated. This elevation in effluent K is attributable both to a decrease in efflux of K from the CSF and an increase in movement of K from brain into CSF. Here, ouabain may exercise an effect upon cellular permeability as well as upon the active transport process. Acetazolamide reduces formation of spinal fluid by approximately one-half, but it has little effect upon the influx of K from plasma to ventricle. The effect of ouabain upon the movement of Ca and Mg is currently under study. In carrying out these calculations of isotope exchange the question may arise as to whether the values obtained describe transport of cation as such. This can be readily checked in the ventriculocisternal perfusion experiments, since the coefficients derived from isotopic studies can be applied to the prediction of the total concentration of cation, when the ventriculocisternal system is perfused with perfusates containing varying initial concentrations. In this computation one applies equation (7) substituting the flux coefficients as appropriate; Jt

+

JPV

4-J B ~ V= Jo f JVP

+

JVBr

C A T I O N E X C H A N G E IN B L O O D , B R A I N A N D

co =

CSF

29 1

Vi Ci iJ r v 1- J J ~~ vkeffit1.u Cip

Vi

+ Vf +

(7) krffiux/z

The values to be used in the numerical solution of this equation are those in Table V. In Fig. 2, the relationship of C, to Ci has been plotted for K, Mg, and Ca using this equation. In each instance, the actual measured values are also shown. It can be seen that there is an excellent fit of the equations to the measured values of Co in each instance. In addition, in the case of ouabain a good fit is also obtained. This would suggest that although ouabain alters both the influx K and the clearance coefficient, that the me-hanism in each case is not basically altered; that is, that efflux is still via diffusion, although at a different rate, and influx via a concentration independent system. The exchange of cations occurred throughout the entire ventricular subarachnoid space. In experiments in which the exchange coefficients had been determined in perfusions between lateral ventricle and aqueduct, lateral ventricle and fourth ventricle, and lateral ventricle and cisteriia magna, the value of the coefficients increases proportionately as the area of circuits exposed to the perfusate increases. This is shown in Table VI. Subarachnoid perfusions in which the area being perfused is difficult to estimate but which has been carried out between the parasagittal space and the cisterna TABLE VI C O M P A R I S O N OF E X C H A N G E C O E F F I C I E N T S I N R E G I O N A L P E R F U S I O N S

Ventriculocisternal Lateral 3rd 4th ventricle (kaolin prepdrdtion) Lateral 3rd ventricle (aqueduct) Ccrebrdl subarachnoid Cisternal-lumbar

0.092

0.026

0.011

0.041

0.022 0.002

0.008 0.0034 0.034 0.012

0.33 0.29

0.06

magna yield clearance coefficients that are several-fold greater than that of the ventriculocisternal perfusicn. A more reproducible system is that of cisternal lumbar sac perfusion in which appreciable exchange is shown along the subarachnoid space enclosing the spinal cord. Again, this is several-fold larger than that seen in the ventriculocisternal region, but the circuit’s area is considerably greater. In the cases of studies of Ca and Mg exchange, the flux between the subarachnoid ventricular space and brain is less than that between CSF and blood. In the case of K, however, there seems to be a very vigorous exchange between brain and CSF, and a slower exchange between brain and blood. This has raised the question as to whether the K i n the extracellular space of the brain may be in equilibrium with CSF K. If such an equilibrium existed, it would certainly be of considerable importance for R r J ; ~ r c n w spp.

293-294

al.

R. K A T Z M A N et

292 8-

76 -

Kout

(rnM/L)

I 0'

1

2

I

I

5 6 Kin ( m M/L)

4

3

I

I

7

2.5-

8

~ 9

10

~

I

I

11

..

_..'

,..' ./.

2.0 -

.. ,.

Ca, (rnM/Ll 1.5-

.I..

,..'

.*.I

* .*

1.0-

&."

..*' i

.:MlAfWOM

EXPERlMfNlS

Fig. 2. Relationship of cation concentration in cisternal effluentto cation concentration in ventricular perfusate. The calculated steady state curves derived from equation (7) utilized the numerical data in Table V.

C A T I O N E X C H A N G E I N BLOOD, B R A I N A N D

CSF

293

CSF K is maintained at the level of 3 mM in all mammalian and many vertebrate species, whereas the blood K may be 50% or more above this. In addition, the blood level is quite variable within different animals of the same species as well as in the same animal from time to time. The CSF level varies only between 2.6 and 3.2 mM. A low extracellular K maintained at a constant level could provide a bias for the resting membrane potential which would lead to a large K equilibrium potential E K and which would, therefore, provide a reasonable membrane potential, even if the membranes were somewhat leaky. It should be noted, however, that the hypothesis that an equilibrium does exist between brain extracellular K and CSF K, while consistent with all known facts, has not been directly determined at the present time. SUMMARY

It has been our purpose to set down in a formal way the simple equations describing isotopic flux during ventriculocisternal perfusion experiments and to relate these equations to the measurement of flux of the parent cation. The effect of variation in cation concentration upon cation flux has been measured. The present evidence suggests that K, Ca, and Mg are cleared from the ventriculocisternal system by a concentration dependent system consistent with simple diffusion. In contrast, the flux from plasma to ventricle is largely concentration independent, presumably involving a carrier-mediated transport system. This asymmetry indicates that the blood-CSF barrier is not a single membrane system. Instead, there may be membranes with different properties which are in series. Alternatively, influx and efflux of cations may occur at separate sites. ACKNOWLEDGEMENT

This work was supported by National Institute of Health grants NB-03356, NB01450, and 5T1 MH-6418, U.S. Public Health Service. REFERENCES

AMES,A., 111, HIGHASHI, K. AND NESBETT, F. B. (1965a) Effect of pC02 acetazolamide and ouabain on volume and composition of choroid plexus fluid. J . Physiol., 181, 516-524. - (1965b) Relation of potassium concentration in choroid plexus fluid to that in plasma. J. Physiol., 181, 506-515. BHATTACHARYA, B. K. AND FELDBERG, W. (1958) Perfusion of Cerebral Ventricles: Effects of drugs on outflow from the cisterna and aqueduct. Brit. J. Pharniacol., 13, 156-162. BRADBURY, M. W. B. AND DAVSON, H. (1965) The transport of potassium between blood, cerebrospinal fluid and brain. J. Physiol., 181, 151-174. BRADBURY, M. W. B., STUBBS, J., HUGHES, I. E. AND PARKER, P. (1963) The distribution of potassium, sodium, chloride and urea between lumbar cerebrospinal fluid and blood serum in human subjects. Clin. Sci., 25, 97-105. CSERR,H. F. (I 964) Excharige of Substances Between Ventricular Fluid, Plasma and Brain with Special Reference to Potassiuni. (Doctoral Thesis), Boston, Harvard University. -( 1965) Potassium exchange between cerebrospinal fluid, plasma and brain. Amer. J. Physiol., 209, 1219-1226. DAVSON, H. AND POLLAY, M. (1963) Influence of various drugs on the transport of and PAH across the cerebrospinal fluid-blood barrier. J . Physiol., 167, 239-246,

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GINSBURG, S. AND KATZMAN, R. (1966) Mg exchange between blood, brain and CSF in cat. In preparation. GRAZIANI, L., ESCRIVA, A. AND KATZMAN, R. (1965) Exchange of calcium between blood brain, and cerebrospinal fluid. Anier. J. Physiol., 208, 1058-1064. GRAZIANI, L. J., KAPLAN, R. K., ESCRIVA, A. A N D KATZMAN, R. (1967) Calcium flux into CSF from blood, brain, and spinal cord. Amer. J. Physiol. (in press). HEisEY, S. R., HELD,D. AND PAPPENHEIMER, J. R. (1962) Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Amer. J. Physiol., 203, 775-78 I . HELD,D., FENCL,V. A N D PAPPENHEIMER, J. R. (1964) Electrical potential of cerebrospinal fluid. J. Neitrophysiol., 27, 942-959. KATZMAN, R. (1966) Effect of electrolyte disturbance on the central nervous system. Ann. Rev. Me& 17, 197-212. KATZMAN, R.,GRAZIANI, L., KAPLAN, R. AND ESCRIVA, A. (1965) Exchange of cerebrospinal fluid potassium with blood and brain. Arch. Neurol., 13, 513-524. KATZMAN, R. AND LEIDERMAN, P. H. (1953) Brain potassium exchange in normal adult and immature rats. Amer. J. Physiol., 175, 263-270. KEMENY, A., BOLDIZSAR, H. AND PETHES, G . (1961) The distribution of cations in plasma and cerebrospinal fluid following infusion of solutions of salts of sodium, potassium, magnesium, and calcium. J. Neurocliem., 7, 218-227. LEIDERMAN, P. H. AND KATZMAN, R. (1953) Effect of adrenalectomy, desoxycorticosterone and cortisone on brain potassium exchange. Amer. J. Physiol., 175, 271-275. LEUSEN, 1. (1950) The influence of calcium, potassium, and magnesium ions in cerebrospinal fluid on vasomotor system. J. Physiol., 110, 313-329. MACINTYRE, 1. AND DAVIDSSON, D. (1958) The production of secondary potassium depletion, sodium retention, nephrocalcinosis and hypercalcaemia by magnesium deficiency. Biochem. J. 70,456-462 NIMS,L. F. (1966) Biologic barriers and material transfer. In: Head Injury, W. F. Caveness and A. E. Walker (Eds.), London, Lippencott. OPPELT, W. W., MACINTYRE, 1. AND RALL,D. P. (1963a) Magnesium exchange between blood and cerebrospinal fluid. Anier. J. Physiol., 205, 959-962. OPPELT, W. W., OWENS, E. D. A N D RALL,D. P. (1963b) Calcium exchange between blood and cerebrospinal fluid. Lqe Sci., 2, 599-605. PAPPENHEIMER, 5. R., HEISEY,S. R. AND JORDAN, E. F. (1961) Active transport of diodrast and phenolsulfonphthalein from cerebrospinal fluid to blood. Amer. J. Physiol., 200, 1-10, PAPPENHEIMER, J. R., HEISEY, S. R., JORDAN, E. F. AND DOWNER, J. (1962) Perfusion of the cerebral ventricular system in unanest hetized goats. Amer. J. Physiol., 203, 763-774. RUBIN, R. C., HENDERSON, E. S., OMMAYA, A. K., WALKER, M. D. AND RALL,D. P. (1966) The production of cerebrospinal fluid in man and its modification by acetazolamide. J. Neurosurg., 25, 430436. SCHAIN, R. J. (1964) Cerebrospinal fluid and serum cation levels. Arch. Neurol., 11, 330-333. WALLACH, S., BELLAVIA, J. V., SCHORR, J. AND REIZENSTEIN, D. L. (1964) Tissue distribution of electrolytes Ca47 and Mg** in acute hypercalcemia. Amer. J. Physiol., 207, 553-560.

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DISCUSSION

D. B. TOWER:I think it is only fair since I rose to congratulate Dr. MCILWAIN that I d o the same for Dr. Katzman. You have been in this particular field for a long time, and I think we owe quite a debt to you, and also to some of the later workers like Held, Cserr, and Pappenheimer who have contributed considerably to the techniques, but perhaps have not been interested in the nervous system. There is one point I would like to comment on, that Dr. Katzman passed over rather quickly, and that is the effect of ouabain. For many years people have been using ouabain in a sort of in vitro system. Not so very long ago we had a conference at the N.T.H., and the main question was: what does it do in vivo? Nobody seemed t o know. Shortly after that there came a publication from Dr. Katzman and I think almost simultaneously one from Dr. Cserr at Harvard, in which they gave an answer to our question. As you saw today, it does have an effect which is - at least qualitatively quite similar to what occurs in vitro. Also, if you give too much for too long a period of time, seizures occur, which is also quite interesting. L. BAKAY: I just want to add one thing: I think this is a most ingenious way to use perfusions for the calculations that were obviously impossible to obtain by injecting tracers into a system with a semi-stagnant cerebrospinal fluid. R. KATZMAN: I would like to point out that even though this kind of analysis leads to equations that do fit the data quite well for points which were not originally included in arriving at the coefficients, that in fact it is not really a pure system because of the existence of heterogeneous interphases. Within the brain we do not have compartmental mixing, but there is mixing in CSF and blood which is much better. L. BAKAY:The main disadvantage of injecting tracers directly into the cerebrospinal fluid system is their unequal distribution in that compartment. There is superficially a much higher activity than in the deeper structures, and the isotopes do not mix very well within a reasonable time along the entire nervous system. unless you use barbotage. By barbotage it can be mixed. However, you don't know quite how much artefact you have induced, because by barbotage you will necessarily reduce the pressure to almost zero when you withdraw the fluid, and involuntarily increase it when you inject it back. You made a remark that with the subarachnoid-cisternal perfusion method it is difficult t o assess the subarachnoid surface with which this fluid is in contact. This is quite true. I think in an animal like the rabbit or rat the effective subarachnoid surface in a short experiment could be considered just the plain surface of the brain, without any consideration for its gyral configuration. From data I have, the circulating cerebrospinal fluid did not seem to go into the sulci. The circulating fluid remained in contact with the top of the gyri so t o speak, at the surface of the brain-and did not go into the spacewithin the sulci very much. I also have very much admired Dr. Katzman's work and would like to ask him whether H. MCILWAIN: he has one particular application of it; that is: to understand the time course of spreading depression. As you know, the phenomena of spreading depression has been attributed to ion movements. One suggestion with respect to it is that there may be a diffusion of potassium ions extracellularly in the brain,*and you may have some information about the extent to which any potassium that is liberated extracellularly from a local source, is removed or diluted. I am wondering if the data fit in with your calculations. R. KATZMAN: T have not made that calculation. Brindley, et a/. (1960, Neurology, 23 : 246) have shown by a local perfusion of the cortical surface that when spreading depression occurs in the area being perfused, the potassium flux from the brain markedly increases. H. MCILWAIN:It would be valuable to try to do it, I think, because there are two candidates as possible intermediaries which are very different: that is glutamate ions and potassium salts.

H. KOENIG: T would like to add my plaudits, to both Dr. Mcllwain and Dr. Katzman. I think it is remarkable that despite the gradient from the ventricular ependymal surface to the depths of the

296

R. K A T Z M A N et

al.

brain, there is such an excellent penetration of radioactivity at 1 mm intervals? Was this your glutaraldehyde fixed preparation or were these frozen materials?

R. KATZMAN: They were frozen materials. H. KOEMG:Have you checked this with your gluteraldehyde-perfused material which you claimed retains and preserves quite precisely the ions as well as other macromolecular constituents? R. KATZMAN: The gluteraldehyde perfusion retained rather well the electrolytes in the brain - that is, within 5 % of the actual barrier. We were not taking any chances.

H. KOENIG:This opens the door to analysis of experimentally induced metabolic lesions which are known to affect neural excitability and function in general. I should like to say a word about the in vitro experiments. I had the privilege a few days ago of seeing an in virro preparation of the pyriform cortex, a system which permits the analysis of synaptic transmission in Professor Mcllwain’s laboratory. The effects of altering and manipulating the chemical environment can be investigated in such a system. There are some structural and biochemical abnormalities in brain slices maintained in vitro. For example, there are quite remarkable changes in the metabolism of purine and pyrimidine nucleotides in brain slices (Ramuz et al., 1964, J . Neurochem. 11 : 827). Despite these changes, neurons in tissue slices seem to behave properly, e.g., they have a proper membrane potential and so on. H. MCILWAIN: Some workers have been doing even more drastic things to neural systems: current studies with giant axons involve the cytoplasm from the interior of the neuronal membrane.

D. B. TOWER:I just want to make a little correction for the record. Dr. Elliott was the first to show a swelling in brain slices in 1946. J. P. SCHADE:In ouabain-treated preparations one finds a drop of the average membrane potential. This may, among other things, be explained by the decreased ATP-content of these brains. Do you have any idea about the membrane potential in your preparations? R. KATZMAN: I have no measurements of membrane potentials. We have not measured these during perfusion, but we have looked a t some behavioral changes. These occur rather rapidly during perfusion.

B. D. WYKE:I am sorry to persist, but there is just one question which worries me. It is really sort of supplementary to Professor Mcllwain’s question about the transcerebral potential. As I understood it from the slide that you just showed, this potential does not oscillate with changes in the potassium concentration in the perfusate. Yet one knows (Tschergi, R. D. and J. L. Taylor, 1958, Amer. J . Physiol., 195 :7; Wyke, B. D., 1963, Brain Function and Metabolic Disorders, London, Butterworths) that if one manipulates the hydrogen ion concentration either in the serum or in the CSF, this potential does change. Do you have any comment about this in relation to what you said about the independence of the transcerebral potential from potassium ion changes? Do you, for instance, get changes in the hydrogen ion concentration of the perfusate which might counteract the possible variations in transcerebral potential due to the potassium change?

R. KATZMAN: The potentials as shown by Dr. Held (Held et a/., 1964, J. Neurophysiol., 27 : 942) change with shifts in potassium very clearly. The point I was making was that with our flux-equations we can account for the changes in terms of concentrations without the electrical factor. There is nowhere that we need to put in a term for the electrical changes that are known to occur. This is very disturbing. The data we have, and the data that Dr. Cserr has, and the data from Drs. Bradbury and Davson are very similar. What seems t o be very reproducible is that the potassium flux looks as if it were simple diffusion from the inside. From the outside of the system it looks like it is a carrier system. This is probably due to the fact that there are membranes in series rather than a single membrane, and it is probably a very complicated system,

297

Distribution of Nonelectrolytes and Electrolytes in the Brain as Affected by Alterations in Cerebrospinal Fluid Secretion D l X O N M. W O O D B U R Y * Department of Pharrriacology, Uiiiversity of Utah College of Medicine, Salt Lake City, Utah ( U.S.A.)

One of the intriguing problems in the field of “blood-brain barrier” physiology is the relation between the cerebrospinal fluid (CSF) and the extracellular fluid of the brain. As has been described by Davson (1956, 1963, 1965) and others (Reed and Woodbury, 1963; Woodbury, 1965b), the CSF, because of its active secretion by the choroid plexus, its bulk flow through the ventricles and subarachnoid cavities, and its exit through the arachnoid villi, acts as a “sink” for substances entering the brain interstitial space from the brain capillaries across the “blood-brain barrier”. Because the transfer rate of most substances across the capillary-brain interface is slower than their rate of egress from the CSF by bulk flow, their concentration in the interstitial fluid of the brain is lower than plasma, but higher than in CSF, provided that the substances do not enter the CSF across the choroid plexus or, if they do, provided that they enter at a rate slower than that across the brain capillaries. Thus, indicator substances normally used to measure extracellular fluid volume cannot be used to measure brain interstitial volume because of this fact. An additional factor that maintains a lower concentration of substances in brain and CSF than in plasma is their active transport out of the CSF across the choroid plexus. Such substances as monovalent inorganic anions, probably potassium, and organic anions and cations are transported out of the CSF in this manner. It is the purpose of this paper to summarize the effects of bulk flow of CSF and of active transport across the choroid plexus on the distribution of nonelectrolytes and electrolytes in the brain. In Fig. I , taken from the work of Ferguson and Woodbury (1967), is shown schematically a summary of the various factors involved in transfer of substances into and out of the brain and CSF as discussed above. In young animals the flow of CSF through the arachnoid villi is sluggish but progressively increases as the choroid plexus matures. Its maturation occurs at about 3-8 days after birth. There is a large extracellular space in the brain of young animals with little or no barrier to entry of substances from the plasma, as will be shown below. Increasing CSF flow, barrier formation between blood and brain, and decreasing extracellular space all contribute to decreased concentration of substances in the brain and a lowering of the brain/

* Recipient of a Public Health Service Research Career Award (No. 5-K6-NB-13,838) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. Ri!Termcrs p p . 312-313

D. M. W O O D B U R Y

298

PLASMA

J

RACH NO ID ViLLI

CHOROI PLEXU

1

PLASMA

r

Fig. 1 . Schematic drawing of the various factors that regulate the exchange of substances between plasma, brain, and cerebrospinal fluid in young as compared with adult rats. The filled arrows indicate the movement of solutes and the open arrows the flow of cerebrospinal fluid. The width of the arrows is proportional to the rate of flow of the solutes across the various boundaries. The thicker boundary line at the brain-plasma interface of the adults as compared to the young is an indication of the greater development of the “blood-brain barrier” in the older group. See text for further discussion. (From Ferguson and Woodbury, 1967.)

plasma ratio for substances found in the interstitial and cellular spaces. However, the brain/CSF ratio continues to estimate the extracellular space as long as no barrier forms between brain and CSF. This aspect will be discussed later. Transport out of the CSF across the choroid plexus also helps to lower the level of substances in the CSF and also in the brain. The concentration of a substance in the CSF and brain is thus determined by the difference between its rate of entrance into brain from plasma and its rate of exit from the CSF, either by bulk flow through the arachnoid villi or by transport across the choroid plexus, or by both processes. Substances such as Na and CI that are transported across the plexus are in approximately the same concentration in CSF as in plasma and hence are little affected by bulk flow. These relationships in developing rats are shown clearly by the data presented in Fig. 2 (Ferguson and Woodbury, 1967). The ordinates are the ratios of CSF/plasma (left side) and brain (cerebral cortex)/plasma (right side) in per cent of [Wlinulin for different aged animals, and the abscissa is time in hours. The uptake data were obtained simultaneously for CSF and brain in animals that varied in age from -4 days (1.2 g) (4 days before birth, or 17 days after conception) to 26 days (60 g) and adults (250 g). It is evident from Fig. 2 that inulin concentrations in the CSF and brain are considerably higher in the young than in the adult animals and that they progressively decrease with age. It is also evident that the brain spaces decrease puri pussu with the CSF spaces as the animals mature. A steady-state distribution is reached in about 24 h in most cases. Since even in the youngest animals the CSF/ plasma ratio is about 0.9, the level in the CSF is not the same as in the plasma. Hence, the inulin space of the brain measured in terms of the plasma still underestimates the extracellular space but is very close to it in the 4-day-prenatal rats. However, as shown in Fig. 3 (Ferguson and Woodbury, 1967), the inulin space calculated from the

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

299

ratio of brain to CSF inulin concentrations probably does measure fairly accurately the extracellular space of animals 16 days of age or younger. The ordinate is the brain/ fluid ratio of [“Tlinulin and [14C]sucrose activity and the abscissa is age of the rats i n days. In fetuses tested 4 days before birth (-4 days old), the brain/plasma and brain/CSF ratios for inulin approach one another, an indication that at this age the two ratios measure nearly the same space. If no CSF flow is present, then the two ratios would be equal. This condition is present in fetuses tested 6 days before birth (15 days post conception), a time also when the choroid plexus first begins to

0 4 4

Fig. 2. The simultaneous uptake of [Wlinulin into cerebral cortex (called brain in subsequent figures) and cerebrospinal fluid (CSF) of rats during maturation as a function of the time after injection of the inulin. The ordinate on the left is the ratio of CSF to plasma water [14C]inulin concentrations 100 (space in per cent); on the right, the ratio of brain to plasma water [14C]inulinconcentrations 100. The abscissa in both graphs is time in hours. All the animals were bilaterally nephrectomized for 24 h. See text for discussion. (From Ferguson and Woodbury, 1967.) 1

form as described by Dr. Virginia M. Tennyson in this symposium. Although both ratios decrease with increasing age, the ratios of brain/plasma and brain/CSF continuously diverge, probably as a result of increasing flow of CSF and of barrier formation to inulin transfer between plasma and brain. These two mechanisms would decrease the brain/plasma ratio presumably without affecting the brain/CSF ratio since there is a rapid rate of diffusion between brain and CSF. Between 16 and 26 days of age the brain/CSF ratio begins to increase and continues to rise to the adult values. The increase at this time probably is due to decreased permeability of the ependymal lining of brain to inulin so that brain to CSF exchange is slowed. Thereafter, inulin does not measure the interstitial volume of the brain. Since the volume of the brain interstitial space in adult rats is about 13-14 % as measured by the technique of ventricular-cisternal perfusion with inulin (Woodward et al., 1967), the curve of the brain/CSF ratio of inulin has been extrapolated to this value (indicated by an asterisk in Fig. 3) and the combined solid and dotted line represents the true extraRsfi.rcnc.rs pp, 312-313

300

D. M. W O O D B U R Y

cellular space of the brain as animals mature. The space decreases from about 48 ”/, in 4-day prenatal rats to 13 to 14 % in adults. The curve for sucrose is at all times higher than that for inulin but parallel to it, a fact that indicates that this substance probably penetrates cellular structures in the brain not penetrated by inulin. Since secretion of CSF and transport across the cells of the choroid plexus profoundly affect the levels of substances in the CSF and brain, and since both processes are active and require energy, it is possible to change the CSF and brain concentrations of these substances entering the CNS across the brain capillaries by inhibition or stimulation of the choroid plexus transport processes. Alterations of this transport system influence the levels either by altering bulk flow rate or by altering movements of the transported ions. If CSF secretion rate is changed then the concentrations of all substances entering the CSF from the brain are also changed. However, if only a single transport system is affected, then the distribution only of the substances transported by that system is changed unless that substance is a necessary part of the CSF secretory system.

g

60-

x

5

::

50-

. c . 2

40-

30-

20-

10-

Fig. 3. Relation between the brain to fluid (CSF or plasma water) ratio x 100 (space in per cent) of [14C]inulin and [14C]sucrose and the age in days after or before birth of rats. See text for discussion. (From Ferguson and Woodbury, 1967.)

Before considering the effects of substances that alter CSF secretion or choroid plexus transport processes, it seems worthwhile to discuss these transport systems of the choroid plexus and their relation to the distribution of ions in the CSF and brain. In Figure 4 is depicted a schematic representation of the choroid plexus cells of the rabbit and the distribution of ions in these cells, in the CSF, and in the interstitial space surrounding the choroid plexus capillaries. Below the diagram is a summary of the observed CSF/plasma ion-distribution ratios for rabbits and dog, the two species for which potential measurements between these two fluids have been measured, and the ratios that would be expected if the ions were passively distributed according to a 5 mV potential (CSF positive) for the dog and a 14 m V potential for the rabbit. The potential measurements in the dog are by Held et al. (1964) and

+

+

CSF

A N D N O N E L E C T R O L Y T E S A N D E L E C T R O L Y T E S IN B R A I N

301

those in the rabbit are by Welch and Sadler (1965). The CSF/plasma ratios are taken mainly from the works of Davson (1956; 1963), Held et al. (l964), and Schain (1964) but are in the same range as for other species including the rat. It is seen that most of the ions listed with the possible exception of Ca++and CI- are not distributed passively. Na t- and Mg++appear to be transported inward from plasma to CSF and K+ appears to be transported outward from CSF to plasma. Of the anions, only CIappears to be inwardly directed and most results suggest that it is passively distributed. However, data to be presented later, and other data (Rall, 1964), suggest that C1is transported actively from plasma to CSF. It appears, therefore, that Na+ and C1are the main ions involved in secretion of CSF. The anions, bicarbonate (HCO3-), iodide (I-), thiocyanate (SCN-) and, as will be shown later, perchlorate ( C D - ) , are transported out of the CSF. In the upper part of the diagram these transport systems are shown in relation to the membranes of the ependymal cells illustrated

CSF

CHOROID PLEXUS

, 1

CELLS

IN1 ERST1 T IAL FLUID

Passive SCN-

1-

c 10;

SCN-

SCN-

N a+

Mgs+ K+ Cat'

ION

C S F l PLASMA

R A T I O FOR PASSIVE D I S T R I B U T I O N

RATIO RABBIT

DOG

14nV RABBIT

5rnV DOG

I 03

I .02

0.59

0.83

0 52

0.75

0.59

0.83

-

I .48

0.34

0.69

co + +

0.72

0.34

0.69

-

1.21

I.I8

1.71

1.21

0.94

0.84

1.71

1.21

1-

$0.05

$0.05

1.71

1.21

SCN-

s0.05

50.05

1.71

1.21

No

+

K+ Mq+

CI

+

HCO 7

Fig. 4. The upper diagram is a schematic representation of the transport systems in, andthevoltage gradients across, the choroid plexus cells. The lower table is a summary of the observed CSF/plasma concentration ratios of the indicated ions in the rabbit and in the dog as compared with the calculated ratios for passive distribution according to the observed potential difference between blood and CSF determined for these two species. The voltage values for the rabbit were measured by Welch (1965); for the dog, by Held ef a/. (1964). See text for discussion. Reji,rences pp. 312-313

302

D. M. W O O D B U R Y

schematically in this figure. In the lower of the three ependymal cells the potential gradients across the cells as determined by Welch and Sadler (1965) are depicted. The ependymal cell membrane on the CSF side has a potential of -64 mV (inside of cell negative) and the one on the interstitial fluid side has a potential of -50 mV. Thus a potential difference of +I4 mV, CSF side positive, exists across the cells. In the ependymal cell shown in the middle of the diagram, the cation transport system is shown with the size of the letters indicating the probable concentration of the substances in the different compartments. K+ and Ca++ are higher on the interstitial fluid side and Na+ and Mg++on the CSF side. In the cell, K+ and Mg++are probably high and Na+ is low, as is the case with most other mammalian cells, although no experimental data are available to support this postulation. In muscle and other cells Mg+ is concentrated (30 : 1) but not as much as would be expected from the musclecell membrane potential of -90 mV and its double positive charge (1000 : I ) ; consequently, Mg++,like Na+, must be pumped out of cells (see Woodbury, 1965a). This is probably accomplished by the same carrier system that transports Na+.However, the transport of Na+ inward and of K+ outward, which process is assumed to be coupled, must take place in the membrane on the CSF side of the cell. Thus the pump of the choroid plexus, assumed to be a coupled Na+ and Mgki for K+ pump, is in the membrane of the CSF side. The movement across the membrane of the interstitial side is probably passive for all three cations since it is along their eleotrical or chemical gradients. The mechanism of transport of Ca++is not known but appears to be passive. In the ependymal cell illustrated at the top of the diagram, the anion transport system is depicted. Chloride ion appears to distribute passively across the membrane on the interstitial side of the cell. It then is probably actively transported into the CSF across the membrane on the CSF side. The outward transport of the anions I-, C104-, SCN- and probably HC03- is thought to be coupled to inward chloride movement. The location of the anion pump with respect to this membrane is necessitated by the observations of Welch (1962a, 1962b) that the choroid plexus can concentrate I- and SCN- ions in vitro. Since this is the case and if only a single pump is present, it must be in the CSF side of the membrane in order to concentrate I- and SCNin cells and still maintain a concentration difference between CSF and plasma. However, a pump in both membranes could also accomplish the same result but there is no evidence for this one way or the other. The consequences of inhibiting the various pumps of the choroid plexus for the concentration of various substances in the CSF and brain can now be considered. In Figs. 5-9 are shown the effects of inhibition of the anion transport system. In Figure 5 , modified from the figures of Reed et al. (1965), are shown the effects of a large load of iodide on the distribution of [l3l]Iiodide ion and [14C]inulin in CSF and brain. Iodide saturates the I- pump and thereby decreases the exit of the I- from the CSF by this route. The major route of I- exit, therefore, is via the arachnoid villi by bulk flow, provided that this flow is not also inhibited by large loads of iodide ion. That bulk flow appears to be little affected is demonstrated by the relatively minor change in CSF [“Clinulin space (which is a measure of CSF bu1kflow)with increasing loads of iodide. The brain space of inulin also increases slightly but in-

CSF

A N D NONELECTROLYTES AND ELECTROLYTES I N BRAIN

303

significantly. The effect on [1311]iodidespace, however, is striking. Increasing doses of iodide progressively elevate the CSF ['3ll]iodide space and concomitantly the brain [131l]iodide space is also increased. Thus the I- space of CSF increases from 2 to 43 % and that of brain from 2 to nearly 12 %. It is clearly evident that saturating the carrier for transport of I- out of the CSF raises not only the CSF but also the brain levels of this anion. The concentrations in these two fluids thus more closely approach each other and also the concentration in the plasma. Since the exchange rates between CSF and interstitial space of brain of this anion are rapid, the ratio between the two (labeled (brain/CSF) 1311 in Fig. 5) is a measure of the volume of distribution of the I- in the brain, especially in the carrier-iodide loaded animals in which the concentrations are more nearly equal in the two fluids. The calculated brain/CSF ratio is 1.04 in controls and levels out at about 0.26 in the carrier-iodide loaded animals. This volume of distribution is equal to that of CI- and suggests that I- distributes like C1- in the brain when 1- transport is blocked. If the final distribution volume is taken as 26 %, then the calculated concentration of I- ion in brain at any one iodide load is its actual concentration in the whole brain divided by the volume of water in which it is distributed, namely, 26 % in this case. Thus the brain concentrations of I- for the four 100-

nosoSO -

7

\

4J-

30

-

csi-I"'

'\, __---------_ _ _ _ _ _--------a ___-

.

a

=.A-

V

20 -

I

\

*' 0

B cR sPil H 1 O l

BRAIN-Ci4-

2'5

lNULlN

I

10 0

50

L L q I N CARRILR l O O l D E I N J L C T F O

Fig. 5. Effects of iodide loading on the 4-h distribution of intraperitoneally-injected[13Ll]iodideand [L4C]inulinin brain and CSF of rats bilaterally nephrectomized for 4 h. The ordinate is the space (brain/plasrna, CSF/plasma, brain/CSF) in per cent and the abscissa is mequiv/kg body weight of carrier iodide injected into theanimals.The figure is drawn from the data of Reed et al. (1965).See text for discussion.

different iodide loads used in this experiment (Fig. 5 ) are

I .9

5.8 =

0.26

1.3,

8.6 = 22.3,

~

0.26

11.7 = 33,

~

0.26

and

_ _ = 45,

0.26

as compared to the corresponding CSF levels of 2, 22, 33 and 43 and plasma levels of 100. Thus in the controls the concentration of I- in brain water is higher than that of CSF but less than that of plasma. However, in the iodide-loaded animals the concentrations in brain and CSF are equal but less than in plasma. These data indicate Ki,J./Privrcspp. 312-313

D. M. W O O D B U R Y

304

that I- enters the CSF from the brain capillaries via the interstitial fluid of the brain and not through the choroid plexus. They also indicate that the barrier to entrance is between the capillaries of the brain and the interstitial fluid of the brain and that there is free exchange of iodide between the brain and CSF, as already mentioned. The effects of intraperitoneal administration of other inhibitors of transport processes on [13ll]iodide and [14C]inulindistribution in brain and CSF are shown in Fig. 6 (Reed et al., 1965). The inhibitors used and the rationale for their use were:

C1‘-INULIN

4 HOUR SPACE

1 0 0 1 0 ~HOUR ~ ~ ~S P4A C E

I. : S i p n ~ l l r o n l l y d i l l e r m l 110.

c

L0”llOl “ O I U e ,

ll

Fig. 6. Effects of various transport inhibitors on the 4-h distribution of intraperitoneally administered [13LI]iodideand[14C]inulin in brain and CSF of rats bilaterally nephrectomized for 5 h. The ordinate is space of brain or CSF in per cent. The Inhibitors used together with their doses are indicated along the abscissa. The data used to construct this figure were obtained from the paper of Reed eta/. (1965). See text for discussion.

perchlorate ion, a competitive inhibitor of I- transport in the thyroid ; acetazolamide, a carbonic anhydrase inhibitor that has been shown to inhibit CSF secretion; and oirabain, an inhibitor of the Na+-K+ active transport system of cells and also of the I- uptake by the thyroid gland. Of the three inhibitors used, only perchlorate ion had a profound effect on the distribution of I-. It markedly increased the concentration of [13ll]iodide in both CSF and brain. The [“Clinulin space was little affected. After intraperitoneal injection, acetazolamide and ouabain, unlike perchlorate, had no significant effect on the distribution of [Wlinulin and [131I]iodide; but, on intracisternal injection, they markedly increased the concentrations of [14C] inulin and [13ll]iodide in CSF and brain (Reed et al., 1965). The changes induced by ouabain and acetazolamide on distribution of intracisternal injected [“Clinulin were greater than with I-, an observation that indicates these agents affect I- distribution by inhibition of CSF secretion with its resultant decreased bulk flow, not only through the CSF but also through the brain substance, a process previously suggested by Reed and Woodbury (1963). An inhibition of bulk flow through brain tissue would be expected to decrease levels in the brain relative to the CSF to the same extent for both I- and inulin even though both diffuse across membranes at different rates. This is precisely what happened after acetazolamide and ouabain. For example, the 4-h

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

305

CSF/brain ratios for [l"l]iodide and [14C]inulin, respectively, after intracisternal administration in controls were 2.5 and 3. I , whereas after acetazolamide they were both 11.2. One of the questions concerning anion transport across the choroid plexus is when it begins developmentally. To answer this, the ability of the choroid plexus to transport I- was tested in 8-day-old rats. In addition, the comparative inhibitory effect of a perchlorate ion load on this system in young as compared to adult animals was also

-

01 0 1 2

I

I

8 TINE

21

I N Houris

Fig. 7. Effects of perchlorate treatment on the [l311]iodide and [*4C]inulin spaces in per cent (ordinbrain in 8-day-old and adult rats as a function of time in hours (abscissa) after intraperitoneal injection of the tracers. The adult rats were bilaterally nephrectomized for 5 hand the 8-day-old rats for 24 h. The duration of nephrectomy, however, only slightly affects the volume of distribution of these substances. See text for discussion.

ate) of

measured. The results are shown in Fig. 7. The brain [13lI]iodide and [14C]inulin spaces are the ordinate and the abscissa is time in hours after injection of [13lI]iodide or [14C]inulin. The uptake experiments were done on 8-day-old control and perchlorate-treated rats and the results were compared with the 4-h brain I- and inulin spaces of adult control or perchlorate-treated rats. The brain uptake of I- is very rapid in 8-day-old rats and reaches a peak of about 5 % in 40 min, it falls off very gradually thereafter. Perchlorate (5 mequiv./kg) elevated the brain space of 8-day-old rats to about 14 "/, at 4 h, compared to the control value of 4.5 % measured at the same time. Thus the brain space was increased 3.1 times by this inhibitor of 1- transport. The levels of I- in the CSF were not measured in this experiment. The uptake of [14C]inulin by the brain of the 8-day-old rats was much slower than that of ['3lI]iodide, but in the controls it reached a larger volume of distribution. In this experiment perchlorate did not affect the volume of distribution or the rate of entrance of inulin in the brain of 8-day-old rats. Since the control I- space is less than the inulin space and the Ispace is increased by perchlorate ion to a value greater than the inulin space, it is apparent that the 1- pump is actively working in these young animals. However, that it is less active than in the adult is evident from the effect of this anion on the Rcfc~rrnccspp, 312-313

306

D. M. W O O D B U R Y

[13ll]iodide space of the adult animals. The 4-h [13lI]iodide space was 1.8 % in control animals, a value less than the corresponding value of 8-day-old animals, but it was increased to 7.2 % by perchlorate in a dose of 3.4 mequiv./kg. This represents an increase of 7.2/1.8 or 4 times the control value as compared to 3.1 times for the young rats in which a larger (5 mequiv./kg) dose was used. Thus the inhibitor exerted a greater effect in the adults than in the young animals. This suggests that the active transport is less active in the 8-day-old animals. The inulin space of the adults was slightly increased by perchlorate but the effect was not significant. In the thyroid gland, anions besides 1- are transported by the anion pump and concentrated in the gland. The next question, then, is whether these same anions are also transported by the choroid plexus system and, if so, whether they inhibit transport across the plexus. In Fig. 8 is shown the effect of perchlorate ion (5 mequiv./kg) on6

0 1 2

4

6

8 12 T I M E I N HOURS

24

Fig. 8. The effect of a perchlorate ion load on the uptake of radioactive perchlorate ion (36CIO~-) by brain and CSF of adult rats. The ordinate is [36Cl]perchloratespace of brain and CSF in per cent and theabscissa is time in hours after intraperitoneal injection of the 36CIOc in rats bilaterally nephrectomized for 24 h. See text for explanation.

the uptake of radioactive perchlorate (36ClO4) by brain and CSF of nephrectomized adult rats. The ordinate is [36Cl]perchlorate space in per cent and the abscissa is time in hours after injection. It is clear that, as in the case for an iodide load, stable perchlorate markedly increased the volume of distribution of 36C104 in both CSF and brain. Inasmuch as the plasma levels remained constant, these increases are due to a marked rise in the concentration of 36C104 in CSF and brain tissue. Since the CSF/ plasma ratio of the control was only 0.1 at equilibrium and since there was no appreciable change in the [Wlinulin space on perchlorate treatment, it is evident that this anion, like I-, is transported out of the CSF and that its transport can be self blocked. Again, an increase in CSF concentration increased the level in the brain such that it approached that of the plasma. Since brain-CSF exchange is rapid for this ion, as it is for I-, the brain/CSFratio of 3sC104 in the perchlorate-treated group measures quite accurately its volume of distribution in the brain. This value is 17.5/54

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES IN BRAIN

307

x,

100 = 32.5 a value close to the chloride space. These data indicate that C104as well as I- distributes in the brain as does CI- ion. The effects on 36CIO~ transport in the choroid plexus of some of the monovalent anions that inhibit iodide transport across the choroid plexus and in the thyroid are shown in Fig. 9 (Chow and Woodbury, 1967). The ordinate is 36C104 space in % of wet weight of brain and skeletal muscle. CSF was not measured, but, as just dei

CONT.

CCHEBrAL CORTEX--SKELtTAL

‘I01

NoSCN

MUSCLE-

Fig. 9. Effects of various monovalent anions (5 mequiv/kg) on the [3RCI]-perchloratespaces of brain and skeletal muscle one hour after intraperitoneal injection of the tracer in adult rats. The anion inhibitors were given 2 hours before sacrifice of the animals. The figure in each bar is the number of animals used for that group. See text for discussion. (From Chow and Woodbury, 1967.)

monstrated, inhibition of anion transport in the choroid plexus by monovalent anions is always accompanied by an increase in both CSF and brain concentrations; hence an effect of a monovalent anion to increase the brain space generally indicates blockade of this transport system. It is evident that iodide, perchlorate, and thiocyanate (in doses of 5 mequiv./kg) all block the anion transport system in the choroid plexus. The order of effectiveness is perchlorate > iodide > thiocyanate. In this experiment the radioactive perchlorate space of muscle was little affected by these monovalent anions. Another question that arises is the effect of a perchlorate-ion load on the distribution of substances other than I - and C104- in brain and CSF. In the schematic diagram of the choroid plexus system shown in Fig. 4 it is indicated that CI- is probably secreted into the CSF coupled to outward transport of I-, C104-, etc. If this is the case, inhibition of outward transport of I- or C I O c with perchlorate ion should inhibit inward CI- transport and decrease the CSF level of CI-. Also, so4=concentration is lower i n the CSF than in the plasma (Van Harreveld, 1966), and the possibility exists that this doubly charged anion might also be transported across the choroid plexus into blood. However, its distribution, like that of inulin, could equally be accounted for by its slow penetration across the capillary-brain interface, coupled with its carriage out of the CSF by bulk flow through the arachnoid villi. If movement of Sod- out of the CSF involves the anion transport system of the choroid plexus, Rc+riwii>\ p p . 312-313

D. M. W O O D B U R Y

308

then a large dose of perchlorate should compete with Sod= for the carrier, as it does for I-, and block outward so4=transport. The result would then be an increase in SO4= levels in the CSF and brain. If only bulk flow is involved in SO4= exit from the CSF, it should behave like inulin when CSF flow is altered. Therefore, the effects of 5 mequiv/kgdoses ofperchlorate ion on the distribution of 36C104, 36C1, 35S04 and ['4C]inulin in CSF and brain were determined in nephrectomized rats. The results of these studies are indicated in Fig. 10. The spaces are shown on the ordinate and the various treatments and isotopes used are presented along the abscissa. The entire uptake curves were determined in both control and perchlorate-treated groups, but only the equilibrium values determined at the times indicated below each isotope listed are depicted in the figure. As demonstrated previously in Fig. 8, a perchlorate load increased the volume of distribution of 36C104 in both CSF and brain. The

Cl%i

24 D W I

c1J6 LI

hOYI,

SJ504 1 how,

Clq

- lwull~ n ~ W I

Fig. 10. Effect of perchlorate treatment on the distribution of 36CI04,36CI,35S04,and [14C]inulinin brain and CSF of adult rats at the times after injection indicated along the abscissa. The ordinate is the space of brain or CSF in per cent. The animals were bilaterally nephrectomized for 24 h. See text for discussion.

increase in the CSF is greater than that in brain. In contrast to its effect on CSF 36c104, perchlorate treatment decreased the concentration of W I in the CSF; but, like 36C104, it increased the 36CI space of the brain. (The stable CI (35C1)space in CSF was also decreased and the 35Cl space of brain increased.) Thus, in this case a decrease in CSF concentration was accompanied by an increase in brain space. Although the changes were not significant, perchlorate slightly and consistently at all time periods increased the 35S04 space of the CSF and also of the brain. It had, however, no significant effect on (14C)inulin spaces in brain or CSF. Since ["Tlinulin spaces were not affected by perchlorate treatment and S5S04 levels were affected slightly, these observations suggest that the low CSF as compared to plasma may to a small extent be accounted for by active transport via the choroid plexus anion system. Further work is obviously necessary to prove this point. The decrease in 36Cl space and the increase in 36C104 space in the CSF induced by perchlorate treatment are in

CSF

AND NONELECTROLYTES AND ELECTROLYTES IN B R A I N

309

harmony with the concept that the two ions are coupled in their transport, as already discussed. As was the case for I-, the C104- space of 9-day-old rats is increased by perchlorate loading but not to the same extent as in the adult (Barham, G. B. and Woodbury, D. M., unpublished observations). This effect is illustrated in Fig. 1 I . The ordinate is

CONTROLS

I}

\Clq-

I

C1j604 S P A C E

)Ci4-INULIN

SPACE!

I N U L I N SPACE

I,/PERCHLORATE I

I

rsr PLASMA

BRAIN PLASMA

9 - DAYS- OLD

ADULTS

Fig. 1 1 . Effect of perchlorate ion (5 mequiv/kg) on the brain/plasma, CSF/plasma and brain/CSF [B8C104]-and[14C]inulinratios (spaces) in 9-day-old as compared to adult rats. The asterisks indicate that the value for the spaces of the perchlorate-treated group marked is significantly different ( P < 0.05) from the corresponding space of the controls. Values plotted from the unpublished observations of G . B. Barham and D. M. Woodbury. See text for discussion.

the 36C104 or [14C]inulin space in per cent. The 9-day-old animals are shown on the left and the adults on the right. Perchlorate spaces are represented by the total height of the bar and inulin spaces by the filled-in areas. The controls are represented by the bar on the left and the treated animals by the bar on the right. In the young rats, perchlorate treatment increased the CSF 36ClO.4 space, which is already high in these animals, from 55 to 88 % (1.6 times) and increased the brain space from 20 to 43% (2.15 times). The [“Wlinulin spaces were also increased by perchlorate: in the CSF from 17 to 26% (1.53 times) and in the brain from 6 to 8.5% (1.42 times). It is evident that blockade of WIO4 transfer by perchlorate ion appears also to inhibit the secretion of CSF as measured by the CSF [“Clinulin space. The anion transport system thus appears to be involved in CSF secretion. In adults, as already described, perchlorate increased the CSF space 5.4 times as compared to I .6 times in the young rats, and increased the brain space 2.5 times as compared to 2.15 times in the young. The inulin space is apparently not altered by perchlorate loads in the adult probably because it is so small that the kind of change observed in the young could not be detected in the adult. It is apparent that anion transport of C104- seems to be less developed in the young, as was shown to be the case for I- in Fig. 7. The brain/CSF ratios give the correct C104- and inulin distribution in brain. In this case, the difference between the 36ClO4 space and [14C]inulin space is a measure of brain intracellular 36C104. In the 9-day-old rats perchlorate increased markedly the brain/CSF 36C104 space and deRcJi.rences pp. 312-313

D. M. W O O D B U R Y

310

creased the [“C]inulin (interstitial) space. Thus intracellular 36C104 levels were increased. In adults the brain/CSF ratio is decreased by perchlorate for the reasons already discussed. However, intracellular 36C104cannot be determined in adult rats from the difference between 36C104and inulin spaces as it can in young rats, because the apparent [14C]inulin space (brain/CSF ratio) is larger than the 36C104space. This situation is generally the case when the blood-brain barrier is developed, as already discussed (see Fig. 3). Although not shown here, the CSF/plasma and brain/plasma CI- ratios were markedly decreased by a perchlorate load in young rats. Hence the reciprocal relation between CI- and C 1 0 ~transfer across the choroid plexus in adults and reflected also in the brain levels is clearly present in the young rats and further supports this hypothesis, especially since perchlorate treatment had little effect on Naf and K+ concentrations in the CSF. Another anion that blocks iodide accumulation in the thyroid and is concentrated by choroid plexus cells (Welch, 1962b) is thiocyanate. Pollay (1966) has shown that large doses of this ion produce the same effects on CSF and brain SCN- and Ispaces as do iodide and perchlorate loads on [13ll]iodide and 36C104 spaces. Thus SCN- is also actively transported out of the CSFacross thechoroid plexus, as indicated in Fig. 4. The data of Pollay are summarized in Fig. 3 (p. 277). We have further evidence to indicate the intimate relations that exist between changes in CSF secretion as a result of transport of substances across the choroid plexus and the concentration of substances in the brain, but the data herein presented adequately illustrate these relationships. One point, however, that needs amplification is the effect of inhibition of CSF flow on the levels of nonelectrolytes and electrolytes in CSF and brain. One facet is shown in Fig. 12. The effects of ouabain and acetazolaamide on CSF and brain inulin spaces in developing rats were studied in order to

‘I

BRAINKSF

20

CSFlPLlSYA

Fig. 12. Effects of acetazolamide and ouabain in the indicated doses on the [%]inulin spaces (brain/ plasma, CSF/plasma, brain/CSF) in rats of different ages after birth. Note that the dose of ouabain used (2 mg/kg ) is considerably lower than the dose necessary to produce an effect in adults (25mg/kg), as seen in Fig. 6. Doses higher than this produced convulsions and death in the young animals, but 25 mg/kg did not produce convulsions or deaths in adults. Data obtained from unpublished observations of R. K. Ferguson and D. M . Woodbury. See text for discussion.

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

311

ascertain their influence in a case where the brain barrier is not developed and changes can be easily detected. The ordinate is the [14C]inulin space in per cent, and the abscissa is age of the animals in days after birth. The CSF/plasma and brain/plasma ratios of the controls decrease progressively from 3 to 16 days of age. Ouabain, which inhibits N a t and K+ transport in both the CSF and the brain, increased the CSF [14C]inulin space markedly at 16 days of age but only slightly at 3 days of age. It had very little effect on the [14C]inulin space of brain as measured by the brain/plasma ratio. In this case the change in CSF concentration was not reflected in changes in brain concentration, probably because swelling of the brain cells, caused by the increased cell Na+ and the decreased cell K+ concentrations induced by the direct effect of ouabain on nerve cells, results in a decrease in extracellular fluid volume of brain in the tight box that encloses the brain. That a decrease in extracellular fluid volume occurs with ouabain is evident from the data for the brain/CSF ratios shown in the top graph of Fig. 12. As discussed earlier the brain/CSF ratio in per cent is a measure of the interstitial space of the brain in animals 16 days of age or younger. Thus, ouabain decreased the brain/CSF space in 16-day-old rats and this was accompanied by a marked increase in water, Na+ and CI- and a decrease in K+ concentrations of the brain, an indication that cerebral swelling had occurred. Inhibition of CSF secretion by acetazolamide (which appears to block CI- transport) increased markedly the CSF [14C]inulin spaces at 9 and particularly at 16 days but had little effect at 3 days. The brain [“Tlinulin space was also increased at all three time periods but the effect was greater at 16 days. However, the brain/CSF ratio was decreased by acetazolamide at 9 and 16 days but not at 3 days. Thus this agent, like ouabain, decreased the extracellular space of the brain, again possibly by causing swelling of nerve cells. These age-dependent effects of metabolic inhibitors of transport on inulin distribution provide further evidence that the transport systems are less well developed in young rats. Acetazolamide presumably acts by inhibiting carbonic anhydrase in the choroid plexus and the brain, and it is well known that this enzyme is low in activity in the brain of young animals (Millichap et al., 1957; Karler and Woodbury, 1960). The relative lack of effect of acetazolamide in such animals is readily explained by the paucity in this enzyme. The lower effectiveness of ouabain, which inhibits Na+-K+ transport in adults, in 3-day-old rats is also probably due to the fact that the cation transport portion of the CSF secretory system is immature at this age. Our studies of CSF electrolytes with age have demonstrated that this system is not completely mature until after 3 days of age. Observations on Naf, K+ and CIconcentrations in CSF and brain at different ages and the effects of ouabain and acetazolamide thereon have clearly shown that the same close relationship between CSF and brain fluids exists for cation and anion transport. Subsequent papers will report these data. SUMMARY

The studies reported in this paper demonstrate that there is an intimate relationship between the CSF and the extracellular fluid of the brain. This relationship was deR(fcrenrcs ppr31.2-313

312

D. M. W O O D B U R Y

monstrated by use of substances that block the formation of CSF, by inhibition of either the cation or the anion transport systems in the choroid plexus, both of which appear to be involved in CSF secretion. Anion transport out of the CSF across the choroid plexus was shown to be present for iodide, perchlorate and thiocyanate ions and possibly for sulfate. This outward transport is thought to be coupled to inward chloride transport, since inhibition of outward anion transport is always accompanied by inhibition of inward chloride transport. Inhibition of anion transport across the choroid plexus resulted in an increase in the CSF/plasma ratio (space) of concentrations of the various anions tested. The increase in CSF/plasma ratio was accompanied by an increase in the brain/plasma ratio of the same anions. Thus a change in the CSF concentration of a substance is accompanied by a corresponding change in the brain concentration. This same relationship between CSF and brain also holds when cation transport in the choroid plexus is blocked. Further evidence for this concept was obtained from studies in developing rats. The formation of CSF in rats begins about 6 days before birth and by the ninth day after birth it has matured, as measured by the ability of the choroid plexus to transport anions and cations. During this period of increasing flow of CSF and increasing active transport of cations and anions across the plexus, the direction of change of nonelectrolytes (such as inulin), anions (such as iodide, perchlorate, thiocyanate, and chloride), and cations (such as sodium and potassium) in the CSF is the same as in the brain. Thus these data in maturing animals again demonstrate the intimate relation between CSF and brain extracellular fluid. ACKNOWLEDGEMENT

Original investigations reported herein were supported by a United States Public Health Service Research Program Grant (No. 5-POI -NB-04553) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. REFERENCES CHOW,S. Y. AND WOODBURY, D. M. (1967) Distribution of C136-perchlorateion in brain and muscle of rats and guinea pigs. Amer. J . Physiol., submitted. DAVSON, H. (1963) The cerebrospinal fluid. Ergeb. Physiol. Biol.Chem. Exptl. Pharmakol., 52, 20-73. -(1965) The extracellular space of the brain. Biology of Neurogliu. E. D. P. De Robertis and R. Carrea (Eds.). Progr. Brain Res., 12, Amsterdam, London and New York. Elsevier Publishing Co. (pp. 124-1 34). R. K. A N D WOODBURY, D . M. (1967) Penetration of C14-inulinand C14-sucroseinto brain FERGUSON, and cerebrospinal fluid of developing rats. With comments on measurement of brain extracellular space with age. Arch. Neurol., submitted. HELD,D., FENCL, V. AND PAPPENHEIMER, J. R. (1964) Electrical Potential of cerebrospinal fluid. J . Neurophysiol., 21, 942-959. KARLER, R. AND WOODBURY, D. M. (1960) Influence of aging on intracellular distribution of carbonic anhydrase. Fed. Proc., 19, 133. MILLICHAP, J. G . (1957) Development of seizure patterns in newborn animals. Significance of brain carbonic anhvdrase. Proc. SOC.Exptl. Biol. Merl., 96, 125-129. M . (1966) Cerebrospinal fluid transport and the thiocyanate space of the brain. Amer. J . POLLAY, Physiol., 210, 275-279,

CSF

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RALL.D. P. (1964) The structure and function of the cerebrospinal fluid. The Cellular Furictioris of Memhratie Tramport. J. F. Hoffman, Editor. Englewood Cliffs, New Jersey. Prentice-Hall Inc. (pp. 269-282). REED,D. J. AND WOODBURY, D. M. (1963) Kinetics of movement of iodide, sucrose, inulin and radioiodinated serum albumin in central nervous system and cerebrospinal fluid of rat. J. Physiol., 169, 816-850. REED,D. J., WOODBURY, D. M., JACOBS,L. A N D SQUIRES, R. (1965) Factors affecting distribution of iodide in brain and cerebrospinal fluid. Anier. J. Physiol., 209, 757-764. SCHAIN, R. J. (1964) Cerebrospinal fluid and serum cation levels. Arch. Neurol., 11, 330-333. VAN HARREVELD, A., AHMED,N. A N D TANNER, D. J. (1966) Sulfate concentrations in cerebrospinal fluid and serum of rabbits and cats. Anier. J . Physiol., 210, 777-780. WELCH,K. (1962a) Active transport of iodide by choroid plexus of the rabbit in vitro. Ar7ier. J . Phvsiol., 202, 157-760. -( 1962b) Concentration of thiocyanate by the choroid plexus of the rabbit in vitro. Proc. SOC. Exprl. Biol. Med., 109, 953-954. WELCH,K. A N D SADLER, K. (1965) Electrical potentials of choroid plexus of the rabbit. J . Neurosurg., 22, 344-351. WOODBURY, D. M. (1965a) Physiology of body fluids. Chapter 45 in Physiology and Biophysics. 19th ed., T. C. Ruch and H. D. Patton (Ed.). Philadelphia, W. B. Saunders Co. (pp. 871-898). -( l965b) Blood-cerebrospinal fluid-brain fluid relations. Chapter 47 in Physiology arid Biophysics. 19th ed., T. C. Ruch and H. D. Patton (Eds.). Philadelphia. W. E. Saunders Co. (pp. 942-950). WOODWARD, D. L., REED,D. J. A N D WOODBURY, D. M. (1967) The extracellular space of rat cerebral cortex. Atner. J. Physiol., 212, 367-370.

DISCUSSION T. Z. CSAKY:What were the animals you used in these studies? D. M. WOODBURY: They were all rats. T. Z. CSAKY:Two mg/kg of ouabain in a rat is, if you calculate the concentrations, about 7 mols. Is not that just too little in a rat to have any effect? D. M. WOODBURY: Too little? I think most people would be amazed at how large the dose was. T. Z. CSAKY: We used two doses: one was 26 mg in one instance, in the second it was 2 mg. D. M. WOODBURY: These are very large doses of ouabain. T. Z. CSAKY:Not for a rat. That is right. Rats are very resistant to ouabain, and large doses have to be given D. M. WOODBURY: to get any effect in the adult (at least 25 mg/kg). However, infant rats are very sensitive to it, and if a dose of 2 mg/kg is greatly exceeded most of the animals die. Even at 2 mg/kg some of the animals have seizures. It is apparent, therefore, that ouabain penetrates into the brain of young rats more readily than into the brain of adults. In the rat, the brain is more sensitive than the heart to the effect of ouabain and if it can enter the brain more readily, as it does in the infant, then it attains a higher concentration and produces a greater effect. The electrolyte changes induced in the brain by ouabain in young rats are also much more marked. Sodium is increased and potassium is lost, and seizures occur. None of these effects on the brain are produced by ouabain in adult rats. The seizures induced by ouabain can be prevented by diphenylhydantoin (Dilantin).

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D. B. TOWER:Dr. Woodbury introduced quite a long time ago the importance of the developmental study. I think that is evident here, because some of the questions that we have about these systems cannot be easily answered unless you can sort out the different cells and membranes as they develop. The question I had was about the chloride, particularly in regard to the effect on the brain chloride as opposed to the CSF. I think that it is quite clear from your data, but if you looked earlier than 9 days, which was the earliest you showed us, did you see a difference in the effect of perchlorate on the apparent brain cellular transport system? The reason I ask this question is to know what cells might be involved in chloride transport in the brain, because as you know very well from Brizzee and Jacob's work, there is a difference in the time of development and the proliferation of neurons versus glial cells. A second very quick question: In your ouabain experiments in the adult it looked as if one could conclude from the brain/CSF ratio that you had a considerable decrease in the inulin-accessible space, and hence presumably some swelling. Is this your interpretation? D. M. WOODBURY: Yes, that is what I assumed. Since there is a marked change in the brain concentration of sodium and potassium outside the inulin space, 1 would assume that the swelling caused a decrease in the extracellular space of the brain. In the young animals, even at 9 days, glial cells are not prominent, and I suspect that most of the changes are neuronal in these young animals. We do get some opposite changes in the adult with perchlorate. I did not have time to go into this, but they suggest that possibly glial cells have a different response, although it is hard to sort this out. R. V. COXON:Could I ask Dr. Woodbury a rather simple technical question? How much CSF can you get out of a 17-day rat, in pl? L. BAKAY: This question occurred to me too. D. M. WOODBURY: We gan get about 10-25 ,d from a 17-day-old rat and about 5-15 111from newborn to 9-day-old rats. In fetuses we get about 2-5 pi. R. V. COXON:From where? From the ventricle?

D. M. WOODBURY: From the cisterna. We use capillary tubes similar to those that are used in pulling microelectrodes and make micropipettes out of them. The CSF is remove from the cisterna of anesthetized animals. The fluid is sucked up by capillary action. CSF is actually easier to obtain than is blood. D. H. FORD:The iodide space which you measured, does this include the iodide in the vascular space and that which would perhaps diffuse out? The reason I ask this is that we have done some studies quite a long time ago, where we did radio-autography after injection of sodium iodide, and I would say that over 90% of the activity in our brains was in the vessels.

D. M. WOODBURY: Blood iodide cannot account for the iodide space we measured since we drain all the blood out of the body from the aorta and residual blood left in the brain cannot account for the amount of iodidL in the brain. We have measured the blood volume under these circumstances and find that it is much smaller than the observed iodide space. R. CUTLER: Some of the older anatomists and neurosurgeons have claimed that young animal and human offspring have no arachnoid granulations. I wondered if you have any data bearing on the bulk clearance of the spinal fluid at this age, or whether the increase in CSF/plasma ratio for inulin and the brain/plasma ratio for inulin has resulted in an increased entry rate in this age group.

D. M. WOODBURY: I don't know about the arachnoid granulations. We have not actually measured the clearance in these circumstances. It is certainly a possibility that the slow flow might be due to lack of development of the arachnoid granulations, but it is more likely, and we have evidence for this, that it is due to the fact that the CSF-secretory process has not yet developed. Maybe some of the histologists or anatomists here know about the development of the arachnoid granulations during this period of maturation.

Factors Influencing Barrier Function

Changes in Barrier Effect in Pathological States LOUIS BAKAY Divisiori of Neitroatrgery, Sture Utiiversitj, of New York a1 BuJyirlo, School of Mediciiie, 462 Gride Street, Bitflulo, N . Y . ( U.S.A.)

Diseases of the central nervous system that are severe enough to alter its structural organization result in localized or generalized increase in the permeability of the blood-brain barrier. From an historical point of view, the principle of the bloodbrain barrier was established by Goldman (1913) who based his theory on two postulates. His “first experiment” revealed that the central nervous system does not stain after the administration of a vital dye into the blood stream. The “second experiment” showed that diffuse coloration of the nervous system occurred when the same dye was injected into the cerebrospinal fluid. Although it was not put in the form of a third postulate, it was accepted from the beginning that lack of staining in the “first experiment” did not include those parts of the brain that were not nervous in structure (choroid plexus, meninges, etc.) or those that were affected by a pathological process. In patho-physiological research, the blood-brain barrier still denotes a hypothetical structure or mechanism with specific vulnerability and almost general rate-limiting importance (Edstrom, 1961). Although a great deal of research, utilizing the latest anatomical and chemical methods, has been carried out over the past years to elucidate the nature of the normal blood-brain barrier, a similar sophistication in aim and methodology was less noticeable in studies of the pathological state. This is perhaps understandable when one considers the enormous impact of any change in permeability of the blood-brain barrier in clinical medicine. The increased permeability or absence of the barrier in various lesions became an accepted fact; the main emphasis was placed on the exploitation of this situation for diagnostic or therapeutic purposes without giving too much consideration to the basic factors that are responsible for its occurrence. From a diagnostic point of view, changes in the blood-brain barrier permeability are used for localization of tumors and other lesions by radioactive brain scanning. The same principle allows for the treatment of cerebral infections by antibiotics and of neoplasms by chemotherapeutic agents ; the substances applied for diagnostic or therapeutic reasons have ready access to the lesion while, on the other hand, they are more or less excluded from the normal brain. A complete review of all pathological conditions affecting the transfer of substances from blood to brain would be prohibitive in size. Consequently, I selected a few specific examples of pathological conditions to illustrate some of the basic problems. Kernicterus and other types of bilirubin pigmentation of the brain were chosen, not R c f i r m w r pp. 336-339

316

L. B A K A Y

only because of their clinical importance and intriguing patho-physiology, but also because they were historically the first type of lesion where the blood-brain barrier as the cause of the disorder was suspected. The various types of cerebral edema represent a pathological condition where increased barrier and membrane permeability are determining factors. Brain injuries are characterized by derangement of the barrier; this group is followed by a review of a much morecomplex subject, namely, the state of the barrier in tumors, Our knowledge of the latter, although still incomplete, was increased lately by a great flow of information derived from radioactive brain scanning. B I L I R U B I N A N D T H E B L O O D - B R A I N BARRIER

(Kernicterus)

Perhaps the first application of the blood-brain barrier theory to a clinical problem was its implication in the development of kernicterus. It was recognized early that bilirubin staining of certain portions of the brain, particularly that of the basal ganglia, can frequently be seen in icteric newborn while, on the other hand, adults, even with severe and long-lasting jaundice, do not show any bilirubin deposits in the central nervous system except for that of the choroid plexuses and cerebrospinal fluid. It was originally thought that bile pigments deposit in the newborn brain because of the physiologically undeveloped and more permeable blood-brain barrier which, when fully developed and "leakproof", keeps them out of the mature brain. This hypothesis was further strengthened by the discovery that areas of tissue damage in the adult brain (infarcts, hemorrhages, contusions, tumors, etc.) do stain with bilirubin in hyperbilirubinemia. It seemed logical to assume that kernicterus represents a clinical variant of vital dye experiments, trypan blue being simply substituted by a biological pigment, bilirubin. However, as time passed by, investigators became increasingly aware that kernicterus of the newborn is caused by a combination of circumstances; the relative importance of the individual factors is not yet known. Space does not permit a complete coverage of the vast literature on this subject. A comprehensive review on kernicterus up to 1959 was edited by Sass-Kortsik (1961). The results of most investigations indicate that staining of brain tissue by bilirubin in infants represents more than a simple change in permeability of the blood-brain barrier, although an increase in permeability might be a predisposing factor. According to Hugh-Jones et a / . (1960), kernicterus is most frequently observed in erythroblastosis fetalis; it occurs only rarely in nonerythroblastotic premature infants in association with severe hyperbilirubinemia. Nevertheless, it is known that kernicterus and increased bilirubin in the CSF may occur in conditions other than hemolytic disease of the newborn in rough proportion to the degree of hyperbilirubinemia (Stempfel, 1955). The variability in the occurrence of kernicterus is considerable; well-developed and undamaged infants might not develop it even in the presence of very high serum bilirubin levels (Zuelzer and Brown, 1961). At the same time, Harris et al. (1958) observed kernicterus in conjunction with relatively low levels of bilirubin in premature

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infants. It is generally agreed upon that the presence of indirect bilirubin in erythroblastosis fetalis is important, although its concentration in the plasma by itself is not unequivocally associated with the development of kernicterus. Local tissue injury is assumed to be present in addition to prematurity, increased barrier permeability, and hyperbilirubinemia to account for the staining of the central nervous system. The development of kernicterus might be promoted by the specific metabolic vulnerability of certain areas of the newborn brain, by anoxic damage, or by the direct toxic effect of bilirubin. Bilirubin is a toxic substance (Day, 1956; Waters and Bowen, 1955; Ernster et a / . , 1957), and Ernster et a / . (1957) showed experimentally that it is capable of inhibiting oxidative phosphorylation in neural tissue. The localization of ATP depletion and impaired respiration within the hyperpigmented brain suggested to Schenker et al. (I 966) that impaired phosphorylation may be an important feature of kernicterus. It is important to remember that most of the serum bilirubin is albumin-bound. Unbound and potentially neurotoxic bilirubin, available for transport into the brain, does not usually amount to more than a fraction of total bilirubin. Although the relative proportion of protein-bound bilirubin in plasma is variable, Odell (1959) as well as Blanc and Johnson (1959) believe that unconjugated, and consequently, more toxic bilirubin is the cause of brain damage because it leads to direct necrosis of the nerve cells. Diamond and Schmid (1966) presented conclusive evidence that only unbound bilirubin is able to cross the blood-brain barrier and that the pigment level of the nervous system and bilirubin neurotoxicity are related to the unbound, rather than to the total, pigment concentration in the plasma. These investigators used 14C-labeled bilirubin which lent itself to more accurate determination in the brain tissue than analysis based on color changes. Diamond and Schmid (I 966) could not reach definite conclusions concerning the regional deposition of bilirubin in kernicterus. They thought that this might be related to the binding and retention of the pigment by already damaged cells, but they also considered the possibility that “selective areas of the brain are a priori more vulnerable to bilirubin and once damaged, retain the pigment more avidly.” Some of their data could be considered in favor of anoxia or respiratory acidosis being responsible for bilirubin deposition because acidosis enhanced the accumulation of bilirubin in the brain, but this conclusion remains only tentative. The clinico-pathological syndrome of kernicterus observed in rats with congenital deficiency of glucuronyl transferase (Gunn’s, 1938, strain of rats) is similar to human kernicterus (Blanc and Johnson, 1959). These rats were used for the experimental study of kernicterus without any difinite answer as to its etiology, although it is clear from the experiments of Menken et al. (1966) that hyperbilirubinemia is necessary, but not alone sufficient, for the uptake of [“C]bilirubin by the brain. The brain tissue of kernicteric animals contained significantly more isotope than tissue from healthy jaundiced animals. Other experimental models yielded equivocal results. Nuclear jaundice and pigmentation of the nerve cells closely resembling that of human kernicterus were produced in healthy newborn kittens by repeated intravenous K~J.jrrcvtr05 pp.

336-339

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L. B A K A Y

injections of a bilirubin-albumin solution (Rozdilsky and Olszewski, 1961). However, these investigators were unable to obtain the same effect in newborn puppies and rabbits. In these two species, additional damage to the nervous system was necessary in order to produce kernicterus. Recently, Rozdilsky (1966) came to the tentative conclusion that under these experimental conditions, albumin counteracts the toxic action of bilirubin on the brain. While some of the experiments indicated the toxicity of bilirubin as the primary noxious factor in kernicterus, Polani (1954) concluded from his experiments in rats that the neurological damage may be associated principally with hepatic damage. The combination of anoxic damage and hyperbilirubinemia has also been considered to be the cause of kernicterus. As Dobbing (1961) pointed out in his critical review on the blood-brain barrier, anoxic damage is a frequent condition in premature infants, and the regional distribution of damage in cerebral anoxia and in kernicterus is somewhat similar. Lucey et al. (1 964):observed that the commonly observed “physiological jaundice” in newborn rhesus monkeys does not lead to kernicterus; neither does the administration of indirect-reacting bilirubin. However, kernicterus can be produced in newborn monkeys by rendering them hyperbilirubinemic after a period of temporary asphyxia. Chen et al. (1966) described electron microscopic changes in kernicterus of newborn rabbits associated with asphyxia as well as hyperbilirubinemia. The ultrastructural alterations suggested that the access of bilirubin to the intracellular compartments of the brain is dependent upon the effect of acute anoxia. They assume that anoxia provokes an increase in the permeability of the cerebral capillaries and simultaneously increases the permeability of the cell membranes. The importance of cerebral anoxia in the development of kernicterus has not been accepted without reservations. For instance, Malamud (1963) studied the distribution and degree of CNS damage in newborns in various types of hypoxemia and concluded that the findings in anoxic damage caused by perinatal trauma or convulsions differ from those of kernicterus. “The lesions observed in convulsive disorders could best be defined as a hypoxaemic encephalopathy and those in kernicterus as possibly a bilirubin encephalopathy. Their occasional coexistence does not detract from their specificity” (Malamud, 1963). In contradistinction to the kernicterus of infants, bilirubin in jaundiced adults might simply mark abnormal brain tissue. The relationship between the duration and severity of hyperbilirubinemia and the transfer of bilirubin into the CNS or CSF is not clear. Although bilirubin is frequently found in the CSF in various types of hepatitis, leptospirosis, cirrhosis, and biliary obstruction, Galambos and Rosenberg (1959) could not establish a definite correlation between the duration of jaundice and CSF bilirubin concentration nor between the bilirubin and protein content of the spinal fluid. Table I, compiled from our own patients with known plasma bilirubin values and cerebral lesions studied at autopsy, demonstrates a certain degree of correlation between the presence or absence of staining of the lesion and the plasma bilirubin level, particularly that of indirect-reacting bilirubin. However, the number of these cases is not sufficient to draw definite conclusions, particularly concerning the relative

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TABLE I B I L I R U B I N S T A I N I N G O F CEREBRAL LESIONS

__

Cerebral Lesion

Caiise

Total

I.

Hepatitis

2 weeks

61.2 mg

2. Ca, liver 3. Cirrhosis 4. Ca, liver

3 weeks 2 months 1 week

23.3 mg 22.0 mg 13.0 mg

5. Ca, liver 6. Hodgkin’s granuloma, liver 7. Colelithiasis

6 weeks

20.4 mg

12 days Transitory, severe for 8 weeks but resolved 5 weeks before death

9.2 rng 14.0 mg

Staining

Indirect 36.9 mg Infarct (approximately 1 month) 14.1 mg Metastasis 13.2 mg Hemangiorna 4.9 mg Old infarct Recent infarct (few days) 7.5 mg Metastasis ? Contusion 2.0 mg Old infarct Recent infarct (approximately 3 weeks)

-1-

+

+ -

+

-

importance of protein-bound or diffusible bilirubin. There is no valid theoretical reason against the assumption that protein-bound bile pigment deposits in most of these gross brain lesions without difficulty. Summary

One can state that at the present time, the exact mechanism of bilirubin transfer and deposition in the brain of the hyperbilirubinemic newborn is not known. It seems to be certain that the process involves more than increased barrier permeability. However, the relative importance of pre-existing cellular damage (most likely anoxic) versus secondary tissue damage due to bilirubin toxicity in kernicteruscannot be assessed, yet. CEREBRAL EDEMA

Edema of the brain is produced by the increase of its water content. In addition, there is a variable increase of solutes ranging from electrolytes to large protein molecules. It is, therefore, obvious that this condition is of considerable interest from the point of view of the blood-brain barrier because the excess fluid and particulate matter originates in the blood plasma and could hardly pass into the nervous tissue without a change in membrane permeability. This is particularly true in those types of brain edema where the edema fluid is proteinaceous; the presence of serum albumin in the fluid is prima facie evidence of increased barrier permeability since, under normal circumstances, the exchange of large protein molecules between plasma and brain tissue is minimal. In addition to the blood-brdin barrier aspect, recent electron microscopic investigations of edematous brains led to important discoveries concerning the extracellular Refirmwr pp. 336-339

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space, a compartment that must always be considered when attempting to measure barrier permeability. Under normal conditions, the space between the cells of the central nervous system is narrow, although the narrowness of these clefts does not necessarily disqualify them as important pathways of fluid and small molecules (Nichols and Kuffler, 1964). It seems that in the gray matter, edema is almost exclusively intracellular. In the white matter, excess fluid might accumulate, either between the cells, thereby greatly enlarging the extracellular compartment, or in the cells. Studies on cerebral edema clearly indicate that osmotic changes in plasma and brain fluids, both extra- and intracellular, result in rapid changes in fluid and solute transport between the various tissue compartments. The effect of these fluxes on barrier permeability should not be underestimated. Recent experiments by Klatzo, Wisniewsky, and Smith (1965) indicate that such reversible changes in plasma osmolality as that caused by intravascular infusion of 30 % glucose result in a highly abnormal permeability of the cerebral capillaries for proteins. The “leakiness’ of the blood vessels is of short duration and disappears as soon as the plasma osmolality approaches normal values. Edema of the brain is not a single entity. Some of the various types of brain swellings differ not only in the localization and chemical composition of the excess fluid but also in the permeability of the blood-brain barrier. The chemical and structural characteristics of various brain edemas were recently described by Bakay and Lee (1965); the brief presentation which follows will be limited to the observations on barrier permeability.

Cold-induced edema The edematous white matter shows vital coloration after intravascular trypan blue administration in an experimental model where the swelling of a hemisphere is produced by freezing of the cortical surface. Similar staining was seen after the application of Evan’s blue (Clasen et al., 1962) and fluorescein-labeled serum proteins (Klatzo et al., 1962). The vital staining of the swollen white matter is subject to the time which has elapsed from the injection of the dye (Bakay and Haque, 1964). The dye extravasates at the marginal zone of the injured cortex and infiltrates the edematous white matter gradually, reaching complete distribution only 24 h after its administration. The same spatial and temporal distribution was found by using RlSA as a tracer (Bakay and Haque, 1964), this corroborates previous theories that many vital dyes, including trypan blue, are protein-bound in plasma, and their recovery in the abnormal brain tissue corresponds to that of plasma proteins. A similar, gradual diffusion from the point of entrance (the marginal area of the cortical lesion with its leaky blood vessels) into the area rendered edematous was observed for eleztrolytes, particularly for sodium and chloride. However, since the relative distribution of these cations and anions inside and outside the cell membrane is not well-known, under these circumstances, the conclusions concerning the permeability of the blood-brain barrier are best based on the entrance into the brain of proteins for which the barrier is normally impermeable. In summary, the bloodbrain barrier was found to be grossly altered and increasingly permeable for large

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particles in cortical freezing at the level of the border zone of the lesion. From there, the extravasated components of plasma penetrate the edematous white matter subsequently; however, the permeability of the blood-brain barrier within the region of the edema, itself, is not perceptibly altered. Edema surrounding cerebral neoplasms and other space-taking lesions has long been known to show coloration with vital dyes and to reveal an increased exchange of other substances including proteins with plasma. The edema of this type as well as the changes in barrier permeability are probably identical with that seen after trauma; the propagation of serum exudate into the surrounding brain tissue is facilitated by the leakiness of the neoplastic vessels.

Other traumatic edemas Vital staining was observed in brains rendered edematous by exposure and manipulation (Prados et a/., 1945), by implantation of dry psyllium seed (Sperl et a/., 1957; Samojarski and Moody, 1957), and by inflating a balloon placed in the extradural space (Ishii et a/., 1959). The dyes applied included trypan blue, acridine dyes, Evan’s blue, and di-iodo-fluorescein. In some of these experiments, tissue proteins and electrolytes were studied. Significant increase of albumin was found in the swollen tissue surrounding implanted psyllium (Hauser et a/., 1963). The greatest concentration was found nearest to the psyllium mass; it decreased progressively as the distance ofthe tissue from the lesion increased. Cutler et al. (1964) studied the movement of 1251labeled serum albumin in brain tissue rendered edematous by balloon compression. They found a close relationship between the penetration of albumin and Evan’s blue, indicating that much of the dye is protein-bound. Just as in cold-induced edema, the passage of albumin from blood into brain tissue occurred at the marginal zone of trauma. From this point of entry, the edematous tissue, which was strictly localized in the white matter, was gradually penetrated by serum albumin, indicating a break in the blood-brain barrier at the level of the lesion but not in the edematous white substance.

Injlammatory edema

In inflammatory edema, which is produced by intracerebral injection of bacterial endotoxins, purified protein derivative of tuberculin, and such additional substances as graphite, the permeability of the blood-brain barrier was increased for Geigy blue (Gonatas e t a / . , 1963). Here, again, the edema is located in the white matter and consists of a protein-rich exudate. There is a considerable freedom in exchange of RISA between plasma and edematous tissue (Katzman et al., 1964). Triethy It in - induced edema Triethyltin-induced edema is of considerable interest regarding the permeability of the blood-brain barrier because it seems to be very different from other types of brain Refiwnccs pp. 336-339

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swelling. Edematous changes are limited to the white matter. The excess fluid, at least in its initial stage, is a non-proteinaceous plasma ultrafiltrate situated in intramyelinic vacuoles which are produced by a split in the myelin sheath (Aleu et al., 1963; Lee and Bakay, 1965). There is no pathological alteration in the ultrastructure of the capillaries, glia cells, or in the size of the extracellular space. It is, therefore, perhaps not surprising that the permeability of the blood-brain barrier for large molecules is not affected. The edematous white matter does not stain with trypan blue and other vital dyes, and large molecules such as albumin do not penetrate the edematous tissue from the blood stream (Magee et al., 1957; Kalsbeck and Cumings, 1962: Katzman et al., 1963; Bakay, 1965). This represents a striking contrast with traumatic and inflammatory edemas. The lack of abnormal morphological changes of the vasculature as well as that of the glia cells is undoubtedly responsible for the normal state of the barrier; unfortunately, this normal state of all structures believed to be instrumental in barrier function does not allow us to draw significant conclusions from triethyltin edema as to the morphological basis of the blood-brain barrier. Edema associated nith cerebral anoxia

Edema associated with cerebral anoxia reflects only one aspect of a complex metabolic disturbance. The exact nature of the changes in tissue structure and metabolism is not adequately known; it is particularly difficult to separate the disturbances pertaining to membrane permeability and to the blood-brain barrier in general from those of anoxic damage to the cells, if such a distinction is, indeed, possible. The complexity of varicus factors involved can be illustrated by the fact that there is very early structural damage in the mitochondria (Bakay and Lee, 1967) that are quite resistant in other types of edema, while on the other hand, the fluid accumulation in anoxia is very moderate. Clearly, edema in anoxic brain damage is only one of many alterations. Studies were directed to investigate the permeability of the capillaries and of the blood-brain barrier, in general, under anoxic conditions because a change in permeability was considered to be essential for the development of edema. The findings obtained with the use of vital dyes were contradictory. Broman’s (1949) experiments on cats showed that complete occlusion of the cerebral circulation caused no disturbance in vascular permeability for trypan blue. Grontoft (1954) came to a similar conclusion, stating that in adults, anoxic injury to the blood-brain barrier cannot be demonstrated with trypan blue, although in infants, barrier damage is closely related to the degree of asphyxia. Becker and Quadbeck (1952) used a more easily permeable vital dye, Astroviolett FF. They were able to demonstrate barrier damage in anoxia produced in a high-altitude chamber and in experimental sub-acute carbon monoxide poisoning at a stage when there was no microscopic evidence of tissue damage. Hodges et al. (1958) used fluorescein to study the relationship between hypoxia and blood-brain barrier permeability with special regard to the relative value of various perfusion techniques in vascular surgery. They found that the appearance of fluorescein in the central nervous system was a sensitive indicator of faulty perfusion technique or inadequate pump-oxygenators with cerebral hypoxia as a result.

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It is, of course, conceivable that permeability changes exist for electrolytes and small molecules, even in the absence of gross changes to vital dyes, because most of the vital dyes used are essentially protein-bound. Factors other than hypoxia, alone, may be responsible for the transfer of substances from blood to brain. Arterial increase in C02 concentration, when severe enough, might be a more important factor. Temporary and easily reversible increase in the permeability of the blood-brain barrier for trypan blue was observed in rabbits by Clemedson, Hartelius, and Holmberg (1957) on inhalation of a gas mixture containing 10 % COZor more. In their opinion, hypercapnia should not be disregarded as a cause of cerebral damage, particularly in asphyxia where the component of anoxia “sometimes seems to have been over-emphasized.” Brierley (I 952) noticed an increase in ~erebral,3~P uptake after inhalation of a C02 and 0 2 mixture, but only when the mixture contained 20 % C02. His tentative conclusion was that the increase in the SZP content of the brain was either due to vasodilatation or to increased capillary permeability, or to both. Goldberg, Barlow, and Roth (1961) showed that exposure to 25 % COZ increased the cerebral concentration of phenobarbital, salicylic acid, acetazolamide, and urea in cats. This increase was not related to changes in blood flow and could not be brought up by inhalation of 5 % C02. Exposure to a high C02 content in the inhaled gas mixture with subsequent severe hypercapnia seems to increase the permeability of the blood-brain barrier, even without additional hypoxia. Goldberg, Barlow, and Roth (1963) studied the effect of 25 % COZ on the cerebral uptake of [35S] sulfate and [14C] urea. They found that although the steady-state sulfate space had not changed, the entry of 35s was greatly enhanced within a few minutes after injection. They also observed regional differences in the cerebral deposition of SSS, including an increased concentration in the central core of the white matter, which suggested irregular changes in vascular permeability. Bakay and Bendixen ( 1 963) separated, experimentally, the various factors involved in asphyxia, namely, anoxia, CO2 retention, and increased venous pressure. They found that real brain swelling occurred only when anoxia was associated with hypercapnia. However, the blood-brain barrier was quite resistant under these circumstances. Although there was an increase in the exchange of electrolytes between plasma and brain tissue, vital staining with trypan blue and significant uptake of albumin by the brain from the blood stream occurred only on extreme hypercapnic hypoxia with arterial 0 2 saturation below 20 % to 25 % and an arterial pH below 6.75. Severe anoxic-ischemic lesions and edema develop following the production of carotid ligation and respiratory hypoxia (Plum et al., 1963). Under such circumstances, there is vital staining and marked swelling of the affected portion of the brain. It is obvious, however, that this experimental model represents infarction and total tissue destruction rather than simple anoxic effect.

Summary Although the amount and distribution of the excess fluid varies in different types of brain edema, they all reveal an increase in tissue water as well as sodium and chloride. Rrjerenrrs pp. 336-339

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However, the exchange of large mole-ules (vital dyes, albumin, etc.) between plasma and edema fluid is limited to those cerebral edemas where structural damage to the small blood vessels can be demonstrated. Furthermore, the point of entry into the nervous system for these large particles is the wall of the injured vessel. B R A I N I N J U R I E S A N D THE B L O O D - B R A I N B A R R I E R

Fresh brain wounds stain immediately with vital dyes injected into the blood stream. They also concentrate various radioactive tracers exclusively or to a much greater extent that the surrounding normal brain tissue. Bakay (1960) found that cerebral lesions exchange sodium with plasma rapidly. This results in an early concentration of z4Na in the injured tissue (Fig. I). However, the ratio of 24Na content between injured

Fig. 1. Transverse section of cat brain (A) and corresponding radioautographs 23 min (B), 70 min (C), and 315 rnin (D)after intravenous injection of NaZ4.(From Bakay, 1960).

and normal brain tissue diminishes during the subsequent few hours because a gradual sodium exchange between normal nervous tissue and blood increases. On the other hand, the 24Na concentration of the injured part remainsconstant and then diminishes due to the fact that the radioactive sodium of the lesion remains in free exchange with that of the plasma, and consequently, decreases in linear proportion to the declining plasma 24Naconcentration. Similar observations were made with 23P (Bakay, 1955). A significantly increased uptake of 32P by the lesion when the tracer was given two hours before death still existed six weeks after trauma. In mild traumas, the increased exchange of substances might be limited to small particles such as the increased uptake of S2Pobserved during temporary concussion by Cassen and Neff (1960). However, injuries associated with visible structural alterations

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of brain tissue usually result in the exchange of serum proteins between plasma and brain. The extent of increased barrier permeability around a lesion, as well as its duration, varies according to the physico-chemical characteristics of the substance used for measurement. Lesions produced by focused ultrasound, as described by Bakay et al. (l956), were used to measure the extent of barrier damage with different indicators. Ultrasonic lesions are very suitable for this purpose because they are spherical and easily reproducible. Furthermore, they are surrounded by normal tissue and are not connected to the surface by needle tract. The results shown in Fig. 2 are based on

32

P

7-7

[I3'I]albumin

trypan blue

Fig. 2. Relative size of increased uptake by spherical ultrasonic lesion in brain tissue of 3zP, trypan blue, and ['3'l]albumin.

measurements of freeze-dried, 25 ,u sections and corresponding contact autoradiograms of cat brains thirty minutes after intravascular administrations of the tracers. Calculations of the volume of tissue with increased permeability are even more revealing. They indicate that the total area of increased uptake of RISA and trypan respectively, of that of 32P. blue is 36 % and 30 The duration of increased permeability also varies according to particle size and other unknown factors. The injured blood vessels seal their walls faster for large molecules than for small particles. As a result, increased uptake of small molecules in the lesion can still be observed at a time when the penetration of vital dyes and protein molecules from the lumen of the vessels is already arrested. Tschirgi (1950) emphasized the importance of protein complexes in the permeability of the blood-brain barrier to dyes. Bakay and Haque (1964) demonstrated the striking similarity between trypan blue and 131I-labeled serum albumin in their exchange between plasma and injured brain tissue. This strongly suggests that the great bulk of trypan blue is protein-bound and that this vital dye can be considered, for all practical purposes, a protein (albumin) tracer. In experiments involving the use of radioactive isotopes, the tracer content of the blood has to be taken into consideration since experimentally-induced cerebral

x,

Rifiwtrces pp. 336-33Y

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lesions often interfere with the blood flow, usually in the direction of stagnation. However, the measurements of Broman et al. (1961) indicated that the false increase in tissue radioactivity caused by pooled blood is less significant than the possible error produced by the rinsing of cerebral vessels. The return to normal of increased blood-brain barrier permeability is gradual, associated with the healing of brain wounds. Normal permeability is established much earlier for large particles than for small molecular substances. Abnormal barrier permeability for serum albumin, an “all-or-none’’ phenomenon , since normally, albumin does not penetrate from the vascular network into the brain substance, was first illustrated by autoradiography in brain injuries by Rozdilsky and Olszewski (1957). Lee and Olszewski (1959) studied the permeability of the blood vessels for RISA at various stages of the healing of brain wounds. The area showing albumin uptake decreased gradually with the passage of time from the injury. The barrier was no more permeable for albumin after three weeks. Abnormal permeability for small molecules, such as electrolytes, exists for a considerably longer time. Similar observations were also made in man in the form of brain scanning of patients with various types of head injuries. It is not surprising that abnormal uptake of various radioactive compounds can be seen in areas of cerebral contusion immediately after the injury (Fig. 3). The process of healing eventually results in a restoration of normal permeability; old scars cannot be visualized by scanning. By using relatively large and metabolically inert compounds, the period of increased permeability can be assessed. Van Vliet et al. (l965), for instance, studied post-surgical brain scans with

Fig. 3. Brain scan in head injury, 4 h after i.v. injection o f 750 pC of [203Hg]~hlormerodrin.Increased concentration of the labeled substance can be seen in the contused frontal lobe.

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polyvinylpyrrolidone (PVP) labeled with l 3 l I to determine the effects of surgical trauma on the vascular permeability of the brain. PVP is an inert substance of high molecular weight which is normally excluded from brain tissue. There was an increased uptake in the crmiotomy site in all patients with cortical incision or resection, or after pressure on the cortex by retraction. The intensity of localized radioactivity diminished as the post-operative period increased. Eventually, the scans became normal ; no positive scan was seen after 79 days following craniotomy. The increasing use of brain scanning in man with different radioactive indicators will contribute, in the future, to our understanding of the spatial and temporal aspects of blood-brain barrier permeability. From a basic point of view, explanations of the blood-brain barrier phenomena can be arranged in two main groups. One theory lays emphasis on the permeability of the capillary wall while the other identifies the selective barrier function with the specific function of the nervous tissue proper. When applied to pathological conditions, this second theory would imply that metabolic, rather than vascular, changes within the altered area of the brain are responsible for the increased concentration of various substances. Although investigative work on the barrier permeability of injured brain tissue involved the use of not only metabolically active but also, inert substances, the relative distribution of the various tracers in the intracellular and extracellular compartments at various time intervals after their transfer into the brain cannot be evaluated from the majority of the published results. The relative importance of the different vascular and cellular membranes remains, therefore, obscure. Hess (1955) thought that a PAS-negative ground substance is responsible for the “blood-brain barrier effect” because this substance, presumably consisting of mucopolysaccharides, disappears in brain wounds to be gradually re-elaborated after a while However, this theory cannot be confirmed by electron microscopy which fails to reveal the presence of an intercellular ground substance. A modified hypothesis (Millen and Hess, 1958) was then put forward that the ground substance immediately surrounding the blood vessels may play an important role in the maintenance of the blood-brain barrier, but this assumption also fails to hold up under electron-microscopic scrutiny. Bakay et a/. (1959) measured the uptake of 32P by normal and injured brain tissue by applying the tracer solution directly to the cerebral tissue by supra-cortical or cisternal application excluding, thereby, vascular channels. During the process of absorption of 32P by the brain tissue, there was no increase in concentration of the isotope within ultrasonically-produced lesions when compared with the surrounding normal brain (Fig. 4). This represented a marked contrast to other experiments that demonstrated high uptake of 32P by the same type of lesions after intravascular administration. The conclusion of Bakay et a/. (1959) was that there is no selective concentration of 32P in the lesion once the isotope has been made equally accessible to injured, as well as normal, tissue. The route by which 32Parrives at the lesion site would be irrelevant if the increased uptake by the lesion werecaused by cellular metabolism alone. Similar observations were made with 24Na; intra- and subcortical lesions did not take up more sodium than normal brain tissue after the direct application of an isotonic 24Na solution over the pia-covered surface. This represents a striking R&rciiczs p p . 336-339

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Fig. 4. Upper: Unstained; dry-frozen section of both upper parietal lobes of cat. Arrow points to vitally stained lesion in the lower layers of the cortex, produced by focused ultrasound 4 h before death. Middle: Radioautograph of same section 200 min after application of isotonic solution containing 32P over thepia-covered cortex. Exposure time: 3 days. Lower: Radioautograph of the same section. Exposure time: 4 days. (From Bakay et a/., 1959).

contrast with the fifteen-to-one ratio between injured and normal brain 24Naconcentration within the first hour after intravascular injection of the isotope (Bakay, 1960). Summary

Structural injury to the brain tissue results in an immediate increase of transfer from blood into traumatized brain of substances that normally are transported slowly or not at all. The abnormal exchange comes to an end at a certain stage of the healing process, earlier for macromolecules than for substances of small particle size. There is some evidence that an increase in vascular permeability characterizes the initial phase of altered barrier permeability; the subsequent migration, retention, and participation of the tracer substances within the nervous tissue is the result of a combination of factors that, at the present time, defy detailed analysis. BRAIN TUMORS

Most brain tumors accumulate various substances, such as vital dyes, various radioactive isotopes, etc., from the blood stream. The exchange of these substances between plasma and tumor tissue is relatively free, or at least much faster than their exchange between plasma and normal brain tissue. The concentration of many radioactive compounds is much greater in the tumor than in the surrounding brain tissue, and thus, a tumor/brain concentration ratio exists which can be measured and exploited for diagnostic purposes. This principle is the basis of radioactive brain scanning for the diagnosis and localization of intracranial neoplasms. In order to determine the possible mechanism responsible for the uptake of sub-

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stances by normal and neoplastic brain tissue, one has to consider differences in vascular permeability, size of extracellular space, and metabolism. Vascular permeability Brain tumors, particularly glioblastomas and metastatic tumors, contain abnormal blood vessels. Some of these vessels take the shape of vascular malformations including tortuous, lacunar, and aneurysmal dilatations, glomeruloids, and arteriovenous shunts. Nystrom (I 960) believes that purely mechanical factors are responsible for the irregularities of the vascular lumen: “Dilatation of the wall in shunts, glomeruloids, and lacunar or aneurysmal vessels was considered to be the result of locallyincreased blood pressure bearing upon a defective, and consequently, weak portion of the vessel wall, the elevated intravascular blood pressure being due to narrowing of lumen distal to the defective portion” (Nystrom, 1960). Such abnormalities could easily be responsible for a change in vascular permeability in terms of increased filtration. In addition, there seems to be a profound metabolic (anaplastic) change in the cellular components of the neoplastic vasculature, at least in malignant brain tumors. This is manifested by endothelial proliferation in the wall ofarterioles, venules, and capillaries. Hagerstrand (I 961) considers the vascular proliferation seen in cerebral metastases as a sign of a specific reaction of the blood vessels of the brain because similar changes cannot be seen either in the primary tumors or in their seedings to other organs but the brain. The possibility of increased permeability across the neoplastic capillary wall by heightened active transport, in contrast with increased diffusion through a structurally deficient vessel, must also be seriously considered. Wright (1963) followed the development of vascularization in artificially-induced ependymoma of mice. Initially, the newly formed arteries were thin-walled and had a poorly defined muscular layer. With the passing of time, the cellular arrangement became more anaplastic; this occurred at the same time as other, profound changes in the vasculature, namely, the development of arterio-venous communications and thin-walled sinusoids that emptied into large, tortuous veins. On electron microscopic examination, the blood vessels of glioblastomas reveal signs of increased metabolic processes in the endothelial cells (accumulation of mitochondria and vesicles) as well as vacuolization and marked variation in breadth of the basement membrane (Nystrom, 1960). Nystrom (1960) also pointed out that the vascular changes were much less pronounced in gliomas of a lesser degree of malignancy. The ultrastructure of the blood vessels of astrocytomas and oligodendrogliomas did not differ essentially from that of normal vessels. This, of course, might be one of the reasons why radioactive brain scan is sometimes “negative” in these gliomas. However, the electron microscopic morphology of the capillaries involved in tumor formation has not been adequately studied, and further investigations are needed. According to Torack (1961), capillary changes vary depending on the involvement of the tissue in the pathological process. In the tumor, itself, the endothelium is hyperplastic, and the perivascular zone is enlarged. In the peripheral zone of the neoplasm, the basement membrane is thickened, R i ~ r r c v i1.5 i pp. 336-339

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fenestrated, and filled with cell processes and dense bodies of lipid material. Torack (1961) related these changes to the increased vascular permeability for electrolytes and colloids, and to the phagocytic activity of the pericapillary cells. Although a parallel between embryonic and neoplastic blood vessels, connoting increased permeability of their walls, is frequently drawn, Tani and Ishii (1963) were unable to arrive at a definite conclusion. As far as their ultrastructure is concerned, embryonic capillaries have a poorly developed basement membrane which might explain their leakiness. However, tumor vessels have many additional features, such as the increased metabolic activity of their endothelial cells and perivascular astrocytes, or the close attachment of tumor cells to the basement membrane, as well as the enlarged extracellular space in the vicinity of the blood vessels; any of these structural peculiarities or a combination of them all could be responsible for the increased exchange of material between plasma and tumor tissue.

Blood content of tumor tissue There is considerable variation in the volume of blood per unit of tumor tissue as well as in the speed of its circulation (Ganshirt and Tonnis, 1956) compared to that of normal brain which averages about 0.5 % in the white matter and 2.5 % in the gray matter. Although the blood content of a mature astrocytoma might not be greater than that of normal brain tissue, most tumors contain more blood vessels per unit of tissue than normal brain. Although we do not have enough data, it is obvious that in a vascular tumor, blood volume is a factor that has to be taken into consideration even without assuming that the vascular permeability is increased. In many tumors, the blood vessels are not only numerous but also distended. Consequently, tracers, including radioactive isotopes, can accumulate in a tumor at a time when their concentration in blood is high. Although the distinction between that part of the tracer which is still contained to the vascular lumen and that which has egressed into the surrounding tissue is probably quite arbitrary after a short interval following its injection into the blood stream, the amount pooled in the blood is important in vascular tumors with a slow transcirculation time, such as some meningiomas. Long et al. (1963) emphasized that those substances which produce a high tumor-to-brain concentration ratio are protein-bound in plasma; this statement implies that a significant portion of these tracers remains in the blood stream. Nevertheless, it seems to me that the importance of radioactive blood contained by the tumor as the main source of some positive brain scans has been over-emphasized in the past. Experimental tumor, such as implanted fibrosarGomas (Matthews and Molinaro, I963), revealed a residual blood volume of only 4.6 % which was less than double that of the normal brain and could hardly be held responsible for the accumulation in the tumor of 1311-labeled albumin, although this large molecular tracer remains in the blood for a considerable length of time. Pinocytosis At this point, it is worth reviewing the possible mechanism of the uptake by tumor of

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substances with large molecular weight. Some of these compounds are very valuable in radioactive brain scanning and in the study of altered barrier permeability. It is also known that they are transferred into the neoplastic tissue from the blood stream by other means than simple diffusion. In contrast, they remain almost entirely within the vascular lumen in normal brain tissue. It is not difficult to explain their presence in necrotic brain or tumor tissue: there, the continuity of small blood vessels is broken, and blood or plasma floods the surrounding tissue, carrying with it almost undiluted amounts of radioactive proteins. However, this mechanism does not apply to neoplasms with reasonably intact vasculature. Few attempts were made to determine the transcapillary exchange of large particles, particularly albumin, in brain tumors ; the conclusions are still somewhat tentative. The exact localization of RISA in brain tumors at the cellular level by light microscopy and microscopic radioautography is all but impossible. Tator et al. ( I 965) were unable to distinguish between vascular and cellular factors in the deposition of radioactive serum albumin in various brain tumors. The incorporation of RISA in cells by pinocytosis is strongly suspected because this mechanism of transfer was observed under other pathological conditions (Klatzo and Miquel, 1960). Incorporation of large molecules into cells of the vascular wall is occasionally seen by electron microscopy in normal brain; the number of pinocytotic vesicles formed by endothelial and glial cells increases greatly in traumatized, and presumably, neoplastic nervous tissue. Raimondi (1964) attempted to localize RISA in human brain tumors at the electron microscopic level. In his opinion, serum albumin is transferred into the capillary endothelium and beyond by pinocytosis. This conclusion is supported by the observation that the number and size of pinocytotic vesicles is increased in tumor tissue, an observation which is also shared by Bakay and Lee (1967). Although pinocytosis is obviously operational in the bulk transport of large molecules, its relative importance in the uptake of these compounds by tumor tissue awaits further clarification. Since pinocytosis is a relatively slow, gradual process, it fails to explain completely the increased barrier permeability that is noticeable immediately after the injection of the tracers.

Extracellular space

It has been suggested by several investigators that the main difference in the uptake of various substances between tumor and normal brain tissue is due to the respective size of their extracellular spaces. This reasoning closely follows the argument that the blood-brain barrier effect in normal brain is not caused by a hindrance to passage through the capillary wall, but rather, at the level of the cell membranes of the central nervous system. The relative impermeability of the normal “barrier” would, then, be based on a functionally inadequate extracellular compartment. An extreme view, no longer accepted, denied the existence of any measurable room between the cells; this theory, however, is contrary to both morphological and physiological evidence. Nevertheless, a comparison of the intercellular space of normal and neoplastic Hifcwnc cs pp. 336-339

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brain tissue should be undertaken to clarify this issue. Unfortunately, there are no sufficient data for this purpose. The exact size of the extracellular compartment is a matter of conjecture even under normal conditions. This subject has been reviewed recently by Bakay and Lee (1965). The present estimate of the extracellular space ranges from 5 % to 20 % of volume; most investigators favor the 15 range. However, there is a difference between the gray and white matter in this respect, and regional differences may exist as well. Although a systematic survey on the size of the extracellular space in brain tumors has never been undertaken, electron microscopic observations by Raimondi el al. (1 962) suggest that an anatomically large extracellular compartment is not likely to be the reason for the increased uptake of different substances by a neoplasm. The intercellular clefts in astrocytomas and glioblastomas are probably not significantly wider than in normal brain tissue, except for those spaces that surround degenerating cells. In meningiomas, which almost invariably concentrate radioactive tracers in high concentration, the cells are positioned closely to one another. Whether the “functional” extracellular space, a compartment that might include some intracellular elements and reflects the distribution of primarily extracellular test substances rather than a visible space, is larger in tumors than in normal brain remains a moot question. Some investigators approached this problem by comparing the uptake by tumor of primarily intracellular and extracellular tracers, respectively. However, such a separation is arbitrary because we are not in a position, at the present time, to determine the relative distribution of these substances in the various compartments of the central nervous system. Analogies taken from their behavior in other organs do not necessarily apply to the brain. Matthews and Molinaro (1963), in their studies on transplanted fibrosarcomas, concluded that “for extracellular substances, tumour concentration depends on extracellular tumour spaces.” However, the weakness of their argument is revealed by the statement that the mean extracellular space of the tumor was 75.5 %; after correction for the Donnan effect and the rdtio of plasma to whole blood concentration, it was 43.6 %. This, although feasible in a few selected tumors, seems to be overly large for members of the glioma group. On the other hand, they allowed only 3.9 for the extracellular space of the normal brain; this estimate is too low because after considering that the residual blood content was 2.4 %, it would indicate a virtual absence of space between the cells. The “extracellular” substances used by these authors included 82Br, BzGa, 95Zr, 1Wb, l311, Ag, 95Nb, and 1311 serum albumin. Although some of these ions probably do not enter the cells, the purely extracellular nature of others, to name 82Br, alone, cannot be accepted. Their conclusion remains, therefore, less categoric: “. . . there also appears to be a ‘barrier’ of some kind to uptake of intracellular substances, and so lack of extracellular space will not explain all the results. Thus, there appear to be two possibilities, either (a) the blood-brain barrier is partly due to lack of extracellular space and partly to some difference in cell permeability or uptake in brain cells compared with cells in other organs, or (b) the blood-brain barrier is due to a physical barrier which is impermeable to extracellular substances but slowly permeable to intracellular substances” (Matthews and Molinaro, 1963).

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Cellular metabolism Although brain tumors do not compare favorably with normal brain tissue in many aspects of their metabolism, such as oxygenation and glucose consumption, metabolism might be responsible for their accumulation of a variety of metabolically active substances. Reasonable as this statement might be, it has to be pointed out that no substance has been found which would achieve a concentration in the brain that would exceed that of the initial blood level or those of such metabolically active tissues as liver or kidney. In fact, as Long et al. (1963) pointed out, the seemingly selective concentration of tracers in brain tumors is due to the low normal background rather than to the abnormally high accumulation in the tumor. There is no conclusive evidence that the concentration in tumors of metabolically active agents is significantly greater than that of inert substances of similar physicochemical properties. Although Selverstone and Moulton (1957) have suggested that the relatively high phospholipid fraction of tumor tissue is instrumental in its 32P uptake, the search for compounds that would selectively concentrate in brain tumors because of their participation in specific metabolic processes has not been successful. The latest investigations of Tator et a/. (1966), for instance, have indicated that the uptake of fatty acids by tumor tissue exceeds that of brain and some other normal organs. The fatty acids are partly oxidized as a metabolic fuel and partly utilized in lipid synthesis by gliomatous tissue. Despite these promising characteristics, no truly significant difference was found between the tumor uptake of [1311]oleic acid and 11:jllIserum albumin except during the initial hour of transfer. Experimental studies furnished important data but failed, so far, to supply us with definite proof concerning the role of metabolism. Mundinger (1965) used in vitro models of tumor cell colonies and investigated the uptake by the cells of various compounds used in radioactive brain scanning before and after the blockage of several metabolic processes by specific inhibitors. His conclusion was that [13lI]albumin was taken up by the cells through pinocytosis; 74As and 1311 were thought to be predominantly extracellular in location, and W u - , 206Bi-, and [2°3Hg]chlormerodrin were mostly intracellular. Some tentative conclusions were reached as to the relative role played by active transport and by diffusion in the uptake of the “extracellular” ions. Interesting as these results are, it should be kept in mind that they were obtained in tissue culture under conditions that could yield only very speculative conclusions when applied to conditions in vivo. Matthews and Molinaro (1963) attempted to correlate the uptake of different radioactive substances by tumor tissue and correlate it with their biological characteristics. The experiments were performed in subcutaneously transplanted fibrosarcomas of rats. Their reasoning in doing this was that any tumor, including brain tumors, has its own vascular structure and metabolism which is comparatively independent from where it is growing. This point, however, can be argued because gliomas, the most frequent type of cerebral neoplasms, possess structural and biological peculiarities which make them, even in their most malignant and anaplastic form, inseparably Refirrirws pp. 336-339

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part of the nervous system. Indeed, one of their characteristic traits is that they do not metastatize into any other organs. Matthews and Molinaro (1963) considered 206Bi-, 43K-, SdRb-, 65Zn-, ”Mnand 2vJHgchlormerodrin] as entirely or predominantly intracellular tracers. However, chlormerodrin is almost completely protein-bound in plasma; we do not know how much of this protein complex is broken down and metabolized in neoplastic brain tissue within the time range involved in brain scanning. Matthews and Molinaro ( I 963) concluded that “for intracellular substances, since tumour concentration is approximately constant, the ratio depends on brain concentration and hence, on blood concentration, and substances which are rapidly cleared from the blood give the best ratio”. However, they point out that no selective concentration of any substance by the tumor relative to other organs was found; the accumulation of the isotopes, even the predominantly intracellular ones, did not depend on some special property of tumor metabolism. Summary

Both structural and metabolic peculiarities could be responsible for the accumulation of substances in brain tumors from the blood stream. The relative importance of the different potential factors involved in this process remains uncertain. The theory that the blood-brain barrier is merely a reflection of cerebral metabolism is rather difficult to reconcile with isotope distribution studies (Long et a]., 1963). The data

Fig. 5. Lateral brain scan, 4 h after i.v. injection of 750 pC of [203Hg]chlormerodrinreveals the presence of a parieto-occipital tumor (glioblastoma).

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Fig. 6. Lateral brain scan, 4 h after i.v. administration of 750 pC of [?03Hg]chlormerodrin.Positive concentration of the isotope in a large area within the right hemisphere; this corresponds to a recent infarct of the brain supplied by the right middle cerebral artery.

obtained, so far, can be readily explained only by the theory that a barrier exists based on selective vascular or cellular membrane permeability, and that this selectivity is reduced or lost in tumor tissue. This assumption is based on the following considerations (Bakay, 1956): 1. Substances which accumulate in brain tumors have different physico-chemical characteristics, such as molecular weight, electric charge, solubility, degree of dissociation, etc., without a common denominator. 2. Inert or metabolically unimportant substances concentrate in the lesion as well, or almost as well, as metabolically active compounds. 3. The concentration of many tracers in tumors is of a similar order of magnitude as in other forms of pathology (brain tissue subjected to trauma, infarction, or infection; Figs. 5, 6). This was also pointed out by Heiser and Quinn (1966) who recentlycompared the brain scan pattern of ischemic infarcts and gliomas by using technetium 99m pertechnetate as a tracer. No significant difference was found between the intensity or homogeneity of the uptake in the two categories. 4. There is no true selectivity in the uptake; none of the agents studied, so far, concentrated in brain tumors to a greater extent than in some other organs of the body. Neither did the tumor concentration ever exceed the initial blood level. 5. Generally speaking, the highest concentration of isotopes is found in nonneurogenic brain tumors; these include a great variety of neoplasms from slow groRcJercnr.e.r f f . 336-339

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wing, benign meningiomas to rapidly expanding, malignant metastases from other organs. Gliomas, the truly characteristic tumors of the central nervous system, do not behave uniformly. Accumulation of tracer substances is most commonly observed in glioblastomas. The uptake rate by astrocytomas, which have a tissue structure that closely resembles that of normal brain, is usually only slightly higher or not higher at all than that of normal brain tissue. The inherent implication of this observation is that really meaningful statements concerning the neoplastic changes of the bloodbrain barrier should be limited to tumors of the glioma group since tumors that are not linked genetically to elements of the nervous tissue could not necessarily share in its organization which includes the blood-brain barrier. ACKNOWLEDGEMENT

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DISCUSSION

D. B. TOWER: I would like to say a word of caution about the presence of ruptured membranes seen in electronmicrographs. When a cell is swollen, one can not necessarily attribute a ruptured membrane to the fact that the cell was swollen beyond its elastic capacity or to other properties of the membrane, because a knife also has had to pass across the section. Certainly, in light-microscopy, knives have been known to create such artifacts. Therefore, I would object to anybody’s being dogmatic about these ruptured membranes. Furthermore, I have a feeling that cells can withstand a tremendous amount of swelling without rupturing the membrane. 1 would also like to suggest that we be cautious about saying that spaces never increase or decrease in size, because I doubt if we understand all the factors influencing these spaces between the time when we sample the tissue and the time when we are able to look at it with the electronmicroscope. I am also disturbed about the apparent constancy which is insisted upon for the size of the spaces between cells. This does not seem quite reasonable from a biological point of view. Finally, I would be cautious concerning ruptured membranes in such conditions as edema, particularly in attempting to interpret where a marker like ferritin might logically be. Dr. Brightman, I remember, called attention to the fact that you can very easily translocate or dislocate ferritin from one place to another if you are not careful with your preparation. I don’t mean this in terms of criticism of Dr. Bakay, or anybody else, but we must be very careful in making statements about these thingswhich are too dogmatic.

L. B A K A Y

L. BAKAY: I can answer this simply. First of all, I don’t think that the rupture of the cell membrane is of any importance. The point remains that the extracellular space increases in size morphologically in edema in the white matter, whether there is a rupture or not, and it does not increase in the cortex where, incidentally, we never see rupture. As far as the artificial nature of the rupture of the cell is concerned, we never see it in unswollen cells, and you don’t see a ruptured cell membrane even if you use a magnification of 10,000 times with electronmicroscopy. It just simply cannot be seen, ruling out the possibility that this is a random artifact. It might be an artifact associated with swelling, but it is an artifact that is never seen in unswollen cells. As far as the size of the space is concerned, when 1 say that there is no evidence of any increase of the extracellular space in the cortex, even in conditions of edema, and there is one seen in the white matter, 1 mean that purely on the ultrastructural morphological basis. This has no bearing on whatever we mean by a functional extracellular space, and if we assume that some of the extracellular space is intracellular as far as the glia is concerned, that is all very well; but we do not see any evidence of an increase in cortical extracellular space size by electronmicroscopical methods. As far as ferritin is concerned, I know about Dr. Brightman’s work, and I fully agree with much of what he said. But ifit would be artificially translocated in these specimens, wouldn’t you expect that it would be translocated from this extremely narrow extracellular space between these enormously swollen cells? If ferritin would be all over the place, you could say that this has been transferred as an artifact. But if you see two extremely large cells and the narrow cleft inbetween, and the material does not go into the very largely hydrophilic cells on both sides, this is not likely to be an artifact. 0. STEINWALL: I would like to comment on the problem of the neonatal brain barr;er and kernicterus. mentioned by Dr. Bakay. The unconjugated bilirubin is very apt to be bound to protein in the blood and there is reason to believe that, even in the embryo, the blood-brain barrier to protein-bound material is effective. This can be illustrated by a quite recent investigation (together with Klatzo, Olsson and Sourander) on newborn rats and embryos (2 weeks or longer gestation) injected with fluorescein-labeled albumin. As far as could be observed by fluorescence microscopy, there was no cerebral extravasation of the tracer. There might exist conditions, however, in which the proteinbinding of bilirubin is loosened. Being a lipophilic compound the free bilirubin would then pass freely into the brain. I wonder if Dr. Bakay would comment on Grontofts work on human fetuses, where he concluded that the cerebral vessels were impermeable under normal conditions while anoxic fetuses showed signs of blood-brain barrier damage. L. BAKAY:I know about Grontoft’s work, and he emphasizes strongly that it is necessary for the brain to be anoxic to permit a trypan blue infiltration into the brain tissue. The difficulty is that in adult animals you can do the most drastic kind of anoxic and asphyxic experiments, but you always will find tissue necrosis, which there should not be in anoxic as versus ischemic anoxia. I don’t know what kind of damage those infants have been exposed to in addition to anoxia. K. NEAME:I would like to ask Dr. Bakay about the effects of pathological states as seen by brain scanning. Is this a change in the so called blood-brain barrier, or is it a change seen in tissues in general? L. BAKAY: That is a very hard one to answer, because it depends on the tissue. One thing is for certain: that there is no selectivity in the increased uptake in a brain scan due to any condition. The amount of maximal concentration you can get in almost any lesion in the brain corresponds to what you get in normal tissue elsewhere. There is no selective concentration. This answers your question: there is no specific change; in other tissues (muscle, liver or spleen), whether tissue is normal or abnormal, it has about the same concentration, although there could be more of course in the case of a pathological process than there would normally be. What you see as a positive lesion in a brain scan is not a selective concentration but a relatively free exchange of the substance with the blood stream, as occurs in other organs, and it becomes selective only because the suirounding brain does not have completely free exchange. How much of this is an intracellular, how much an extracellular process is a long story which I would like to go into, but do not have the time.

C H A N G E S IN BARRIER EFFECT I N P A T H O L O G I C A L STATES

341

J. DOBEING: About the spread of edema throughout the white matter of the hemisphere which is being damaged at a focal site in the cortex, I want to ask Dr. Bakay if he thinks that this has any lessons for experimenters who routinely mutilate the brain in the course of the experiments. The question - which is impossible to answer - is, what size of lesion, what extent of trauma is necessary to produce the effect throughout the white matter of the hemisphere? Would ventricular perfusion do it? Would needles, passing through thecerebrum into a ventricle (in most oftheselaboratory animals it is only a potential space anyway) cause such edema? Therefore, the evidence that one is in the ventricle, at least in a small animal, is likely to be at the expense of at least some trauma to the ependyma as well as throughout the tract of the needle? Certainly pharmacological intracerebral injections, which are horrible from the point of view of structures and edema, but will probably suit the purpose of the pharmacologist, would be likely to do this. The question is as to what extent can you inflict damage to the brain before it becomes necessary (particularly when investigating blood-brain barrier effects with drugs and substances concerned with edema) to provide controls rather better than with trqpan blue to discover whether you have this spreading edema from the trauma of your procedure or from the test substances employed.

L. BAKAY: I shall give you an extremely dogmatic answer to this. I don’t know about ventricular perfusion. If it is done without changing tht. pressure conditions and if the solute is not very different in osmolarity or very definitely toxic, I suppose a careful perfusion might not cause any damage, at least on theoretical grounds, in the surrounding brain. Whatever is introduced into the brain causes edema: how much is unpredictable. But you cannot put a lesion in the brain and you cannot put even a simple needle tract in the brain without having a certain amount of edema around it. A. LOWENTHAL: You said that there are two types of edema: one type with an increased protein content and another type with a less increased or with a reduced protein content. Could you say which are those two types?

L. BAKAY:There might be a great number of different types of edema. For example, we don’t know what category anoxia would fit in. The two fairly well defined edemas so far are: ( I ) the traumatic inflammatory peritumoral focal type of edema which seems to be characterized by a fairly free exchange of serum albumin and an obviously greatly disturbed capillary permeability, and (2) the type, which is really only characteristic of traumatic edema and seems to be an intramyelinic accumulation of protein-free plasma ultra-filtrate with no evidence of capillary damage. These are the two extremes, and we don’t really have any other categories as yet.

This Page Intentionally Left Blank

343

Clinical Importance of Alterations in Barrier G .QUADBECK Heidelberg (Germany)

Before discussing the problem of alterations of the blood-brain barrier, I have to define what I mean by the term blood-brain barrier (bbb). Anatomically, the bbb includes all structures between the capillary lumen and the central nervous tissue: the endothelium of the capillaries, the basement membrane, and the surrounding glial foot processes. Functionally, the bbb should be regarded as a system limiting the free exchange between blood and brain; it further exerts its function on transport processes essential for the nutrition of the brain and on those in the opposite direction, from the brain into the blood. The bbb is also effective as an intermediate system in humoral regulations of peripheral vegetative functions. Alterations of the bbb with increased permeability cause enhanced exchange between blood and brain which is dependent on the intensity of the disturbance; by this means the penetration of viruses and bacteria from the blood into the brain may be facilitated. This increased permeability may be followed by cerebral edema and by an increased predisposition for epileptic seizures. In his paper Dr. Bakay has amply demonstrated the increased permeability of the bbb in pathological conditions. Therefore, I will mostly discuss pathological conditions involving a reduced exchange between blood and brain. Under normal conditions glucose is the most important if not the only source of energy for the brain. Because glucose cannot enter the brain from the blood by a process of diffusion, glucose uptake is independent of the glucose level of the blood, over a wide range. The nutrition of the brain depends on a transport mechanism specific for glucose in the bbb system. The glucose uptake of the brain can be measured in man by the method of Kety and Schmidt (1948). With this method, one may determine the cerebral blood flow, the cerebral uptake of glucose, oxygen, and amino acids; and at the same time the output of COz, lactic acid, pyruvic acid, and other metabolic products of the brain. A reduced glucose uptake by the brain can be brought about by the following mechanisms : (a) reduced blood flow caused by disturbances of the blood circulation. (b) reduced glucose transport across the bbb. (c) reduced cerebral metabolic activity. (d) markedly reduced glucose levels in the blood. In my opinion, only point b is related to the bbb system. With reduced cerebral blood Rrferences p . 347

G. Q U A D B E C K

344

TABLE I CEREBRAL BLOOD FLOW

( C B F ) A N D C E R E B R A L G L U C O S E U P T A K E ( C G L U ) IN E L D E R L Y M E N M E A S U R E D BY D I F F E R E N T G R O U P S

( % of the normal value)

Gottstein et a/., 1962 Gottstein et a/., 1964 Dastur et a/., 1965 Becker and Hoyer, 1966

N = 13 N = 21 N = 17 N = 26* N = 18

CBF

CGIU

95 953 70 93 105

71 80 62

I7 63

* Normal elderly men without mental disturbances flow, the brain has the possibility of increasing the glucose uptake from the blood. Erbsloh et al. (1958) found, however, that with blood glucose levels ranging between 56 mg % and 25 I mg % there was no difference in the uptake of glucose by the human brain. It is usually assumed that in cerebral arteriosclerosis the reduced bloodflow is the cause for the mental disturbances seen clinically. However, at autopsy the clinical diagnosis of cerebral arteriosclerosis often cannot be confirmed. There seems to be no correlation between the autopsy findings on the brain vessels and the intensity of the mental disturbance in older men. Table I shows the results of measurements of cerebral metabolism in patients with mental disturbances and in older men without great mental disturbance. All groups had the same results. The glucose uptake in older men more frequently showed the greater alterations in metabolic values than in bloodflow and oxygen uptake. Many studies by Erbsloh have indicated that the lactic acid output by the brain is not increased in patients with reduced oxygen uptake. This would be expected if the oxidative metabolism of glucose by the brain were inhibited and the glucose metabolized by the glycolytic pathway. Such an increase of lactic acid production by the brain may occur in brain tumors, as we found, where the values were 20 times the normal output. In patients with failure of the metabolic processes of the brain, one may differentiate between the pathological mechanisms and arrive at a correct diagnosis by means of measurement of cerebral metabolism. It has been found by Becker and Hoyer (1966) that a reduced cerebral glucose uptake can be found not only in older mentally disturbed patients but also in younger patients with mental or neurological disorders. In earlier experiments we were able to demonstrate that some drugs used in geriatric patients influenced the glucose transport across the bbb by increasing the cerebral uptake. In normal animals (mice) this can be demonstrated only in very high doses, since the glucose uptake by the brain is an excellently regulated mechanism which can be affected only by extremes. For example, drugs effective in animal experiments were nicotinic acid, centrophenoxine, and pyrithioxine,

345

A L T E R A T I O N S IN B L O O D - B R A I N B A R R I E R

Pyrithioxine

The latter was given to patients with reduced glucose uptake by the brain in daily doses up to 1200 mg. Under this treatment the cerebral glucose uptake rose together with a clinical improvement of the neurological or psychic condition (Table 11). The time needed for the measurement of all the data in one patient by this method is so great that only two patients can be measured each day by one person in the laboratory. We, therefore, looked for a simpler screening method to diagnose disturbances of the bbb permeability, and remembered a study of L. Doust published some years ago. Doust (1960) found different frequencies of the rate of vegetative regulations in some patients. In epileptics the frequency increased as compared with the normal, whereas in schizophrenics a marked reduction of this frequency was found. TABLE I 1 C H A N G E S O F CE RE B RAL METABOLISM U N D E R TR EA TMEN T W I T H P Y R I T H I O X I N E

(from Becker and Hoyer) a: Man ( 5 1 ) with clinical diagnosis: cerebral arteriosclerosis before treatment

33 days treatnient

62 % 29 7” 64 7;

98 % 120% 84 %

Blood flow Glucose uptake 0 2 uptake

b: Man (35) with clinical diagnosis: Jackson seizures be$ treatin. 12 days treatni. Blood flow 244 74 133% Glucose uptake 36 % 86 % 02 uptake 173 83 %

<:

26 days treatin. 150% 165% 1027;

c- Wife (25) with clinical diagnosis: amnestic syndrome before trealnienf

Blood flow Glucose uptake 0 8 uptake

136 % 25.5 % 89 7;

8 days

no freatnienf 135 % 25.3 % 59 %

14 days treatment

92.4 % 76.6 % 97.3 04

36 days

treatment 99 % 82.204 81 04

If we assume that the function of the bbb in the humoral regulation of vegetative functions is comparable to a shock absorber, it should then be possible, with a high frequency and high amplitudes of changes in vegetative functions, to demonstrate that there is a failure of the shock absorber function of the bbb caused by a pathologically increased permeability. A reduced frequency together with low amplitudes could indicate an increase in the “shock absorber” effectiveness caused by a reduced permeability of the bbb, Rcfcrcnccs p.

347

346

G. QUADBECK

Fig. 1 . Registration of the 0 2 pressure of the venous blood. Normal registration of a young man (20) without nervous or mental disease.

Fig. 2. Venous oxygen pressure in a schizophrenic (46).

We have registered the changes of the oxygen pressure in the cubital vein, using the Beckman Physiological Gas analyzer. The registration curves of healthy students were similar, although each showed individual characteristics (Fig. 1). The type of registration curve could not be influenced markedly by the time of day, smoking, sleeping, or by chlorpromazine injection. The greater part of schizophrenics had very flat and slowly changing curves (Fig. 2). One half of the epileptic patients studied by this method had curves with high frequency and high amplitudes (Fig 3.) The hebephrenic schizophrenics had without exception normal curves. It is too early to suggest that measurement of the venous oxygen pressure could be a valuable method for bbb diagnosis. If our assumption concerning the “shock absorber” effect should prove to be correct, we could demonstrate only one part of the bbb function by this method. Furthermore, we have studied only about 230

ALTERATIONS I N BLOOD-BRAIN BARRIER

Fig. 3. Venous

0 2

347

pressure in an epileptic man 28 (posttraumatic epilepsy).

persons until now, a number too small for definitiveconclusions. I t is possible, however, that this method will become a useful diagnostic method. REFERENCES BECKER,K. A N D HOYEH,S. (1966) Hirnstoffwechseluntersuchungen unter der Behandlung mit Pyrithioxin. Derrt. Z. Nervenheilk., 188. 200-209. DASTUR,D. K., LANE,M. H., HANSEN,D. B., KETY,S. S., BUTLER, R. N., PERLIN,S. AND SOKOL ~ F F ,L. (1965) Effects of aging on cerebral circulation and metabolism in man. In: H~rtiiairAgitig, Public Health Service Publication No. 986, Washington, DC. DOUST,J. W. L. (1960) Spontaneous endogenous oscillating systems in autonomic and metaholic effectors: their relation to mental illness. J. Nerv. Metit. Dis., 131, 335-347. EHRSLBH.F., KLARNER, P. AND BERNSMEIER, A. (1958) Uber die Bilanz des cerebralen Zuckerstoffwechsels. K h . Wsclir., 36, 849-852. Gomsi-tiN. U.,BERNSMEIER, A. A N D STEINER, K. (1962) Die Wirkung von Nicotinsaure auf Hirndurchblutung und cerebralen Stoffwechsel des Menschen. Klbi. Wschr., 40. 772-778. GOTTSTEIN, U., BERNSMEIER, A. A N D SEDLMEYER, 1. (1964) Der Kohlenhydratstoffwechsel des menschlichen Gehirns I I . Kliti. Wschr., 42, 310-313. KFTY,S. S. A N D SCHMIDT, C. F. (1948) The nitrous oxide method for the quantitative determination of cerebral blood flow in man. J. d i n . Invest., 27, 476. DISCUSSION R. KATZMAN: I would like to ask Dr. Quadbeck a question. It was not clear to me what thecorrelation was between the change in blood flow and the change in glucose uptake. I had the impression, or you left the impression, that the blood flow was reduced and glucose uptake was increased. G. QUADRECK: In all cases in which the blood flow was primarily increased prior to therapy and the glucose uptake reduced at the same time, the blood flow diminished with therapy and the glucose uptake increased.

R. V. COXON:I wonder if anybody knows what the relationship is between anoxia and glucose in the brain of the rat. G. QUADBECK: We have carried out many experiments in animals. When we exposed rats in a decompression chamber to reduced oxygen pressures, all medications which brought about an increased uptake of glucose were simultaneously active in raising the resistance to oxygen lack.

348

G . QUADBECK

B.D. WYKE:Could you tell us definitely whether the increased gulcose uptake produced by this drug is or is not independent of a change in cerebral blood flow?

G. QUADBECK: The increase in the glucose uptake by the action of pyrithioxine is certainly independent of the volume of blood flow.

R.KATZMAN: Is it independent of oxygen utilization? G . QUADBECK: No, if the utilization of the oxygen and the uptake of glucose are reduced before treatment, then these two quantities are increased at the same time by pyrithioxine.

H. M. ADAM:How long does it take for this drug to produce this effect? How long is the effect maintained on a single dose? G. QUADBECK: In animal studies, as in man, a single dose of this drug has no effect. Therefore, the preparation must be administered in both animals and in man for a t least a week before an effect is seen. As far as we have been able to determine until now, after therapy is discontinued for a specified time, the pathologic state observed in the patients prior to therapy returns. A. LAJTHA: If I interpreted correctly from Dr. Crone’s talk, the glucose pump is fairly saturated below the physiological blood levels. I thought this might be a safety factor in that the brain then should not react to changes in blood glucose concentration since it has a constantly saturated pump. Then the only way I could explain the decrease in cerebral uptake in the old patients is by a decreasingly efficient glucose pump. So perhaps in old age we don’t have a thicker wall but have a less than optimal glucose pump.

G. QUADBECK: Yes, I fully agree with Dr. Lajtha. A. LAJTHA: Then it would be very interesting to know how you get the answer with the drug. Would the drug cause the formation of new carrier molecules?

G. QUADBECK: The question is difficult to answer, since we do not know even today, how glucose is transported from the blood into the brain. Perhaps you might like to suggest a suitable theory.

349

Drug Infuence on the Barrier G. QUADBECK Inhritrrt .fiir Pnthochernie rrnrl Allgertieine Nertrochemie, Pathologisches Institut cler Universitat Heidelberk, Heidelberg (Germany)

The blood-brain barrier (bbb) as a controlling and regulating mechanism is efficiently stabilized under normal conditions. Therefore, it is very difficult to alter the permeability of the bbb by drugs in doses comparable with those used in clinical therapy. If one wishes to influence the bbb of healthy animals, which is not already pathologically altered, one is obliged to use very high doses of drugs. The results of such experiments are of no value from a quantitative point of view. They can only show qualitatively the direction of an effect. Emil Behring 68 years ago was the first who tried to influence the bbb by drugs. He attempted to make the bbb in man more permeable for tetanus antitoxin by morphine, chloral hydrate, and amyl nitrite, and believed that he had somesuccesswith amyl nitrite. In the years following the basic experiments by E. E. Goldmann to demonstrate the bbb with trypan blue, this dye remained the only marker in bbb research. Some years ago, however, we could demonstrate that this dye itself affected the bbb by decreasing its permeability. With this marker it was nearly impossible to study changes in the bbb permeability by drugs, because only a breakdown in this system enabled this dye to enter the central nervous tissue from the blood. Using acid dyes with less acidic groups i n the molecule, Frohlich and Zak could demonstrate an increased permeability of the bbb by theophylline. Kelentei and Bosczko reported an increased permeability of the bbb for penicillin under the influence of aminophylline. From a therapeutic point of view one should avoid a general increase or decrease of the bbb permeability, because this is a physiological system necessary for the normal function of the brain. The essential question is, therefore, not how to disorganize the bbb permeability and its effectiveness, but how to normalize its function, if this system is changed pathologically. In our studies on the influence of drugs on the bbb we used as markers ofthe bbb function the following substances: [32P]Phosphate [24Na]Sodium chloride [14C]Glucose Acridine orange Quinine sulfate Our experimental animals were mice and rats. The marker was given intraperitoneRc,Jrrmws p. 353

350

G. Q U A D B E C K TABLE I

Drug

A.

INCREASE OF THE

bbb-PERMEABILITY WITH 3'P

AS MARKER

120 1122

200 15 90 50 40

Nicotinic acid Persantin Aminophylline Chlorpromazine Prothipend yl Perphenazine Chlorprothixene But yrylperazine Fluopromazine Reserpine Tetrabenazine Lithium chloride 2,4-Dinitrop henol

150

126 132 129 128 1 I9 139 118

7 daily 4 weeks 50

20 25 2 50 500

155

218 116'

20

B.

DECREASE OF THE

Hydergin Pyr i t h ioxine Decort ilen Diphenyl hydantoin

*

bbb-permeability $: of the control group

Dosage (mglkg rat)

bbb-PERMEABILITY WITH 33P AS

MARKER

85 * 80 87 84

0.2 200 20 200

The increase was significant in the cerebellum only. T A B L E I1

Drug

Dosage (mglkg rat) A.

INCREASE OF THE

Persantin Pyrit hioxine Centrophenoxine 0-Pyridyl-carbinol Nicotinic acid Methamphetamine

B. Chlorpromazine Amitriptyline Prothipendyl Perphenazine Chlorprot hixene Reserpine Tetrabenazine Imipramine Opipramol Iproniazid

DECREASE OF THE

bbb-PERMEABILITY WITH 24Na AS

bbb-permeability

7; of the control group MARKER

15

136

200 50 200 200 2

115

I I7 I l3* l08** 123

bbb-PERMEABILITY WITH 24NaAS 50

20 40 12.5 12.5 0.7 4 weeks daily 50 5 50

200

4 weeks daily

MARKER

76 85 70 63 75 83 82 87 84 91

35 1

D R U G INFLUENCE O N THE BARRIER

Orphenadrine ATP 2,4-Dinitrophenol

81 89 89

50 20 20

* The difference was significant in the cerebellum only. * * The difference was not significant.

T A B L E 111 bbb-permeability

Dosage (ttiglkg rat)

Drug

A.

INCREASE OF THE

bbb-PERMEABILITY FOR

7: of the control group ACRIDINE ORANGE

140 193 178 141 129

2 50 4 25 200

Reserpine Phenmetrazine Amphetamine Flup henazine Boric acid B.

REDUCION OF THE

bbb-PERMEABILITY FOR

ACRlDlNE ORANGE

12.5 50 2.5 200 200

Perphenazine Chlorpromazine KCN NHdCI Rutine

59 72 86 72 76

Drugs without effect on the acridine transport across the bbb. Centrophenoxine(50 mg/kg).Cort isone(20 mg/kg).Dexamzthasone (20 mg/kg).Decort ilene (20 mg/kg). Prednisone (20 mg/kg). Nicotinic acid (200 mg/kg). Lithium chloride (200 mg/kg).

TABLE IV INFLUENCE

OF2

MG K E S E R P l N E i K G O N T H E T R A N S F E R O F Q U I N I N E A N D A C R I D I N E O R A N G E FROM T H E BLOOD INTO THE B R A I N I N DIFFERENT STRAINS OF R A T

(Concentration of the marker in the brain in Treattnetit

N o treatment Reserpine No treatment Reserpine

* Quadbeck,

Marker

quinine quinine acridine orange acridinc orange 1956.

ReJerences p. 353

7” of the concentration in blood)

Wistar-rat

straiti with a high susceptibility to audiogenic seizures*

5.0 f 1.2 4.95 f 1.2 27.8 10.0 32.4 8.6

5.3 f 1.9 8.7 5 2.4 26.6 f 7.0 37.6 f 13.1

+ +

352

G. QUADBECK

TABLE V INFLUENCE OF CHLORPROMAZINE O N THE PHOSPHATE A N D SODIUM TRANSPORT ACROSS

THE bbb Effect of a single dose of 50 mg chlorpromazine/kg rat and a daily dosage of 20 mg/kg during 28 days. A : Single dose. B : 5 h after the last dosage of chlorpromazine. C: 4 days after the last dosage of chlorpromazine. D: 8 days after the last dosage of chlorprornazine. The [32P]phosphate has been given 2 h and the radioactive sodium 1 h before killing the rat (Quadbeck and Sachse)

radioactive marker:

azP (orthophosphate) 24Na (sodium chloride)

brainlblood ratio of activity in

A 126 16

E

loo* 90 *

of the control group

C 88 * 89

D 75 19

* The difference between the experimental group and the control group was not significant. ally or in experiments with glucose, intravenously. The animals were killed by decapitation at a time when the level of the marker in the blood was higher than in the brain. The most suitable time for each of the markers has been studied in earlier experiments and was found to be 2 h for phosphate, I h for sodium(both in rats), 10minfor glucose (mice) and 1 h for acridine orange or quinine (rats). The radioactivity of the brain and the blood was measured and the ratio, blood activity/brain activity, was calculated. Acridine orange and quinine were extracted by phenol, the extracts purified to avoid quenching effects, and the concentration of the fluorescent substances measured by fluorescent spectrophotometry. If the bbb were a simple, membrane like-structure, an effect of a drug on this membrane would be in the same direction for all markers and perhaps only dependent on the electrical charge of the marker. The following results of our experiments show that the effect of a drug on the bbb depends not only on the marker used, but also on the strain of rat. In addition, we found differences between one dosage of a drug and the repeated administration of the same drug. Some drugs had an after-effect opposite to that seen directly after the initial administration (Tables I-V). We must assume, therefore, that the bbb in its effectiveness is a very complicated system with regulatory and counter-regulatory effects. Generally, one can say that any form of sedation is primarily associated with a reduction of the sodium exchange between blood and central nervous tissue. On the other hand, an increase of sodium exchange across the bbb seems to be an indication of cerebral stimulation. It could be demonstrated that all drugs increasing glucose transport or the transfer of [1*C]glucose activity under our experimental conditions also made the rat brain more resistant to reduced oxygen pressure. We found one exception to this rule, however. Analeptics, for example metamphetamine, increase the glucose uptake by the brain but decrease the cerebral resistance to oxygen deficiency. Most of the drugs studied under our experimental conditions were without any effect on the bbb. So far as we know drugs with an action on the central nervous system only have an influence on the bbb permeability. The mechanisms of action of the drugs mentioned above cannot be stated and for now must be left unexplained.

D R U G INFLUENCE ON THE BARRIER

353

REFERENCES BEHRING, E. (1898) Thaisachliches, Historisches und Theoretisches aus der Lehre von der Giftimmunitat. Deut. Me(/. Wschr., 24, 661. FROHLICH, A. AND ZAK,E. (1927) Theophyllin und seine Gewebswirkung als Mittel zur Potenzierung von Giften und Arzneien. Arch. e.rpt/. Puthol. Phurmukol., 121, 108. GOLDMANN, E. E. (19 13) Vitalfarbung am Zentralnervensystem. G. Reimer, Berlin. KELENTEI, B. AND BOCZKO,M. (1956) Studies of the Hemato-Encephalic Barrier. Cot$ Neurol., 16,15. QUADBECK, G. ( 1956) Die Auswertung anticonvulsiver Verbindungen am audiogenen Krampf der Ratte. Arch. e.rpr/. Parhol. Phurtnukol., 228, 178. G. AND SACHSSE, W.(1961) Beeinflussung der Blut-Hirnschranke im Dauerversuch. Arch. QUADBECK, Psychiut. Z . ges. Neurol., 201, 580.

DISCUSSION

T. Z. C S ~ K Y May : I suggest that before we evoke any barrier we consider that the observed phenomenon may in many instances be explained by simple cellular metabolism. I may give a simple example where we actually know what happens: We have studied the effect of reserpine on the intestinal transport of glucose and found that if we reserpinize the animal, the intestinal transport rate of glucose increases. There is nothing magical about it: no barrier is involved; reserpine decreases the serotonin content in the intestinal wall and parallel with this, the relative intracellular concentration of potassium versus sodium increases. Whenever the potassium content increases in the intestinal epithelium, the rate of glucose metabolism also increases rapidly, and this can explain the more rapid disappearance of glucose from the lumen. Now with your data, it is quite remarkable that in the chronically reserpinized animal you find a decrease in sodium content and an increase of the glucose uptake, just like we found in our study in the gut. Based on our experiments, I would venture to explain the effect of reserpine simply on the basis that y o u have increased the intracellular glucose metabolism, consequently more glucose enters the system. Thus, there is no magic reduction in barrier function. Simply more glucose was needed, more was metabolized, and more was transported.

B. D. WYKE:I hope you will forgive me if as one of the very few surgeons attending this meeting I would like to remind the group that there are some very, very old observations that if you want to alter the permeability of the so-called blood-brain barrier, the cheapest and simplest way to d o it is to put an air bubble in the ventricle. Another simple way is to give the animal a tap on the head. D. H. FORD:I would like to comment on these reserpine experiments, because I think the use of reserpinized animals can be misleading. At one time we did a study on the effect of reserpine on the metabolism of thyroid hormone by the nervous system. It was observed a t the end of our experiments that there were significant changes occurring in all parameters of triiodothyronine uptake and metabolism as compared with controls. Then we noticed that the animals looked somewhat “scrawny”, and it occurred to us secondarily to watch the reserpinized animals. We found they were so tranquilized that they had stopped eating. Then we repeated the experiment all over again and put the animals literally on the top of their food. Once they started to eat all the significant differences previously seen disappeared. So I wonder about your effect on the barrier, as to whether the results are due to the direct action of the drug or not. G. QUADBECK: When we began with our reserpine experiments we had our animals in automatic cages, and they had t o open a vent in order to drink. But under reserpine they could not drink, and so they died. Later they had a tray of water in the cages, and then they lived. D. M. WOODBURY: And they ate? G. QUADBECK: They could eat all the time; they had to open a vent only for drinking.

3 54

G. QUADBECK

D. M. WOODBURY: When studies are done with radioactive sodium, more than one point should be measured, because it takes about 8 to 16 hours to equilibrate in the brain, and it penetrates into at least three different compartments: interstitial, glial, and neuronal, and at different rates. The entire uptake curve then consists of three components which can be resolved by graphical analysis or by a computer. The first component represents penetration into neurons, the second transfer from brain capillaries into the interstitial space, which is the location of the so-called “blood-brain barrier”, and the third and slowest component penetration into glial cells. Therefore, one cannot draw any conclusions about the rate of entrance of sodium across the barrier, which is the second component of the uptake curve, on the basis of one point at 2 or 3 hours. One does not get any kinetic information at all with isotopic sodium or chloride or other such substances unless the entire uptake curve is measured and then resolved by the graphical or computer techniques. From these resolved curves, in addition to the rate constants for each compartment, one can also obtain the total amount of isotope in the compartment from the zero-time intercept of each component. Therefore, to say that a substance has an effect on the barrier when the conclusion is based on only one point is obviously dangerous. I think this is an important point that should be considered in all these types of isotope studies. N. M. VAN GELDER: I just wanted to add that some of these drugs will also change the body temperature, e.g., like chlorpromazine. And I think it should be taken into account when one studies something like this. G . QUADBECK: Yes, but in this case all animals were at 28°C. during the experiment. Therefore, the reduction of the temperature would not be very important.

N. M. VAN GELDER: I don’t think that the room temperature always reflects the actual body temperature. G . QUADBECK: No, but in order to lower a patient’s temperature in the hospital you also have to reduce the temperature in his room. Otherwise it is impossible to reduce his temperature.

P. MANDEL: You found that you get a glucose uptake of 4572 with chlorpromazine? You should then also get a decrease of the entire metabolism.

G. QUADBECK : Yes, for the brain it was 45 %. P. MANDEL: Do you have any control for this?

G. QUADBECK: Yes, we put the animals under low oxygen pressure, and the chlorpromazine had no effect on the survival time. If there was only a reduction in the glucose uptake, then the animal would die in this experiment. A. LAJTHA: May I give a brief comment on the effect of chlorpromazine? This drug seems to inhibit the flow of amino acids in both directions, that is, it inhibits uptake as well as exit. It causes an amino acid put in the circulation to get into the brain more slowly, and once injected into the brain to exit more slowly. Since this effect is much larger if chlorpromazine is combined with hypothermia, and since chlorpromazine lowers the body temperature, part of its effect may be by causing hypothermia. However, if animals are put into a 30” incubator that keeps the body temperature normal, chlorpromazine still inhibits amino acid flux.

GENERAL DISCUSSION

A P O S S I B L EFFECT E O F VITAMINBe O N BRAINB A R R I E R S Comment by DR.C. F. BAXTER on DR.QUADBECK’S paper The similarity in structure between pyrithioxine and Vitamin Be has already been pointed out. However, Dr. Quadbeck stated that Vitamin Be had no effect upon the blood-brain barrier criteria which he has measured.

D R U G INFLUENCE O N THE BARRIER

355

Preliminary observations in our laboratory might lead to an opposite conclusion and we would sugg. st that under certain conditions Vitamin Be might play a role in blood-brain barrier mechanisms. Some years ago we were engaged in experiments with rats, in which thiosemicarbazide (TSC) was injected intraperitoneally and convulsions prevented, by the administration of pyridoxine or pyridoxal some time later. (Baxter and Roberts, 1962). Measurements were made of glutamic acid decarboxylase (GAD) the enzyme synthesizing y-aminobutyric acid (GABA), GABA levels and y-aminobutyric-u keto-glutaric transaminase (GABA-T) the enzyme system degrading GABA - all in the brain tissues of the treated rats. G A D and GABA-T activities were measured in virro without the addition of pyridoxal phosphate. While TSC administered in vivo decreased levels of GABA in brain and inhibited GAD, it had no apparent effect on GABA-T. When TSC treated animals were injected with pyridoxal, the levels of GABA in the brain continued to decline while G A D activity appeared reactivated and GABA-T remained unchanged. There were various ways to interpret these results. It had been reported in the literature (Gammon et a/., 1960) that the blood-brain barrier to GABA was altered in rats injected with methoxypyridoxine. It seemed possible that in our experiment a similar alteration in the brainblood barrier had occurred as a result of TSC plus pyridoxal treatment. The continued decrease in cerebral levels of GABA at a time when GABA synthesis was accelerated, would then be explained by a leakage of GABA out of brain tissues into the rest of the body. This hypothesis was tested using 3 groups of 4 adult male Swiss mice. At zero time of the experiment, group 2 and 3 received TSC intraperitoneally at a level of 12 mg/kg body weight. At 15 minutes, group 3 mice received an anticonvulsant dose of pyridoxal HCL (40 mg/kg). At 30 minutes, 10 1 of 2 [ W ] GABA (containing about 2 p C) was injected into the left brain ventricle of each mouse. Fifteen minutes after the isotope injection (and before the TSC treated animals could convulse) all mice were quick-frozen in liquid nitrogen. Whole body sections were prepared and radioautographs made using the techniques pioneered by Ullberg and his colleagues (Ullberg, 1954). In control animals, most of the radioactivity remained in the brain. A little activity can be seen in the bladder and some in the liver. In mice treated with thiosemicarbazide, the picture was about thc same. In mice rdeiving TSC plus pyridoxal HCI, however, an increast in the radioactivity in the liver was observed in 3 out of 4 animals. Although the number of animals tested precludes a definitive conclusion and quantitative data are yet to be obtained, these preliminary experiments suggest that the administration of pyridoxal to TSC treated mice increased the outflow of isotopicallv labeled material from the ventricle or brain tissue into other tissues of the body. This increased flux may reflect n change in permeability of the brain-blood barrier in response to Vitamin Be administration.

I BAXTER,C. F.,

A N D ROBERTS, E. (1962) Effect of 4-Methoxymethylpyridoxine and CarbonylTrapping Agents on Amino Acid Content of Mammalian Brain and Other Tissues. In Amino Acid Pools, J. T. Holden (Ed.), Elsevier, Amsterdam, London and New York (pp. 499-508). 2 GAMMON, G. D., GUMNIT, R., KAMRIN, R. P., AND KAMPIN, A. A. (1960) The Effect of Convukant Doses of Analeptic Agents upon thc. Concmtration of Amino Acids in Brain Tissue. In hihibition in the Nervous System in Guninla Aminobutyric Acid, E. Roberts et al. (Ed.), Pergamon Prm (pp. 328-330). 3 ULLBERG, S. (1959) Autoradiographic Studies on the Distribution of Labelled Drugs in the Body. Progress in Nuclear Energy - Series 6, Vol. 22 - Biological Sciences. Pergamon Press, London, Oxford, New York and Paris, (pp. 29-35).

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357

Transport Inhibition Phenomena in Unilateral Chemical Injury of Blood-Brain Barrier OSKAR STEINWALL Dqxirtriietit of Nerrrology, Uiiiversity of Giitehorg, Giiteborg, Sweden

A damage to the blood-brain barrier (BBB) is commonly discussed as a state of abnormal outflow of plasma constituents into brain tissue. This phenomenon, which is the basis of conventional methods for the indication of BBB damage, is no doubt of decisive importance in many experimental and clinical situations, for instance in the development of brain edema (Bakay 1966; Klatzo et al., 1966). More complex aspects of BBB pathophysiology arise, however, from the current view that certain barrier effects may be linked to mechanisms for specific and directed transport in the bloodbrain interphase. The studies reviewed in this paper have especially dealt with such aspects. A main object has been to attain in living animals a model BBB damage, where the anomalous behaviour of different blood-borne tracers indicates that transport inhibition is a predominant or contributory factor.

CNS: Organic ions (waste products, ‘indicators‘)

_ - _ _ _ _ .-

a _ _ _

Brain

>

Acids

Bases .

- ._ - -.

Glucose (nutrients)

Blood

BBB

--

+ c-? - Indicator --

4 KIDNEY:

Biood

Tub. ce!ls

Urine

The initial working hypothesis, discussed earlier together with some experimental results (Steinwall, 1961), is represented by the diagram in Fig. 1. It proposes a system of transport mechanisms within the frame of the BBB, selectively conveying different categories of substances inward or outward. In teleological terms these mechanisms would serve for the uptake of nutrients and the excretion of metabolic waste products through a system of membranes otherwise practically impermeable to lipid-insoluble material. Table I shows these ideas inserted in a simplified outline of transfer and barrier conditions with respect to differences in physicochemical and biological characteristics (Steinwall, 1964). The hypothesis has been approached experimentally along two main lines. Of these the attempts to prove that a “counter”

* Head professor Tore Broman. References

p . 363-364

358

0. S T E I N W A L L

TABLE I SIMPLIFIED I N T E R P R E T A T I O N OF B L O O D - B R A I N P A S S A G E A N D BARRIER EFFECTS I N RELAT I O N TO P R O P O S E D T R A N S P O R T M E C H A N I S M S

Passive diffusion

No barrier effect

I . “Nutrients” (glucose, amino acids, etc).

Specific transport* blood CNS

2. “Waste products”, organic ions (conjugates, indicator dyes, etc.)

Specific transport* blood + CNS

3. Nonmetabolizable and

No transfer

Barrier effect above max. transport capacity Barrier effect Partly due to the “counter” transport Barrier effect “passive”

1. LIPOPHILIC SUBSTANCES

11. HYDROPHILIC SUBSTANCES

--f

noncharged solutes, (inulin, sucrose, mannitol, etc.)

* Carrier mediated - limited transport capacity; inhibited by competitive overloading or by toxic influences. transport from brain to blood might be involved in the barring of acid dyes and other organic ionized material have not led to conclusive results. More convincing findings have been obtained in the experiments designed to determine if decreased brain uptake of nutrient tracers can occur in a BBB damage commonly interpreted only in terms of abnormal extravasation of acid dyes. EXPERIMENTAL TECHNIQUE

The basic experimental procedure employed in the studies discussed here was designed to meet the following conditions: (a) Injection of noxious agents within the cerebral vessels in living animals with satisfactory control of concentration in loco and of application time. This allows for the determination of critical levels for bringing about barrier dysfunctions. (b) Restriction of the damaging influence to one hemisphere in order to get a control hemisphere for direct comparison in each experiment. (c) Avoidance of circulatory disturbances after the application of the injurious agent by adequate choice of chemical agent and concentration. The injection of the noxious solutions was performed according to a technique developed by Broman and Olsson (1948, 1955) and modified by Steinwall (1958). Rabbits (under nembutal and/or urethane anaesthesia) were used because their cerebrovascular supply make them particularly suited for this experimental procedure as they have no rete mirabile anastomosis between the internal and external carotid arteries. The various steps of a standard experiment are illustrated in Fig. 2. The solution was injected through a plastic catheter secured in one common carotid artery after ligation of its proximal part and external trunk as well as detectable extracerebral branches from the internal carotid artery. By keeping the injection pressure at the blood pres-

UNILATERAL DAMAGE OF BLOOD-BRAIN BARRIER

359

Fig. 2. Main steps in the experimental procedure for unilateral perfusion with barrier-damaging agents and subsequent demonstration of the effects by means of intravenously given tracers. (EEG records from a study by Flodmark and Steinwall, 1963a.)

sure level the solution displaces the blood of the ipsilateral hemisphere as controlled by inspection of the pia, vessels through the intact transparent dura in a trephine opening. If the injection (application) time is kept within narrow limits (generally about 30 sec), the concentration will be decisive for the effect of each chemical agent. Afterwards the injected hemisphere was recirculated via the circle of Willis, which in rabbit is supplied with blood predominantly from the vertebral arteries. As tested in some studies on brain uptake of nutrients the blood flow in the experimental hemisphere after mildchemical injury was practically equal to that in the control (see also Flodmark and Steinwall, 1963a). For the detection of the barrier dysfunctions induced by the unilateral perfusion, suitable tracers were given intravenously and comparatively determined in the two hemispheres. The presence of staining was estimated by visual inspection and radioactivity assayed by conventional methods for counting or by autoradiography. Organic ion transport

The proposed transport from brain to blood of ionized “waste” material, which was also thought to impede the extravasation of blood-borne organic ions (e.g. acidic or basic indicators of BBB damage), has as yet but fragmentary support from experimental observations. Data in favour of this theory are summarized in the following statements: 1. Different types of noxious agents which are known to inhibit the renal tubular transport also cause BBB dysfunction (Steinwall, 1961). 2. In adequate concentrations a number of organic acids give rise to reversible BBB “inhibition” as tested with acid dyes. The barrier inhibiting potency roughly parallels References p. 363-364

360

0. S T E I N W A L L

their respective tubular Tm, i.e., their ability to occupy maximum transport capacity (Steinwall, 1961). 3. Acid tracers, such as sodium fluorescein, which have permeated into the brain in reversible BBB impairment are eliminated within a few hours of restored function of the BBB (Flodmark and Steinwall, 1963 b). A corresponding phenomenon was recently reported by Werdinius (1966) who found that acid monoamine metabolites, produced within the brain, appeared to be secreted into the blood. It is, however, still an unsettled problem if the organic ions are eliminated directly through the bloodbrain interphase or have to make a detour via the choroid plexus, where such transport events are convincingly shown to take place. Obviously, the hypothesis of a brainblood counter transport of organic ions as contributor to the BBB effect needs more experimental substantiation. Brain uptake of nutrients in BBB damage

In earlier investigations mercuric ions in low concentrations (of the order of 10 pM) were found to impair the blood-brain barring of ionized organic acids or bases (Steinwall, 1961; Hansson and Steinwall, 1962). From studies on other membraneous systems it was known that this agent could be a potent inhibitor of the specific transfer of glucose and other nutrients (Passow et al., 1961). It was therefore used in the experiments aimed at bringing about a BBB damage characterized by simultaneous decrease in blood-brain transfer of tracers of the nutrient type and extravasation of normally barred organic ions such as acid dyes. All these experiments were of short duration (5-15 min) in order to minimize the possible influence of unequal metabolic events in the damaged and control hemisphere. The following nutrient representatives were studied separately : [14C]glucose, [14C]methyl-O-glucose, [14C]cycloleucine and [75Se]selenomethionine. In the first study [14C]glucosewas used and assayed by counting of plated trichloroacetic acid extracts of brain (Steinwall, unpubl. data). The results (Table 11) suggested TABLE I1 U P T A K E O F [ ' 4 c ] G L UCO S E FROM B L O O D I N U N I L A T E R A L B B B D A M A G E C A U S E D BY MERC U R I C IONS. CO M P ARAT I VE ASSAY O F ''CACTIVlTY I N EX P ER IMEN TA L A N D C O N T R O L HEMISPHERE. DE G RE E OF S T A I N I N G W I T H A C I D D Y E D ETER MIN ED BY GR OS S I N S P E C T I O N

No.

HgClz conc. pcM 50 50 50 40

80 80 Saline

Staining of experimental hemisphere

14Cratio experinlentat

weak weak weak weak medium strong none

0.8 0.7 0.7 0.6

/control

1.o 1.7 1 .o

UNILATERAL DAMAGE O F BLOOD-BRAIN BARRIER

36 1

that an inhibition of glucose uptake occurred and that it was best detected in mild injuries, while in more severe barrier damage with strong staining from extravasated dye the experimental hemisphere seemed to contain equal or higher amount of 14Cactivity than the control. This tendency illustrated a diffuculty implicit in the experimental situation. Evidently, the diminished [ “T]glucose uptake caused by inhibition of a specific transport mechanism might be countered and overpowered by a passive leakage in more pronounced BBB damages. Later experiments confirmed that the most distinct indication of blocked nutrient uptake was achieved in very weak BBB impairments with respect to extravasation of acid dyes. In another series (4 experiments) with [ 14C]glucose, sections from brain slices, frozen immediately after removal, were used for autoradiography (Steinwall and Hansson unpubl. data). In two of these brains the decreased uptake in the hemisphere perfused with HgC12 (20 pM)was quite evident (Fig. 3) and the counting assay showed an experimental/control ratio of 0.61 to 0.65. These studies on [14C]glucose did not include any attempt to identify the radioactive material within the hemispheres. Thus, the possibility that hemispheric dif-

Fig. 3. Autoradiogram of cross sections of a rabbit brain (14Cactivity white). The left hemisphere was perfused with 20 /tM HgClz (40seconds) and [‘4C]glucose and an acid dye (Prontosil soluble) given intravenously. The left-sided BBB dysfunction was indicated both by extravasation of the dye and by decreased amount of I4Cactivityas compared with the control hemisphere. References p . 363-364

362

0. S T E I N W A L L

ferences in glucose metabolism influenced the results could not be ruled out, even if the short duration of the experiments made it less likely. By replacing glucose with [14C]methyl-O-glucosean attempt was made to refine the situatiton with regard to the transport problem. According to studies on other organs this glucose analogue is transferred by a specific glucose transport mechanism without being further metabolized (Csaky, 1958, Jorgensen et al., 1961). In the experiments with this tracer, a method was employed which allowed a sensitive fluore-photographic image to be made of extravasated fluorescein-Na in the same sections that were used for autoradiography. As reported in detail elsewhere (Steinwall and Klatzo, 1966) there was in 2 of the 4 animals a markedly reduced uptake of [14C]methyl-O-glucose in the experimental hemisphere in regions with scarcely visible fluorescence, while an accumulation of radioactivity appeared in small spots which in the photography showed a rather bright fluorescence. The studies with [“C]cycloleucine (Steinwall and Snyder, to be publ.) were also based on the assumption that this amino acid shared the transport meachanism of its natural relative leucine (Christensen, 1962; Lajtha et al., 1963). In series of 4 experiments all brains showed a considerably diminished uptake of the amino acid in the perfused hemisphere (ratios of 0.29 to 0.71). Chromatographic analysis verified that the radioactivity in the brain was derived solely from unchanged cycloleucine. In 3 similar experiments the blood flow factor was tested by applying the rapidly permeating compound [14C]penthothal, which was recovered in equal amounts from the two hemispheres in each brain. In the last series [75Se]L-selenomethionine was used as nutrient tracer. According to Blau and collaborators (see Blau, 1964) this amino acid behaves biologically like the natural L-methionine with regard to transport in the intestines as well as in uptake in the pancreas. In these experiments a direct control of circulatory symmetry could be achieved by administering to some of the animals both the amino acid and the blood flow tracer, [131I]antipyrine (Oldendorf and Kitano, 1965). The differences in half life of these 2 gamma emitters, conveniently assayed by well scintillation counting, permit the deduction

-

[ESejL selenomethionine

pI]

4-iodoantipyrine

11 11 ‘I1

Fig. 4. Brain uptake of [7Y3e]~-selenomethionineand concurrent blood flow test with [1311]4-iodoantipyrine in unilateral mercurial BBB damage. Left column : experimental hemisphere. Righr colitnm : control hemisphere (arbitrary unit in each pair). Decreased amount of 75Se activity in the experimental hemisphere is apparent also when the blood flow tracer shows a practically symmetrical deposition.

UNILATERAL DAMAGE OF BLOOD-BRAIN BARRIER

363

of the activity emitted by each of them. From this series, still in progress, results are given in Fig. 4, showing a markedly diminished uptake of the amino acid in the barrier damaged hemisphere without significant asymmetry of the blood flow. CONCLUSION

The presented experiments, although crude and at best semiquantitative, were able to illustrate a BBB damage in which an abnormal ouflow of conventional tracers like acid dyes might occur concomitant with a decreased transfer of other tracers such as glucose or amino acids. With regard to the extravasation of acid dyesitisan open question whether or not this reflects in part the blockage of a counter transport mechanism for organic ions, or if it is solely due to increased passive permeability. In any case, the latter factor does not explain the diminished uptake of the nutrient tracers, a finding that can be postulated to reflect an inhibition of specific transport mechanisms. An increasing number of data from other investigations corroborate that such mechanisms operate in the blood-brain interphase (for reviews, see Lajtha, 1962; Crone 1965; a.0.). In the general discussion of BBB damage the pathogenic significance of transport inhibition might deserve more attention than usually given.

REFERENCES

L. AND LEE,J. C. (1965) Cerebral Edema. Charles C. Thomas Springfield, Illinois. BAKAY, BLAU,M. (1964) Pancreas scanning with 75Se-Selenomethionine.Medical Radioisotope Scanning Vol. 11. International Atomic Energy Agency, Vienna (p. 275). BROMAN, T. AND OLSSON, 0. (1948) The Tolerance of Cerebral Blood Vessels to a Contrast Medium of the Diodrast Group. Acta Radiol., 30, 326-342. -, (1956) Technique for the Pharmaco-dynamic Investigation of Contrast Media for Cerebral Angiography. Acfa Radiol., 45, 96-100. CHRISTENSEN, H. N. (1962) Biological Transport. W. A. Benjamin, Inc. New York. (p. 54). CRONE, C. (1965) Facilitated Transfer of Glucose from Blood to Brain. J. Physiol. (London), 181, 103-113.

CSAKY, T. Z. (1958) Active Intestinal Transport of 3-0-Methylglucose. In: 4th International Congress of Biochemistry. Infern. Abstr. Biol. Sci., Supp. p. 80. Vienna. FLODMARK, S . AND STEINWALL, 0. (1963a) Differentiated Effects on Certain Blood-Brain Barrier Phenomena and on the EEG Produced by Means of Intracarotidally Applied Mercuric Dichloride. Acra physiol. Scand., 51, 446453. -, (1963b) Reversible Blood-Brain Barrier Alteration Induced by Certain Organic Acids and Indicated by Means of EEG and Dye Tests. Acta Physiol. Scand., 58, 368-375. HANSSON, E. AND STEINWALL, 0. ( I 962) Abnormal Blood-Brain Passageof aQuarternary Phenothiazine Derivative (S35-labelled AprobitR) Induced by Chemical Agents. Acta physiol. Scand., 54, 339-345.

J ~ R G E N SC. E NE., , LANDAU, B. R. AND WILSON, T. H. (1961) A Common Pathway for Sugar Transport in Hamster Interstine. Amer. J. Physiol., 200, 1 1 1-1 16. KLATZO, I., STEINWALL, 0. A N D STREICHER, E. (1967) Dynamics of cold injury edema. Proc. Symp. on Brain Edema, Vienna, Sepf. 1965. I. Klatzo and F. Seitelberger (Eds.), Springer-Verlag, WienNew York. LAJTHA, A. (1962) The Brain Barrier System. Neurochemistry. Thomas Springfield, Illinois. (p. 399). LAJTHA,A., LAHIRI,S. AND TOTH,J. (1963) The Brain Barrier System-IV. Cerebral Amino Acid Uptake in Different Classes. J. Neurochem., 10, 765-773. OLDENDORF, W. H. A N D KITANO, M. (1965) The Symmetry of P1 4-i~doantipyrineUptake by Brain after Intravenous Injection. Neurol., 15,994-999.

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0.S T E I N W A L L

PASSOW,H., ROTHSTEIN, A. AND CLARKSON, T. W.(1961) The General Pharmacology of the Heavy Metals. Pharmacol., Rev., 13, 185-224. 0. (1958) An Improved Technique for Testing the Effect of Contrast Media and Other STEINWALL, Substances on the Blood-Brain Barrier. Acta Radiol., 49, 281-284. -, (1961) Transport Mechanisms in Certain Blood-Brain Barrier Phenomena. - A Hypothesis. Acta psychiat. neurol. Scand., 36, Suppl. 150, 314318. -, (1964) Blood-Brain Barrier Dysfunction: Some Theoretical Aspects. A c f a nertrol. Scmid., 40, SUPPI. 10, 25-29. STEINWALL, 0 .AND KLATZO, I. (1966) Selective Vunerability of the Blood-Brain Barrier in Chemically Induced Lesions. J. Neuropathol. Exptl. Neurol., 25, 542-559. WERDINIUS, B. (1968) Effect of Probenecid on the Levels of Monoamine Metabolites in the Rat Brain. Acta pharmacol. toxicol., 25, 18-23.

DISCUSSION T. Z. CSAKY:Dr. Steinwall, with regard to your 3-methyl-glucose experiments, I would like to ask if you followed this experiment up with a quantitative determination of 3-methyl-glucose, and what did you find? 0. STEINWALL: This has not been done.

T. Z. CSAKY:Where is the label in your 3-methyl-glucose? 0. STEINWALL: In the methoxyl group.

T. Z. CSAKY:Is it in the methoxyl group? This is very important, because there are two preparations available: one is prepared from uniformly C-14 labeled glucose which is methylated. This is always contaminated with radioactive glucose. The other is prepared from inert glucose by methylation with radioactive methyliodide. The reason for my asking about this is that some five years ago we published experiments on the distribution of 3-methyl-glucose in rats. If the methyl-glucose was injected into nephrectomized animals, after about two hours, equilibrium was reached in the body. If we then determined the relative concentrations of 3-methyl-glucose in the various organs, the brain always contained very low amounts. With cardiac muscle and liver we obtained an almost complete equilibrium between the organs and the blood. The muscle contained less water and in the brain the ratio was less than 1 : 10. Moreover, if radioactively labeled 3-methyl-glucose was injected into a rat, it was excreted in the urine. After 48 hours we still found some radioactivity in the brain, which means that the exchange between the blood and the brain is extremely slow in the case of 3-methyl-glucose. 0. STEINWALL: As I said, we did not d o quantitative estimations; however, the brain uptake of our methyl glucose was very convincing as judged from the autoradiographic findings in comparison with such carbohydrates as mannitol and sucrose, studied under corresponding conditions. T. Z. CSAKY:But mannitol probably does not penetrate the brain at all. 3-methyl-glucose does, but at an extremely slow rate, so it certainly does not behave the same way as glucose. 0. STEINWALL: One would not expect that. But there may be enough uptake under normal conditions to reveal this inhibition under the abnormal conditions that we created in the left hemisphere.

H. M. ADAM:If you damage the blood-brain barrier with mercuric ions. then can you reverse the process? 0. STEINWALL: That might perhaps be possible by special means. The spontaneous course of this mercurial barrier damage, however, seems to be of long duration and progressive rather than regressive in nature (Flodmark & Steinwall, Acta physiol. scand., 1963, 57, 446453). The mercuric ions are probably tightly fixed to their site of action.

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365

H. M. ADAM:Did you estimate the amount of mercuric ions that was captured by the cerebral tissue? 0. STEINWALL: No. A. LAJTHA: I would just like to make some general philosophical remarks for the concern of the

morning session. Perhaps every scientist should be entitled to his own concept of a blood-brain barrier and perhaps one of the functions of a meeting such as this could be to simplify somewhat the matter. I t needs to be emphasized that this is a living brain with more than just one blood-brain barrier. We could classify, as the first step, the test-substances into at least three classes: (1) substances that do not penetrate membranes; (2) substances that penetrate membranes by diffusing through membrane constituents, and (3) substances that penetrate membranes by carrier or mediated mechanisms. Now, if we realize that one may effect one kind of passage (such as mediated transport) without effecting another (such as ditfusion) we may perhaps use the term blood-brain barrier somewhat more selectively. Definitions of the blood-brain barrier would also depend on how we define the brain. May I add one difinition? My own is that the brain is the nucleus of the neuron. This is of course said only as a joke, but with this I would like to emphasize that between the blood and the brain there are many more membranes than the capillary membrane; that these would include the neuronal membranes and perhaps even the mitochondria1 membranes. In fact the cell membrane by altering the concentration in the cytoplasnia, must have an important influence on the concentration of substances in the nucleus.

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367

Changes in Blood-Brain Permeability during Pharmacologically induced Convulsions ROBERT W. P. CUTLER, A N T O N I O V. L O R E N Z O

AND

C H A R L E S F. BARLOW

Neurology Service of the Childreds Hospital Medical Center, Peter Bent Brigham Hospital, and the Department of Neurology, Harvard Medical School, Boston, Massachusetts, U.S.A.

INTRODUCTION

Disturbances of the normal functions of the “blood-brain barrier” are well known following a large variety of pathological stresses. Relatively less attention has been directed towards the transient alterations in solute exchange between blood and brain that may accompany less destructive stimuli. Such alterations may be significant in the pathogenesis of certain disorders of central nervous system function. In the experiments to be reported, temporary alterations in the entry of [35S]sulfate into brain were found to accompany pharmacologically-induced convulsions. Marked concomitant changes in brain surface pH, oxygen and carbon dioxide tensions were recorded by direct measurement during the convulsion. As a result of these observations, the influence of hypercapnia and acidosis on brain vascular permeability was studied in some detail. [35S]sulfateand [125l]albumin were employed as indicators. The former solute is a diffusible anion with an intermediate rate of entry into brain. A steady concentration in all regions of brain is reached one to two hours following intravenous injection. While sulfate is predominantly confined to the extracellular compartment of brain, a portion is incorporated into metabolites, permitting an evaluation of alterations in sulfate metabolism on brain uptake. Such factors as enlargement of the sulfate space, enhanced metabolism of sulfate, expanded capillary surface area from vasodilatation, or increased cerebral blood flow may contribute to an accelerated early entry of [35S]sulfateinto brain. By contrast, iodinated albumin is confined to the brain vasculature, with no evidence of entry into brain under normal conditions (Barlow et al., 1958). Therefore, extravasation of albumin may be considered to reflect a pathological change in barrier mechanisms. A . The efect of pharmacologically induced convulsions on the penetration of [35S]sulfate into cat brain 1. General methodology. Adult cats were anesthetized locally with xylocaine, immobilized with gallamine, and artificially respired at carefully controlled rates and minute References p . 378

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volumes. The convulsant drug was administered intravenously. Following the appearance of electroencephalographic seizure activity, carrier free [35S]sodium sulfate, 1 mC/kg, was given intravenously. After a five minute circulation period, the animals were anesthetized with pentobarbital, heparinized, and were then perfused through an aortic cannula with 200 ml isotonic saline. The brain was removed, frozen, and sliced freehand for gross autoradiography. Fifteen regions of brain, CSF, and plasma were assayed for 35s radioactivity. [3jS]metabolites were separated by paper electrophoresis (Goldberg et al., 1963) to permit expression of the data as a tissue to plasma ratio for [35S]sulfate. The experimental procedures will be published in detail elsewhere (Lorenzo et al., 1967). Control animals were treated in a fashion comparable to the experimental animals, except that the convulsant agent was omitted. The results of this series of experiments are presented in Fig. 1 and Table I.

Fig. 1. 35Sautoradiograms made from frozen coronal slices of cat brain at the level of thalamus and medulla. [35S]sulfatewas injected intravenously five minutes before sacrifice.

2. Pentylenetetrazol (metrazol) in a dose of 30 mg/kg produced increases in the fiveminute uptake of [35S]sulfate generally throughout the brain. However, distinct regional accentuation of sulfate uptake was observed, with an approximate 12-fold increase in the thalamus and lateral geniculate ganglion, an 8-fold increase in the medial geniculate ganglion, and a 4-fold increase in the cerebral cortex and white matter. There was no increase in penetration of sulfate into the cisternal cerebrospinal fluid. In addition, the percentage of 35s incorporated into metabolites was not significantly increased. When metrazol was given to animals that had received [35S]sulfate after ureter ligation six hours previously, no change in the steady-state concentration of [35S]sulfate was observed. 3. The eflect of strychnine (0.5 mg/kg) on sulfate uptake, while less pronounced than metrazol, was nonetheless distinctly regional. The highest uptake, approximately four times control, was observed in the lateral geniculate body, followed in order

369

BLOOD-BRAIN PERMEABILITY

TABLE I cpm/gm wet tissue 35S04 Uptake by Brain

~

-

-

_x 100

_

J

\ cpm/ml plasma Region

Cerebral cortex Cerebral white Thalamus Lateral geniculate Medial geniculate Caudate Hippocampus lnferior colliculus Cerebellar cortex (paleo) Cerebellar cortex (neo) Cerebellar white Central medulla Cervical cord CS F

Coritrol

Metrazol

Strychnine

( 9 )*

(5 )

(10)

Methionine Sulfoximine (6)

0.38 f 0.02 0.11 0.02 0.18 i 0.02 0.19 f 0.01 0.18 i 0.02 0.23 t 0.03 0.22 1 0 . 0 3 0.28 f 0.01 0.35 f 0.02 0.35 f 0.02 0.10 0.01 0.14 0.01 0.45 10.05 I S O & 0.20

1.61 0.34 0.49 f 0.09 2.19 0.45 2.34 f 0.67 1.43 i 0.26 1.37 f 0.17 0.86 & 0.16 1.02 & 0.13 1.02 & 0.14

0.55 f 0.05 0.16 f 0.01 0.44 f 0.03 0.79 f 0.08 0.35 f 0.02 0.27 f 0.03 0.34 f 0.04 0.31 f 0.03 0.80 f 0.12 0.49 f 0.06 0.16 f 0.02 0.43 f 0.04 0.74 f 0.10 1.80 f 0.20

1.10 f 0.18 0.34 f 0.06 0.69 f 0.16 0.96 & 0.24 0.70 f 0.13 0.54 f 0.09 0.78 f 0.20 0.69 f 0.09 0.49 f 0.05 0.55 f 0.04 0.27 f 0.04 0.38 rt 0.10 1.02 f 0.14 1.33 f 0.18

*

*+

_

_

0.28 :k 0.03 0.59 & 0.12 0.96 f 0.21 1.89 0.45

~ _ _ _

*

Number of animals

by the thalamus, the cerebellar paleocortex, and the central medulla. Less, but significant increases in sulfate uptake were observed in most other regions, while the cerebrospinal fluid showed no change (Lorenzo and Barlow, 1967).

4. The data for a series of cats convulsed with methionine sulfoxamine are tabulated in the last column of Table I. A dose of 5-20 mg/kg was given twenty-four hours prior to the onset of the study. This drug produces a seizure disorder characterized by both psychic and motor activity. Upon the appearance of a clinical seizure, the five minute sulfate uptake was measured as described. A more generalized increase in sulfate penetration was found, particularly prominent in the cerebral and cerebellar cortex and hippocampus. In addition, autoradiography revealed a pronounced uptake in the substantia nigra (arrow, Fig. l), an area ordinarily inaccessible to direct radioassay. While detailed comparisons were lacking for methionine sulfoximine, the regions of increased sulfate penetration during metrazol and strychnine convulsions were comparable to the regions which exhibit enhanced electrical excitation in depth electrode studies (Starzl et al., 1953; Johnson, 1955). It seemed reasonable to propose that the enhanced regional uptake of anion in these drug-induced convulsions was a consequence of heightened neuronal activity, rather than of direct alterations in permeability produced by the drugs. Several observations are consistent with this hypothesis. Previous studies (Lorenzo et al., 1965) have demonstrated specific increases in sulfate uptake in the visual and auditory systems upon photic and acoustic stimulation, indicating that neuronal activation by more physiological stimuli may be accompanied Rcfivriiri~sp. 378

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by alterations in [35S]sulfate exchange. In addition, sub-threshold doses of the drugs employed produced no changes in sulfate penetration. Furthermore, when blindfolded cats were convulsed with strychnine, the tissue-plasma ratio for [35S]sulfate was 0.61 f 0.03, as compared to 0.19 for control animals. When the eyes were exposed to laboratory illumination during the strychnine convulsion, however, the [35S]sulfate tissue-plasma ratio in the lateral geniculate increased significantly to 0.91 f 0.1 I . This observation was consistent with the recognized ability of strychnine to facilitate neuronal activation by an afferent stimulus. The alterations in sulfate penetration induced by metrazol and strychnine convulsions were rapidly reversible. In experiments in which the sulfate was injected one hour after the cessation of seizure activity, brain [35S]sulfate uptake had returned to control values. In the case of metrazol, convulsions were stopped by the intravenous injection of trimethadione. With strychnine, convulsions were abolished with methocarbamol and trimethadione. Such rapid reversibility lends additional support to the concept of heightened neuronal metabolic activity as a factor in the production of permeability changes. B. The effect of metrazol on the penetration of['3lI]albumin into cat brain In this series of experiments, cats were convulsed with metrazol for five, fifteen, thirty and sixty minutes, and the uptake of [131'l]albumin (100 pC/kg) during the seizure was studied. There was no detectable tissue penetration of iodinated albumin in control animals, nor in those convulsed for five minutes. After fifteen minutes of seizure activity, patchy extravasation of protein was observed in the thalamus, which by thirty minutes became extensive and confluent (Fig. 2). By one hour, the amount of

Fig. 2.

1311

autoradiogram made from frozen coronal slice of cat brain at the level of the thalamus, following a thirty-minute metrazol convulsion.

albumin in extravascular compartments of the thalamus was five to six times the amount normally contained in the blood vessels of that region, while the cortex and white matter remained relatively impermeable, as illustrated.

37 1

BLOOD-BRAIN PERMEABILITY

C. Additional physiologicaI alterations accompanying metrazol convulsions Blood pressure, blood pH, p0z and pC0z were recorded during a five minute pentylenetetrazol convulsion. Cortical pH, oxygen tension, carbon dioxide tension, and the electroencephalogram were recorded from electrodes on the exposed cortical surface. Cortical temperature (cerebral blood flow) was measured by a thermistor. Craniec-

/

I

...

Wood pC0,

Fig. 3. Continuous in vivo recording of the physiological parameters indicated prior to and during a live-minute metrazol convulsion. The interruption of the time pulse recording at the top of the polygraph signifies the period of injection of rnetrazol (from Lorenzo et a/., 1967). Refiriwws p . 378

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tomy and electrode placement was performed under ether anesthesia, and recordings were begun thirty to sixty minutes after anesthesia was discontinued. The results of a representative experiment are illustrated in Fig. 3. Thirty seconds following the intravenous administration of metrazol, seizure discharges appeared on the electroencephalogram, accompanied by tachycardia and systemic hypertension. Cortical temperature rose promptly and remained elevated during the entire convulsion. There were no significant alterations in blood gases or hydrogen ion concentration. By contrast, changes on the cortical surface were quite marked. Brain oxygen tension fell from 16 mm Hg to 0 mm Hg, while carbon dioxide tension rose from a control value of 22 mm Hg to 85 mm Hg. The pH fell from 7.30 to 7.10. It was apparent that the state of increased neuronal metabolism during a convulsion was sufficient locally to generate excessive quantities of carbon dioxide, with a concomitant fall in tissue pH. D . The effect of hypercapnia on the penetration of sulfate andalbumin into the brain 1. I n a series of cats artificially respired with 20

% COz : 80 % 0 2 , marked alterations

in the five-minute uptake of [35S]sulfatewere observed. While somewhat greater variability was noted in the hypercapnic animals than in the convulsed animals, the most significant increases in uptake occurred in the thalamus and brain stem (Fig. 4). As

Fig. 4. The influence of hypercapnia on [35S]sulfatepenetration into cat brain.

shown previously (Goldberg et al., 1963), hypercapnia affected the rate of entry of sulfate without altering the steady state tissue-plasma ratio or the degree of metabolic incorporation.

2. A more detailed study of the effects of hypercarbia on brain vascular permeability was carried out in guinea pigs, employing [125I]albumin as a tracer (Cutler and Barlow, 1966). Because serum albumin enters brain to a very limited extent, if at all, the ratio of brain tissue~[125l]albuminto plasma [125l]albumin serves as a measure of brain plasma volume in the normal animal, while extravascular penetration of radioiodinated albumin into brain under experimental circumstances reflects an alteration in brain vascular permeability. The methods employed in this series of experiments were as follows: anesthetized

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BLOOD- BRA1 N P E R M E A B I L I T Y

guinea pigs were artificially respired with room air or various mixtures of carbon dioxide : oxygen. In the first series of animals, [1251]human albumin was injected intravenously, and circulated for one hour. Animals were sacrificed at the end of one hour either by decapitation with rapid immersion of the head in solid carbon dioxide: acetone at -6O"C, or by vascular perfusion with 100 ml isotonic saline. Frozen brains were dissected into nine regions and assayed with plasma for 1251 radioactivity. pH and p C 0 z were measured in samples of jugular venous blood drawn from a cannula inserted cranially. Histological autoradiograms were prepared from fixed, paraffin embedded tissue sections dipped in liquid photographic emulsion. T A B L E 11 METHOD O F DETERMINING ['"I]ALBUMIN

( A ) C02 Decapitate

(B ) Control Decapitate ( C ) COZPerfuse

*

A - [B I C]

=

D I S T R I B U T I O N I N BRAIN

Nornial Vascular Volutne

Exfravascular Volume

Change In Vascular Volume*

+ +

+

t

0

0

-t

0 0

Change in Vascular Volume.

By separate analysis of experiments terminated by either vascular perfusion or rapid freezing, the iodinated albumin activity in the brain may be partitioned into normal vascular, dilated vascular, or extravascular compartments, as summarized in Table 11. In order to equate tissue plasma volume with tissue vascular volume, it was necessary to assume that regional changes in hematocrit did not occur under conditions of hypercapnia. This assumption was supported by the lack of change ofjugular venous blood hematocrit in hypercapnic animals as compared with controls. Penetration of serum albumin into the guinea pig brain under normal conditions was not detectable. The slight amount of radioactivity remaining in the normal brain after perfusion may be identified within vessels by histoautoradiography. Upon exposure to high concentrations of carbon dioxide, marked changes in vascular permeability were observed. There was a consistent extravasation of albumin into the thalamus, hypothalamus, mid-brain, medulla and spinal cord. By contrast, there was little evidence of abnormal permeability in the cerebral cortex, cerebral white, caudate nucleus or cerebellum, as illustrated in Fig. 5, and in column C,Table 111. During more prolonged exposure to carbon dioxide, for periods up to eight hours, protein extravasation continued at the same rate in vulnerable regions, while the cortex and white matter remained resistent to permeability alteration. It was evident that the state of altered vascular permeability persisted as long as the animal remained hypercarbic, and that no adaptation to this stress occurred, at least during an eight-hour interval of time. However, the changes in permeability were rapidly and completely reversible. When animals which were exposed to twenty-five percent COz for one hour and then room air for ten minutes were subsequently injected with radioiodinated Refirc~rtri~s p. 378

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w. P. C U T L E R et al.

Fig. 5. 1251 autoradiograms of frozen slices of guinea pig brain following a one-hour exposure to twenty-five percent COZ.(a) mid-sagittal plane; (b) coronal, mid-thalamus; (c) coronal, mesencephalon; (d) coronal. medulla. (From Cutler and Barlow, 1966). T A B L E 111 ONE-HOUR BRAIN-PLASMA RATIOS*

C COz-Perfitse

(5)t

B ControlDecapitate (6)

5.40 f 0.30 2.86 f 0.20 4.09 f 0.44 5.58 f 0.77 6.70 f 0.80 4.60 f 0.61

4.00 f 0.17 1.83 f 0.34 2.85 f 0.27 3.52 f 0.13 4.80 f 0.46 2.86 f 0.34

0.34 f 0.06 0.22 0.02 0.37 0.06 2.25 f 0.65 2.44 f 0.55 2.03 f 0.50

A COz-Decapifate

Cortex White Matter Caudate Thalamus Colliculus Medulla

*

t

(6)

A Vascular Volume

( A -[ B t C l ) -I 1.06 0.81 0.87 -0.19 i-0.06 -0.29

+ +

CPM/mg dry tissue

x 100 CPM/,uI plasma number of animals.

albumin, no penetration of albumin into the brain was found after a one hour circulation. With prolonged hypercapnia (eight hours), relatively large quantities of plasma albumin escaped into the tissues. In the thalamus and medulla, this amounted to seven times the normal quantity contained in the vasculature of these tissues. While the

BLOOD-BRAIN PERMEABILITY

375

Fig. 6 . [1251]alburninhistoautoradiogram showing high silver grain density overlying a neuron in the reticular formation of the medulla. Hemdtoxyhn. X 560. resolution of light microscopy was insufficient to permit precise localization of the extravasated albumin by autoradiography, it was apparent that some of the protein had entered cells, notably into large neurons of the reticular formation (Fig. 6). By ultracentrifugation it was found that approximately thirty percent of the [125I]radioactivity was sedimented with subcellular particles. In animals exposed to twenty-five percent COZ for one hour, it was possible to determine the amount of /125I]albumin within the brain vasculature, as well as in extravascular compartments, as outlined in Table 11. The results of this analysis for six brain regions are presented in Table 111. I n this table, valuesincolumn Crepresent the amount of extravascular protein, while the values in the last column are a measure of expansion or constriction of the vascular compartment. It was apparent that the greatest permeability changes occurred in regions in which the vascular volume remained unaltered. Conversely, enlargement of the vascular compartment, either by vasodilatation or by opening new vascular channels was not associatedlwith pathological permeability. When extravascular protein concentration was expressed as a percentageof the normal vascular concentration andplottedlagainst changes in vascular volume, a consistent inverse relationship was found for the nine regions of brain assayed (Fig. 7). Additional experiments were performed to assess the effects of lower concentrations of carbon dioxide and of metabolic acidosis. With ten percent carbon dioxide Rcfcrmri~rp. 378

376

w. P.

R.

w z

3

9

50

40 30

p

20

u)

w +I0 z

(3

z

40 .

et al.

60

3 3 0

CUTLER

o

- 10 s

0

0

0 0 0 0 0 0 fi

1

10 20 30 40 50 60 70

RELATIVE PROTEIN PENETRATION

Fig. 7. The relationship between protein permeability and vascular volume changes in hypercapnic guinea pigs. Each point represents one of nine brain regions sampled. (From Cutler and Barlow, 1966).

exposure, the jugular venouspCO2 rose from 48 mm Hg to 79 mm Hg, but no protein extravasation was found. Similarly, in animals made acidotic by HCI infusion (mean pH 7.02, mean pC0z 53 mm Hg), there were no alterations in protein permeability. It was apparent that the relatively high levels of carbon dioxide tension produced by exposure to twenty-five percent COz (mean pH 6.94, mean pC0z 136 mm Hg) were required to produce pathological protein permeability. DISCUSSION

Alterations in [35S]sulfate penetration into the brain during convulsions have followed a distinct pattern for each of the pharmacological agents studied. The observation of enhanced sulfate uptake in regions shown to be electrically activated during metrazol (Starzl et al., 1953) and strychnine (Johnson, 1955) convulsions provides evidence that local acceleration of neuronal metabolism may be accompanied by heightened solute exchange. Neither the degree of metabolic incorporation of 35s nor the size of the steady state sulfate pool were altered by the convulsions. Theenhanced [35S]sulfate uptake may be considered as a partial consequence of increased delivery of the anion to the tissues secondary to capillary vasodilatation or increased blood flow. In support of this thesis is the close correlation of enhanced regional sulfate penetration (Lorenzo et al., 1965) and increased regional cerebral blood flow (Sokoloff, 1957) in visual centers of photically stimulated cats. However, as sulfate enters the brain relatively slowly (one to two hours for maximum levels to be reached following intravenous injection in the cat), its rate of entry cannot be closely regulated by regional blood flow. Alternatively, or in addition to change in blood flow, the enhanced penetration of [3%] sulfate may be a more direct consequence of regional changes in vascular permeability to sulfate. The finding of regional entry of iodinated albumin during more prolonged convulsions is strongly in support of this argument. Because this tracer is normally excluded from the brain even after long circulation times, its- entry under these circumstances must imply altered vascular permeability.

BLOOD-BRAIN PERMEABILITY

377

Changes in arterial blood pH, oxygen tension, and carbon dioxide tension were not observed during a brief metrazol convulsion, while the same recorded values from the brain cortical surface were markedly altered. Carbon dioxide is one of the most active agents in the regulation of cerebrovascular tone and cerebral blood flow (Sokoloff, 1959). Its influence on brain vascular permeability was first demonstrated by Clemedson et al. (1958) who found the brains of hypercapnic animals stained by trypan blue. In the experiments reported here, hypercarbia had a profound effect on vascular permeability in some regions of the brain while sparing others. There were no demonstrable defects in vessel walls on light microscopy, nor other neuropathological change. Diapedesis of erythrocytes was not an associated feature in our studies as in the experiments of Clemedson et al. (1958). From direct measurement of vascular volumes, we have concluded that vasodilatation was not a necessary factor in producing increased permeability. While the selective vulnerability of brain vessels to a systemically administered agent was emphasized by these results, the reasons for the selectivity were not forthcoming. It is of interest that during metrazol convulsions, increased entry of sulfate was observed in a number of regions of the cat brain, while protein penetration was predominantly restricted to the thalamus. Similar observations were made by Lee and Olszewski (1961) following repeated electroshock convulsions in the rabbit. It is reasonable to expect that a continuous rapid production of metabolic COZby excited neurons occurs during more prolonged convulsions. Regional increases in carbon dioxide tension of sufficient magnitude to produce protein extravasation would be anticipated. However, protein penetration would be detected only in those regions which both generate large amounts of carbon dioxide and are susceptible to the effects of hypercapnia. Therefore, in the case of metrazol, while both the thalamus and cortex are under excitation, the pathological permeability changes to albumin would be expected only in the thalamus. The techniques of autoradiography and quantitative regional tissue assay used in this study may provide a general method for the identification of physiologically or pharmacologically activated neuronal groups which are functionally interrelated. In this regard, attention is called to the autoradiograms depicting methionine sulfoximine convulsions in Fig. I . As indicated by the arrow, there is an apparent high uptake of 3% in the region of the substantia nigra, which could be followed in serial sections to its caudal extent. This region of brain is not ordinarily sampled for direct radioassay or probed by the recording electrode. The finding of a high autoradiographic density in these studies should provide a stimulus for investigating this nucleus with electrophysiological techniques. ACKNOWLEDGEMENT

This work was supported in part by grant NB-05172 of the National Institute of Neurological Diseases and Blindness. Rcfcrcnws p. 378

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REFERENCES BARLOW, C. F., SCHOOLAR, J. C. AND ROTH,L. J. (1958) An autoradiographic demonstration of the relative vascularity of the central nervous system of the cat with iodine 131-labeled serum albumin. J. Neuropathol. Exptl. Neurol., 17, 191-198. CLEMEDSON, C. J., HARTELIUS, H. AND HOLMBERG, G. (1958) The influence of carbon dioxide inhalation on the cerebrovascular permeability to trypan blue (the blood-brain barrier). Acta Pathol. Microbiol. Scand., 42, 137. CUTLER,R. W. P. AND BARLOW,C. F. (1966) The effect of hypercapnia on brain permeability to protein. Arch. Neurol., 14, 54-63. GOLDBERG, M. A., BARLOW,C. F. AND ROTH, L. J. (1963) Abnormal brain permeability in C02 narcosis. Arch. Neurol., 9, 498-507. JOHNSON, B. (1955) Strychnine psroxysms in brain stem. I. Anatomical distribution. J . Neurophysiol., 18, 189-199.

LEE,J. C. AND OLSZEWSKI, J. (1961) Increased cerebrovascular permeability after repeated electroshocks. Neurol., 11, 515-519. LORENZO, A. V., FERNANDEZ, C. AND ROTH,L. J. (1965) Physiologically induced alteration of sulfate penetration into brain. Arch. Neurol., 12, 128-132. LORENZO, A. V., BARLOW, C. F. AND ROTH, L. J. (1967) Effect of metrazol convulsions on S-35 entry into cat nervous system. Amer. J. Physiol., 212, 1277-1287. LORENZO, A. V. AND BARLOW, C. W. (1967) Effect of strychinine convulsions upon the entrv of V5 sulfate into the cat central nervous system. J. Pharmacol. Exptl. Thrrup. (in press). SOKOLOFF, L. (1957) Local blood,flow in neural fissues. New Research Techniques of Neuroanatomy. W. F. Windle, Editor. Springfield, Charles C. Thomas (p. 51). - (1959) The action of drugs on the cerebral circulation. Pharmacol. Revs., 11, 1-85. STARZL,T. E., NIEMER,W. T., DELL,M. AND FORGRAVE, P. R. (1953) Cortical and subcortical electrical activity in experimental seizures induced by metrazol. J. Neuropathol. Exptl. Neurol., 12, 262-276.

DISCUSSION

D. B. TOWER: Two things, Dr. Cutler. First of all I would like to make a plea in regard to methionine sulfoximine. It is nice to see people using this agent. This providesavery interesting seizure preparation because it is somewhat more chronic than many of the convulsant agents that one normally uses. When you give a convulsant dose of metrazol it generally gives a completely generalized kind of acute convulsion unless you fractionate the dose rather carefully. But in the case of methionine sulfoximine, the development is, as you indicated, over quite a period of time unless you inject it directly into the central nervous system. Furthermore, you tend to see more different forms of seizures (which one would like to relate by analogy to clinical situations in patients) in that you may have partial seizures that may develop into a generalized convulsion. The running behavior, the salivation and chewing movements are characteristics of four-footed animals and can be seen in other types of seizures, and are not peculiar to methionine sulfoximine at all. It is just that here is an agent that is showing many more manifestations because of the longer period over which they develop, and is in fact a sort of a semi-chronic affair. The one question I had in mind regards your perfused preparation: How much of a factor in analyses of the type you are doing is an asphyxia1 change in the cells of the central nervous system during this time? In other words: if you alter the inflow of nutrients, oxygen and glucose and so on o the nervous system, does it take very long for some of the cells to swell, as many people have shown. R. CUTLER:There was no demonstrable protein in control animals perfused in the same manner. One comment I might make about the methionine sulfoximine: agree that one cannot call this a perfect model of a temporal lobe seizure.

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379

W. W. TOURTELLOTTE: I would like to speak about perfitsion of the bruin to remove blood “coniaminants.” Perfusion, while it removes all contaminating blood, may introduce complications. For example, published data (Hogeboom, G . H., Schneider, W. C., and Stribbich, M. J.: J. Biol. Chem. 196, I 1 I , 1952; and Luch, J. M.: J. Biol. Chem. 115: 491, 1936) indicate that as much as 20-30% of liver nitrogen is removed by perfusion. On the other hand, estimations of the vascular space in the rat liver (ml of bloodikilogram of tissue) can vary from 99 to 178 to 270 depending on whether the rat was sacrificed by “slow” exsanguination (Caster. W. O., Simon, A. B., and Armstron, W. D.: Am. J. Physiol. 183: 317, 1955). sacrificed by ether (Friedman, J. J.: Am. J . Physiol. 196: 420, 1959) or sacrificed by rapid immersion in liquid nitrogen (Gibson, J. G., Sebgman, A. M., Peacock, W. C., Aub, J. C., Fine, 5.. and Evans, R. D.: J. C/in. Invest. 25: 848, 1946). Since the concentration of nitrogen in whole blood is very close to that of liver, the large nitrogen losses as a result of perfusion suggest considerable losses from extravascular liver substance. The best solution to these difficulties would seem to lie in a method for the determination of whole blood in fresh tissue rather than in any attempt to remove or stabilize this variable “contaminant.” To check this possibility in regards to brain, we have studied (unpublished observations) immunoglobulin-G (IgG) concentration (see chapter by Tourtellotte for discussion of the technical aspects of the immunochemical IgG assay) before and after perfusion of the guinea pig brain with 100 ml of 0.1 5 M sodium chloride. Furthermore, the blood content of the brains was determined (see Chapter by Tourtellotte for discussion of the technical aspects of brain hemoglobin determination); hence, it was possible to calculate the IgG concentration contributed by blood in the brain as well as the extravascular brain values. It was found that the IgG concentration, corrected for residual blood IgG concentration of the brain after 30 minutes of pentobarbital anesthesia (70 mg/kg, intraperitoneally) and quick removal of the brain after guillotining, was 0.78 f 0.04 gm/kg of brain (five guinea pigs). After perfusion with 100 ml of isotonic saline in 10 minutes, the brain IgG content, corrected for residual blood IgG, was 0.1 I t 0.01. Therefore, perfusion of the cerebral vessels under the conditions mentioned above removed 86 percent of the extravascular brain IgG. Since our results suggest that perfusion with isotonic saline of the brain immediately after death can reduce the concentration of rather large molecules, such as immunoglobulin-(?, would it not be more accurate to include in your experiments a determination of the amount of blood in the brain, (rather than to try to remove the blood “contaminant” by perfusion) and make a calculated correction for the extravascular concentration of the tagged substance you are studying? The volume of the blood in the brain and its “contaminating” substance can be estimated rather simply and precisely (Tourtellotte, W.W., and Parker, J. A.: Science 154: 1044, 1966). R. CUTLER: This whole problem of perfusion is a difficult one to know the real answer to. In answer to the last question: in that study we were dealing with sulphate, which has an extremely high concentration in the blood after five minutes, relative to the concentration in the tissue. If we don’t perfuse out the blood we won’t see any changes. With regard to the technique of determining haemoglobin, it seems much easier and more accurate for us to use a radioactive labeled protein that is excluded from the brain during this time period.

Did the [1311]albuminmove into the brain and hence interfere with your brain W. W. TOURTELLOTTE: blood volume determination?

R. CUTLER: Not in five minutes, though. Our method for determining blood flow per capillary surface area is to give an injection of iodinated albumin, determine the cardiac output in an individual animal, and let the albumin circulate for five minutes. Then we give an injection of 1311-labelled antipyrine, let that circulate for one minute, chop off the head and fix the vascular contents for dissections at -30”. The tissue is assayed for its iodinated antipyrine content, which can then be related to the cardiac output. This gives you a direct measurement of milliliters per gram of blood flow in an area. In the normal animal one can calculate the capillary volume; from the volume determine the capillary length, assuming a mean capillary diameter; then in the experimental situation further assume that the capillary length does not change, but that the capillary diameter will be the parameter that will change, and then see what the flow-surface re1ationship:is. W. W. TOURTELLOTTE: The only substance for sure that does not moveout ofthevascular space into the brain is hemoglobin. Hence, it would appear that the measurement of hemoglobulin to determine the extent of the contribution of blood “contamination” in the brain is on sound grounds.

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L.BAKAY:I would just like to make one statement as far as determination of the blood content is concerned. The determination of blood volume in any small area of brain tissue by determining haemoglobin is very difficult, because many of the small vessels of the white matter for example are very small indeed, In a cerebral capillary the haematocrit is onlyabout 16 to 18%. In otherwords, 70 to 8076 of the blood in the small vessels is plasma. This means that the determination of plasma proteins or any other plasma constituent is far superior to determination of the haemoglobin, which reflects directly only on the number of the blood cells which is identically distributed in the small capillary vessels. J. P. S C H A D ~I :would like to make a short remark about methionine sulfoximine. We have been studying with the light microscope the effect of methionine sulfoximine on the dendritic organization. Even after 24 hours, a severe degeneration in both cerebral and cerebellar cortex was found, resulting in a reduction of the dendritic plexus.

D. B. TOWER: You can get severe lesions from methionine sulfoximine, but you can also have a long period of seizures in which you cannot demonstrate any pathological change under the light-microscope by the usual neuropathological procedures. Therefore, I think that this is a question which depends apparently on the severity of the seizures, since it is well known that changes secondary t o the seizures themselves can occur after very severe seizure manifestation. I would like to make one comment in relation to what Dr. Bakay has just said: He has reminded me of one of the things which has not been mentioned here which is perhaps something that neurosurgeons see from time to time. This is a sort of “bloodless” capillary. When the cortex is exposed and proximal flow is temporarily occluded or sluggish, you can watch a capillary fill up with fluid in which there is absolutely no blood at all. D. P. RALL:I would like to make two points. One is just to get the record straight; I think Dr. Cutler well recognizes that you cannot really equate a p H of 7 caused by an increase in COZwith a pH of 7 caused by infusion of hydrochloric acid. With the COz-induced pH, your intracellular and cerebrospinal fluid-pH will be the same as your plasma-pH. This is a very different story from the situation in which you have induced this acidosis by HCI. In that instance the CSF-pH will be, if anything, higher than normal, and the intracellular pH will probable not have changed at all. This can lead to a very different set of circumstances. Secondly, a number of people have mentioned the use of iodoantipyrine as a method of following body water. I do not know the reference, but it is clear that iodoantipyrine does not act like nonlabeled iodopyrine. It is more ionized and less soluble, and furthermore, it can be metabolized; so as a convenient agent for tracing body water iodopyrine with its problems of determination is very good, but iodoantipyrine, I am afraid, is not. GENERAL DISCUSSION

I just wanted to make a comment on the remarks of both Dr. Bakay and Dr. Dobbing. R. KATZMAN: I quite agree with them that in so-called physiological measurements in ventriculo-cisternal perfusions one really has to consider the possibility of artifacts such as edema occurring. The only evidence in this regard is the fact that in chronic repeated perfusions, animals d o quite well, and d o not have any increase in intracranial pressure. However, I am not aware of any specific studies as to the existence of a minimal amount of cerebral edema in association with such perfusions, and it certainly ought to be studied. One thing that is quite clear, however, is that many movements of substances from blood to brain are controlled by COZ,and perhaps the experimenters who do ventriculocisternal perfusions should be monitoring the COz-level of the blood in their animals. L. BAKAY:I believe that changes occur in perfusion only if the pressure is drastically changed, or if the tonicity of the perfusate is way off, or when it is toxic. 1 don’t assume that there are changes under other circumstances than that. R. V. COXON:What do you mean, Dr. Bakay, by “pure hypoxia”? Would this mean that the animals were breathing a low oxygen concentration in nitrogen? Because if that is the case, they would also be hypocapnic.

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L. BAKAY: They were not hypocapnic. As far as this situation is concerned, when I spoke about pure hypoxia, I meant just that. In other words: the animals were breathing a mixture low in oxygen, but with a normal minute volume, or a slightly increased minute volume occasionally. There was only a very slight drop in the pH.

M. BRIGHTMAN: I cannot share Dr. Bakay’s unconcern about the presence of broken membranes or even an absence of membranes in the electronmicrograph if that micrograph is used as a basis for ascertaining whether edema has occurred or not. In order to determine whether the compartment is intracellular or extracellular, the least you must have is a membrane separating the two compartments. It is even more difficult to say whether a protein marker has got there through a membrane, or whether it was simply an artifact. The effect of fixation on undamaged tissues is difficult enough to ascertain, especially if there are any signs that damage has occurred during the process of fixation. But when chemical fixation is superimposed upon some sort of damage before that, it is cven more difficult. I fully concur with that. The fact remains, that when it comes to edema ofthe white matter, L. BAKAY: you do occasionally see ruptured cell membranes. I d o not believe that that necessarily is the sole reason, or perhaps even the principal reason for the fact that the extracellular space in the white matter expands.

D. P. RALL:I would like to come back to Dr. Dobbing’s question. You could perhaps suggest that there might be two problems with the perfusion of the ventricular system. One is related to the introduction of the needle, which I will not comment on. The second concerns what the fluid does to the brain, and I can comment on this. We have put indwelling capillaries into patients to treat malignant processes within the brain, and we have perfused these patients repeatedly. We use a solution, called Elliot’s B-solution. These patients suffer no ill effects. They do not have headaches, and they are not disturbed in any way. If you use the living human brain as an indicator of the state of function of that brain, eight hours of perfusion at normal pressures has no deleterious effect. P. MANDEL: Is there a physiological control too?

D. P. RALL:Yes, rather simple EEG’S, and they are normal. L. BAKAY: Everybody with a modicum of arterial hypertension has a measurable amount of cerebral edema, without having any ill effect, or any headache, or anything whatsoever. So the fact that a person is perfused and is able to talk and does not have headache does not necessarily mean that he does not have a slight amount of edema. We have been doing ventriculo-cisternal perfusions in rats. In a situation where D. M. WOODBURY: we measured thc water content in the brain during various times of perfusion, there was no change at all worth mentioning, and there was no cerebral edema. As far as inulin movement was concerned: we sampled the brain at some points where the perfusions were made and as well as in other portions of the brain. There was very little difference in the content of inulin in these different areas.

K. A. C. ELLIOTT: Long ago I did a series of experiments to comparethemetabolismofvariousexcised tissues. I t was pointed out that brain and testes were more alike than any other tissues. I would like to ask Dr. Bakay a question. In his remarks on kernicterus, he mentioned that any relation to it with the undeveloped brain-barrier system could not be dismissed, but he did not give us a reason why. L. BAKAY: The reason why most people dismiss the theory in terms of kernicterus is because there is a striking discrepancy between the high incidence of kernicterus in premature babies with erythroblastosis fetalis, and a very low incidence, or almost absence of the same condition in premature infants, who have icterus of another nature. This strongly suggests that there is something specific about the unconjugated bilirubin in erythroblastosis fetalis. If this were simply a matter of membrane or capillary permeability, there should be more of a similarity in these various forms of icterus in the newborn babies. J. FOLCH-PI:In the literature it is mentioned that there is a kind of blood-brain barrier system in the testes quite similar to that for the brain.

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L. BAKAY:I did not want to insinuate that this was a statement of generalized implication. I personally d o believe that the barrier, whatever it is, whether in the form of increased membrane permeability, or in the form of an increased size of extracellular space, is different in newborn and that it develops as the time goes on. It is just in relationship to kernicterus that we don’t understand what we see. Here is a point which is so difficult: One can equally well reason theoretically that kernicterus occiirs in the basal ganglia and not in the rest of the brain because of differences in vascularity or metabolism or in other ways. One point is very striking. This is that the region in which kernicterus develops is also where the anoxic damage in carbon monoxide poisoning occurs. However, whether this is due to a matter of membrane permeability or to a special metabolic factor, 1 don’t know.

J. FOLCH-PI:The blood-brain barrier has definite regional differences and is essentially a dynamic situation. A dynamic situation requires an input of energy which is probably controlled in some way or other feed-back information on the needs of the tissue. Another point is that weallshould remember that the barrier does not necessarily work in only one way. L. BAKAY:I really can’t answer most of the remarks that Dr. Folch made. But one answer, which is also pertinent to what Dr. Lajtha said, is that 1 applied a great variety of markers or tracers, some of them for the sake of technical convenience in pathological conditions. It was not elaborated which of them was metabolically active and which was not, and which might go into the brain by passive diffusion and which by a carrier mediated active transport. The fact remains, that when it comes to a pathological condition, there is hardly any difference in the majority of cases, as to whether the tracer is an inactive structure or one which is a highly metabolically active agent. There is very little selection in the distribution between metabolically active or inactive or even small or large particle-sized tracers between what we consider normal and abnormal brain tissue. R. CUTLER: Just a short comment. It is the unconjugated non-protein bound moiety of the bilirubin that enters the brain. This never occurs in an adult because one never reaches such high levels of unconjugated non-protein bound bilirubin as occur in the newborn infant. But, if you infuse Cl4--labeled unconjugated bilirubin into an adult animal, it will enter the brain and cause kernicterus.

L. BAKAY:I happened to have cases with hepatitis with a very high concentration of unconjugated bilirubin in the human adult. And while it did stain the lesion it did not seem to go into the normal white matter brain tissue. A. LAITHA:I just want to add a very short remark to what Dr. Folch said. We have some evidence that developmental differences also exist as far as active processes in the barrier systems are concerned. The pumping-out mechanism, although not completely absent, is not quite as well developed in the early stages, so that this may also be an explanation.

D. B. TOWER:I would just like to add a comment to what Dr. Bakay said about how in various kinds of pathological conditions the brain responds in the same way. We have to recognize that from a neuropathological point of view the range of reactions is extraordinarily small. Many different conditions produce the same end-result, at least in a gross sense. Perhaps, looking a t this philosophically, it has some relevance to the subject we are discussing here. Normally the brain is, so to speak, protected from many insults and therefore has not been stimulated to develop a versatility for reacting to insults. W. W. TOURTELLOTTE: It is rather striking to see at postmortem examination of the brain an acute cerebral infarct in a jaundiced patient. The infarct stains yellow and the surrounding “normal” appearing tissue is unstained. Furthermore, with formalin fixation the bilirubin is oxidized and the infarct turns green. L. BAKAY: You misunderstood me. In the person in whom I found a high unconjugated bilirubin and who died, there was an infarct that was stained, but the surrounding brain was not stained. But, to add more complications, I must add here one more factor to confuse us. A patient with severe long-lasting icterus, has bilirubin in his cerebrospinal fluid, that is low in protein. So, you can’t say that all the bilirubin is albumin-bound. The main problem is that here we have a patient with a high serum bilirubin, a high cerebrospinal fluid-bilirubin and an unstained brain, except for the ependymal area.

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D. H. FORD:I have a comment on this problem of the differential uptake of amino acids between brains of very young and adult animals. A point which is very seldom made in regard to this transfer of amino acids from blood to brain, is the tremendous differential in growth and synthesis of new protein in these young neurons which must increase in size a hundred-, two hundred-, or a thousandfold in some instances. If one then examines a group of rats injected intraperitoneally with [35S]cystine and determines the uptake or incorporation of cystine into the brain areas, e.g. at 5 weeks of age - a time when most people consider the blood-brain barrier to be more or less mature - one finds that the accumulation of cystine by various areas of the brain more closely resembles that occurring in young rats rather than in adults. Since growth of neurons is still progressing a t this time, it seems more likely that the higher than adult uptake of 35S-labeled compounds is more related to growth than to an incomplete formation of a blood-brain barrier. B. D. WYKE:Several papers during this session have emphasized the importance of monitoring the arterial P c o ~ or , the necessityfor it. lfwe slip needles into brains of patients or animals to take biopsies, we d o this under anaesthesia. Under general anaesthesiawith any agent, the arterial PcoZ is elevated, unless the animals or the patient is given a relaxant and ventilation is controlled. If you slip a needle into a brain of an animal with a normal arterial Pco,, there is not any spreading edema of the sort that Dr. Bakay described. We showed in our laboratory some years ago that if you combine hypercapnia with a small cerebral lesion, made either by repeated needling, or by biopsy, then you get a spreading edema through that hemisphere which develops quite quickly. T. Z. CSAKY:May I discuss the paper of Dr. Quadbeck in some detail? Three basic facts stand out: ( I ) There is a low capacity transport mechanism for glucose in the brain, yet, the need of the brain for glucose is large. (2) If a non-metabolized sugar is offered to the brain the rate of uptake is extremely low. (3) Certain drugs, as we have just learned, can increase the uptake of glucose by the brain. How can we reconcile these various findings? A partial answer may be offered in the following: When we talk about the uptake of glucose by the brain, this is not necessarily equivalent t o transport. Uptake in case of glucose means that the sugar is pouring into a very active metabolic sink. A relatively low capacity carrier will be sufficient to mediate this in-pour. If a sugar is offered which uses the same carrier but is not metabolized, the system will soon be “saturated”, an equilibrium reached, and the further uptake will slow down. If a drug is administered which increases the glucose metabolism of the brain cell this will simply increase the metabolic disappearance of the free glucose and hence facilitate the in-pouring of more sugar. I suspect that this is what Dr. Quadbeck’s drug does. What we measure in this case is really the clearing of glucose from the blood. So I think that we can reconcile these different facts by assuming a sugar carrier mechanism with relatively low capacity which carries glucose from the blood into the brain cell but, because of the rapid metabolic disappearance of the sugar, the reaction will proceed in the direction of uptake. As long as the metabolism of glucose is vigorous, a large amount of the sugar can be cleared from the blood.

G. QUADBECK: With metamphetamine it is possible t o stimulate cerebral metabolism. Metamphetamine in animal experimentswith oxygen deficiency, hohevcr, fails to improve the glucose supply to the brain. In patients with a reduced uptake of glucose the drug does not bring about an increase. Consequently, I have to believe that the effect of the drug we have observed is on the pump mechanism and not an expression of increased requirements of the brain for glucose.

D. B. TOWER: Dr. Quadbeck pointed out that this compound is structurally related to the pyridoxine group of vitamins. 1 would like to know from Dr. Quadbeck whether he has evaluated the effects of the 8-6 group of vitamins, and 1 say “group” because it is evident now that one cannot do this with pyridoxine alone. In certain circumstances you have to evaluate the whole group in order to make sure that you have not overlooked the effect of pyridoxal, or pyridoxal phosphate, and so on. It is conceivable, as Dr. Csaky has pointed out. that you are dealing with a situation here in which the cells are somehow unable to consume glucose, and that the effect of this drug is at a cellular level rather than at some other level which has to d o generally with the transport of glucose into the central nervous system. Pyridoxine has such myriad co-enzyme functions that such possibilities must be immediately suspect.

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G. QUADBECK: Pyridoxine and pyrithioxine differ markedly in their effects.The latter has novitamin effect. Ifweadminister large doses of pyridoxine to an animal, transport of phosphate into the brain is increased. With an equivalent dose of pyrithioxine, the phosphate transport is reduced. Consequently, I am sure these two compounds are not related in their biological actions.

D. B. TOWER: I don’t think that answers the question, Dr. Quadbeck. Because 1 am not quite sure that one can make a complete parallel here. So, I would like to know whether you have tried the effects of pyridoxine, pyridoxamine, pyridoxal, and pyridoxal phosphate on the apparent depression of glucose utilization in these patients. G. QUADBECK: We have not studied pyridoxine with the Schmitt-Kety method. We have never seen with pyridoxine, however, an effect in older patients which is comparable to animal experiments where a disturbance of the glucose transport had to be assumed.

C. CRONE:Before we enter into too much of a detailed discussion of the mechanism of action of this substance, I would like to ask Dr. Quadbeck a question on the accuracy of the methods of measurement. If you have a situation where the cerebral blood flow rises with a factor of 2.5, it means that if the cerebral glucose uptake stayed constant under these conditions, the normal arterial-venous difference of 10 mg per 100 ml would go down to 4 mg per 100 ml. If the glucose uptake were reduced further, as was shown on the slide, down to 36%, it would mean that you would now have a difference of about 1.2, or 3 mg %. I would hesitate very much to put too much weight on differences based on this sort of calculation. In order to put a concrete question to you, I would like to ask: How many times were the controls controlled before the substance was given? G. QUADBECK: In one patient we were able to make measurements a week apart without intervening therapy. No significant differences were noted in these measurements. If we determined the glucose uptake of the brain after treatment for several weeks, then we found a distinct effect. D. P. RALL:As far as Dr. Steinwall’s presentation is concerned, I want to reemphasize that if we allowed paraminohippurate to diffuse into the brain, it would be able to move in 10 mm, in a way consistent with passive diffusion. The concentration of paraminohippurate was very low, deep in the brain. Any active transport at the capillaries should not have severely altered the shape of this curve. I think this sort of evidence, almost virtually conclusive, suggests that there is no active transport of this sort of compound in the brain. 0. STEINWALL: I hope my presentation made it clear that I was not as pleased with my results on this point as with the hypothesis. R. V. COXON:I take it for granted that Dr. Rall does not claim that this is not active transport through this particular region? He would not deny that at least a transport against the concentration gradient could exist? D. P. RALL:At the choroid plexus, probably, yes. I would be in conflict with myself if I denied this. A. LAJTHA:May I ask Dr. Steinwall whether he investigated any compound that is not actively transported under the same conditions? I would almost expect an opposite effect. Perhaps this would be a nice demonstration that there is more than one kind of barrier. A metabolic inhibitor by decreasing available energy may inhibit active uptake or transport of substances. The decrease in energy may at the same time have an effect on the membrane itself which may increase the penetration of nutrients entering by diffusion alone.

0. STEINWALL: In many experiments we found a seemingly opposite effect, as there could be an extravasation of normally barred dyes such as sodium fluorescein simultaneously with the decreased uptake of the nutrient tracer in the hemisphere perfused with the mercuric solution. I think this indicates the coexistence of more than one kind of barrier function.

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The Effect of Hypothermia on Electric Impedance and Penetration of Substances from the CSF into the Periventricular Brain Tissue I G O R K L A T Z O , C H O H - L U H LI, D O N M. L O N G , A N T H O N Y F . B A K , M I R O S L A W J. M O S S A K O W S K I , L E V O N 0. P A R K E R A N D LOIS E. R A S M U S S E N Branch of Swgical Neurology, National Institute of Neurological Diseases and Blinclness, National Institutes of Health, Public Health Service, Departinen! of Health, Education and Weljare, Bethescla, Marylarrd 20014 ( U S A )

1NTRODUCTION

Lowering the temperature of the brain can affect it in either a beneficial or an adverse manner. The ability of mild hypothermia to reduce brain volume has been extensively used by neurosurgeons in alleviating increased intracranial pressure and cerebral edema. On the other hand, a number of adverse effects have been reported following cooling of the brain below 28°C. At these temperatures numerous clinical complications (such as cerebral edema or hypoxic brain injury) and experimental disturbances (such as a breakdown of the blood-brain barrier) have been reported (Brendel et al., 1966). A further elucidation of the basic changes due to lowering brain temperature appears to be of considerable importance. Hypothermia causes an increase in the electric impedance of brain tissue in the rabbit (Collewijn and SchadC, 1962, 1964). This increase was thought to be a result of changed physical properties of the tissue since a similar change was found in electrolyte solutions at corresponding temperatures (Collewijn and SchadC, 1962). Recently a series of experiments were designed in our laboratory (Li et al., 1966) for the study of physiological changes in the brain under hypothermia. These experiments show that impedance changes recorded from the grey matter at temperatures above 20°C were in accordance with temperature coefficients of the blood serum and NaCl solution. Below this temperature, the changes were greater than those recorded from serum and electrolyte fiuid. These observations probably reflect changes in the electrolyte and extracellular compartments, as has been reported to occur in asphyxia (Van Harreveld, 1957). It was then conjectured that alteration in such basic properties of nervous parenchyma should influence penetration of various substances from the cerebrospinal fluid (CSF) (Davson and Spaziani, 1962). After crossing the ependymal barrier the substances can migrate further in several ways. Generally, the inert, extracellular compounds, such as inulin, may spread primarily by passive diffusion through extraR I ~ PPII( I P B p. 396

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cellular spaces (Rall et a/., 1962), whereas, an active transport mechanism has been implicated in penetration of diverse substances such as histamine (Draskoci et al., 1960), amino acids(Lajtha, 1962; Levin et a/., 1966), and albumin (Klatzo etal., 1964). The purpose of the present study is to correlate some of the electrophysiological data obtained after brain cooling with observations on periventricular penetration of substances, selected for their association with either diffusion or active transport. Materials and Methods

For the study of electric impedance changes, 35 cats under Fluothane anesthesia and extracorporeal hypothermia were used. The temperature and the blood flow i n the brain were regulated by extracorporeal circuits connected with the common carotid arteries. This method is similar to that described by Kristiansen el a/. (1960) and will be reported in a separate communication (Ohta rt a / . , 1966). In brief, the blood flow through the carotid artery was constant at a rate of 7.3 ml/kg/min and the temperature of the cerebral hemisphere was gradually lowered to 13-12°C. The perfusion pressure was continuously measured at the entry of the extracorporeal catheter into the carotid and the general systemic blood pressure was taken from the abdominal aorta. Temperatures of various structures of the brain and of the rectum were likewise continuously recorded. Before, during, and towards the end of the cooling experiment blood samples were obtained from the abdominal aorta for determination of Pco,. PO, and pH. Square pulses of low intensity current were applied through two Ag-AgC1 electrodes across the brain while recordings were made with two microelectrodes in the tissue. The difference of potential recorded was taken as a measure of impedance between the two recording electrodes. These electrodes were also capable of recording membrane resting potentials and action potentials of the brain tissue. Impedance changes at various temperatures were also measured in 0.9% NaCl solution and cat’s blood serum. Observations on passage of various substances from the CSF into the periventricular brain tissue were carried out on 20 cats using ventriculo-cisternal perfusion. Palmer’s intraventricular cannula was introduced into the right lateral ventricle through an opening located 8 mm from the midline and 5 mm posterior to the fronto-parietal suture. An outflow needle was inserted into the cisterna magna. An adjustable flow rotary pump was used to regulate the rate of flow through a polyethylene tube connecting the intraventricular cannula with a reservoir of perfusing fluid. The latter consisted of Elliott’s fluid containing one of the radioactive substances and sodium fluorescein which was used as a standard marker for comparison. The radioactive compounds and their concentrations in the perfusing fluid were as follows: ( I ) 0.014 mM [14C]inulin1, specific activity 2.7 mC/gm ; (2) 0.201 mM [14C]sucrose2, specific with specific activity 34.1 mC/ activity 9.0 mC/mM; (3) 0.0009 mM [14C]~-leucine3 mM. In each case approximately 210,uC of the individual radioactive tracer in mixture

3

New England Nuclear Corp., Boston, Mass. Intern. Chem. and Nuclear Co., Los Angeles, Calif. Tracerlab, Waltham, Mass.

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with sodium fluorescein was perfused for 45 min at the rate of 0.15 ml/min. Following passage of the tracers the ventricular system was rinsed for 10 min at the same perfusion rate with Elliott's fluid after which the animals were sacrificed by rapid exsanguination. In hypothermic animals the ventriculo-cisternal perfusion was started about 30 min following the onset of cooling when the brain temperature had been lowered to approximately 15°C. In this hypothermic group, 5 cats were perfused with [14C]inulin, 3 with [14C]sucrose and 2 with [14C]~-leucine.An equal number of animals were perfused with the same tracers at the normal body temperature. Following the sacrifice, the brains were immediately removed and a standard coronal block at the level of lateral ventricles, corpus callosum and putamen was rapidly frozen on a metal holder. From this tissue l o p sections were cut in the cryostat. Using a previously described (Steinwall and Klatzo 1966) double tracer technique it was possible to compare individually in the same section the areas of penetration of sodium fluorescein and the distribution of the radioactive tracers by subjecting single sections consecutively to photography under the U.V. light and radioautography. For assessment of the ultrastructural effects of these manipulations the following procedure was utilized. Three cats underwent brain cooling to 15-12"C, ventricular perfusion with Elliott's solution for 55 min, and then were fixed by direct carotid perfusion with 1000 ml 2.5% glutaraldehyde in a 440 mM phosphate buffer at the same flow rate as utilized in the blood perfusion. Three animals were similarly fixed at normal body temperature after undergoing only ventricular perfusion. Following fixation the tissue was diced (1 x I mm), exposed to 1 % osmium in the same buffer for one hour, and then embedded in epon according to standard technique. RESULTS

The time course of the change in temperature of the cerebral hemisphere under investigation was almost identical in all experiments as shown in Fig. 1. In about 28 rnin the temperature dropped from 36-34°C to 15°C and, thereafter, was maintained at a level of 13-12°C. Measurements of perfusion pressure, systemic arterial pressure, PO,,PCO, and pH at various temperatures obtained from different animals were found to be similar. Fig. 2 shows the average values of these measurements. In all cases the oxygen tension in the blood was increased with a decrease of brain temperature. It is to be noted that the body temperature as taken from the rectum ranged between 31 and 30°C while the braintemperaturewas 15-12°C. If the increase in PO, is due to a decrease in oxygen consumption of the body tissue as a result of hypothermia, the decrease must have been more marked in the cooled cerebral tissue than in other tissues. In any case, there was no indication of hypoxia in any of these animals. During the cooling procedures resting membrane potentials were measured from 87 polarized elements in the cortex of the cooled hemisphere. At temperatures above ITC, 60% of these elements showed injury discharges with a sudden shift of potential to negativity. In the absence of injury discharges the negative potential may represent resting potentials recorded from glial elements (Li, 1955 and 1959). At temperatures RpJi,renci,s p. 396

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Fig. 1. Time course of change in brain temperature under extracorporeal hypothermia.Arrows indicate beginning and end of ventricular perfusion.

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Fig. 2. Perfusion pressure, blood pressure, Po2. PcoZ and pH measurements at various brain temperatures.

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Fig. 3. Resting membrane potentials of 87 polarized cortical elements at various brain temperatures.

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Fig. 4. Impedance changes simultaneously recorded from the grey matter (first tracing) and white matter (second tracing) at various brain temperatures (indicated at right upper corner of each record)

below 17"C, none of the penetrated elements showed injury discharges. The mean value and the standard deviation of these measurements are shown in Fig. 3. Since there is no way of identifying neurons from neuroglia by electrophysiological methods at low temperatures, the values given in Fig. 3 must represent the mean values from Hejorrnccs p . 396

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Fig. 5. Impedance changes of the grey matter, white matter, blood serum and NaCl solution.

all polarized elements in the cortex. It follows that no change in the resting potential occurs in these polarized elements at temperatures between 30 and 11°C. Impedance changes recorded at low temperatures consistently revealed a discrepancy in the measurements from the grey matter and white matter. An example is given in Fig. 4, which shows a gradual increase in the electric resistivity of the cortex and little or no change in the white matter when the temperature of the hemisphere was lowered from 33 to 13°C. These measurements of impedance changes obtained from each experiment were converted into proportional values and the average values at a given temperature obtained from all experiments were plotted in Fig. 5. It can be seen that in the white matter there is very little change in the impedance at temperatures above 20°C; and a gradual increase to 10% at temperatures between 20 and 10°C. This is in contrast with the findings from the grey matter from which an increase of over 60 % at 15°C and 70 % at 12.6"C were recorded. Fig. 5 also shows the average values of impedance measurements recorded from cat's serum and NaCl solution. At temperatures above 20"C, these values are comparable to those recorded from the grey matter; but below 20°C they are significantly smaller. It also shows that these values are much higher than those recorded from the white matter throughout the entire range of temperatures tested. The penetration of various substances from the ventricles into the surrounding parenchyma revealed the following features. In animals subjected to selective cooling of the brain sodium fluorescein, ["C]inulin and [14C]sucrose showed a striking reduction of periventricular spreading in comparison with cats perfused with these substances at normal temperature. On the other hand, the effect of hypothermia on the passage of [W]~-leucineappeared to be rather insignificant. At normal temperatures all indicators extended for approximately 3-4 mm into the putamen, whereas the spread into the corpus callosum seemed to be more limited (Fig. 6a, b). At lower brain temperatures the reduction in penetration of the first three indicators was very conspicuous, and was usually more pronounced in the putamen where only a narrow rim of subependymal tissue showed the presence of

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Fig. 6. (a) Cat subjected to ventriculo-cisternal perfusion with sodium fluorescein and [14C]inulin at thc normal temperature. The 101‘ section photographed under the U.V. light. The fluorescence of sodium fluorescein is seen extending into brain tissue adjacent to the lateral ventricle; (b) Radioautograph showing the distribution of [14C]inulinon the same section. Fig. 7. (a) Ventriculo-cis‘ernal perfusion with sodium fluorescein and [“Tlinulin at 13°C. Thesame anatomical level as in Fig. 6. The penetration of sodium fluorescein from the lateral ventricle is very restricted, especially into the putamen; (b) Radioautograph showing the passage of [W]inulin on the same section.

the tracer (Fig. 7a, b). Although it appeared that the passage of [14C]~-leucine was also affected by lowering the temperature the reduction was small and sometimes even difficult to ascertain (Figs. 8, 9). Electron microscopy revealed no major abnormality in either normothermic or hypothermic brain. Preservation of tissue was adequate. An occasional pyknotic neuron was seen, but in general nerve cells were not abnormal. Definite astrocyte R&riwrca p . 396

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Fig. 8. (a) Ventriculo-cisternal perfusion with sodium fluorescein and [14C]~-leucine at the normal temperature. This section photographed under the U.V. light shows the distribution of sodium o n the same fluorescein; (b) Radioautograph showing the periventricular penetration of [14C]~-leucine section. Fig. 9. (a) Ventriculo-cisternal perfusion with sodium fluorescein and [14C]~-leucine at 14°C. Fluorescent photograph shows the reduction in passage of sodium fluorescein as compared with that at the normal temperature; (b) Radioautograph of the section shown in 9a. The reduction of L-leucinc passage into periventricular brain tissue appears to be insignificant when compared with Fig. 8b of a cat perfused at the normal temperature.

swelling or shrinkage was not seen, and there were no major changes in the extracellular space in grey or white matter (Fig. 10). There were no obvious differences between the two groups. These preliminary observations are not complete, however, and measurement analysis has not been applied to the tissue. While inspection reveals no obvious abnormalities which might explain the observed phenomena, it is not possible to compare exactly cellular, process, and space sizes in the two groups until quantification is complete.

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Fig. 10. Cat caudate nucleus fixed at 12°C by perfusion with 2.57; glutaraldehyde in PO4 buffer. A normal appearing neuron ( N U ) with a dendrite ( D )are shown. Another dendrite (D, right) crosses the field. No dramatic change in dendrite size. Cellular elements of neuropil (NP) and extracellular space do not appear different than normal. x 10 300.

DISCUSSION

Under normal brain temperature, the electric current applied to the brain for measurement of impedance changes is primarily carried by electrolytes in the extracellular space. From experiments on circulatory arrest (Van Harreveld and Ochs, 1956), it was estimated that the blood, which flows through the brain tissue, is responsible for about 1 I ofthe total conductivity of the brain. Thecellular elementscontributelittleornone to the change in brain conductivity owing to the high resistance of their limiting membrane. In the present investigation, impedance changes recorded from the grey matter at temperatures above 20°C were found to be comparable to those recorded from NaCl solution and blood serum. This can be attributed to the temperature coefficient of the electrolyte fluid in the extracellular space, which is in complete agreement with Rrfercnms p. 396

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et al.

Collewijn and SchadC (1962, 1964). However, at temperatures below 20°C the impedance changes of the grey matter became greater than those recorded from NaCl solution and serum, suggesting that impedance changes of the grey matter at temperatures below 20°C are not entirely determined by temperature coefficient of the extracellular fluid. One may argue that the excessive increase of grey matter impedance is due to a decrease in extracellular space as a result of an increase in the volume of the cell (Van Harreveld et al., 1965). It was shown (Reulen et al., 1966) that a significant increase in the uptake of sodium and water in brain tissue occurred at temperatures below 10°C. Above 10°C there was no significant increase. It is also known that cells in brain become swollen in experimental animals under asphyxia (Van Harreveld, 1957). I n the present investigation the brain temperature was reduced, but not below 11°C; and, in all instances, there was no indication of hypoxia. The measurements of resting membrane potential are comparable to those recorded from cortex of normothermic cats (Li, 1955 and 1959) and brain slices of the guinea pigs (Li and Mcllwain, 1957). The consistent values obtained at low temperatures are in agreement with the findings from single skeletal muscles of the hypothermic frogs (Li and Gouras, 1958) and rats (Li, 1958). The lack of change in resting membrane potential indicates not only an absence of disturbance in the differential distribution of ions between the extracellular and intracellular compartments but also an absence of excessive accumulation of water in the tissue. The indication that cell swelling did not occur is supported by the preliminary findings from electron microscopy. Despite the regulated blood flow in the common carotid artery of our experimental animal, the actual volume of blood circulating in the cooled hemisphere is not known and this variable remains to be determined. In experiments (Li et al., 1966) designed for the study of systemic blood pressure and brain impedance changes, it was found that the lower the blood pressure the lower is the tissue impedance. This observation implies that the impedance change of the grey matter at temperatures below 20°C should be lower than the change caused by the temperature coefficient of the extracellular fluid alone; but this is not the case. The above arguments leave an alternative that at temperatures below 20"C, the increased impedance recorded from the grey matter is due to a decrease of the extracellular space. This suggestion, however, cannot be applied to the changes recorded from the white matter of the brain. The conspicuous inhibition of the passage of sodium fluorescein, inulin, and sucrose may well be related to a reduction in extracellular space. Thedistributionof these tracers (especially the last two) is generally believed to be extracellular. Restriction of the space available for migration should inhibit their rate and extent of spread. Since temperature has a direct influence upon rate of diffusion, the inhibition of migration of inulin in our study was compared with the inhibition to be expected if the effect was simply one of temperature reduction on diffusion. The quantitative technique of Rall et al., (1962) was utilized for us by Dr. Fenstermaker of that laboratory. Preliminary results indicate the reduction in inulin migration from the ventricular surface

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markedly exceeds the predicted reduction if the results were related to temperature coefficients alone. Reduction of the extracellular space available for diffusion is a plausible explanation of this discrepancy. We expected that hypothermia would have a definite effect upon the active transport mechanism which supposedly is responsible for the movement of L-leucine from the CSF. While the results were not unequivocal, the reduction in passage of L-leucine was small at best, and difficult to ascertain. This is unexplained, and additional study of this phenomenon is planned. The ultrastructural analysis of the tissue is not complete. However, it can be stated that no obvious tissue damage or large differential increase or decrease in any tissue element occurred. I t is entirely possible that exact measurements may reveal a significant change in some volume component, however. This possibility cannot be fully evaluated until measurements are complete. At least there is no serious cellular damage or destruction of tissue which should markedly influence the results.

SUMMARY

Study of impedance changes and resting membrane potentials as well as the rate of passage of various substances from the cerebral ventricles to the brain tissue in hypothermic cats has led to the following results and suggestions: 1 . At brain temperatures above 20”C, impedance changes recorded from the grey matter were comparable with those recorded from blood serum and NaCl solution at corresponding temperatures. This can be attributed to temperature coefficient of the electrolyte fluid in the extracellular space. 2. At temperatures below 20”C, impedance changes recorded from the grey matter became greater than those from serum and NaCl solution. It is suggested that at these low temperatures there is a reduction of the extracellular space. 3. At temperatures above ITC, there was no indication of hypoxia and no change in the resting membrane potential of the cellular elements in the cerebral cortex. These findings imply that swelling of cells and disturbance of differential distribution of ions did not occur at these low temperatures. 4. Impedance changes recorded from the white matter were much smaller than those recorded from the grey matter. The mechanism by which this discrepancy exists remains to be determined. 5 . Using fluorescence and radioautography the effect of lowering the brain temperature on periventricular passage from CSF was assessed with regard to sodium fluorescein, [14C]inulin, [14C]sucrose and [“C]~-Ieucine.Sodium fluorescein, [“Wlinulin and [14C]sucrose showed a marked reduction of periventricular passage at 15-1 2”C, whereas only a small effect was observed with regard to [“C]~-leucine. 6. These data indicate the possibility that at 15-12°C there is a reduction in extracellular compartment without appreciable shift in electrolytes which may account for the increased impedance values in the grey matter and for the observed reduction in pasage of the mentioned compounds. R&wwc,s

p. 396

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REFERENCES BRENDEL, W., MULLER, CH., REULEN,H. J. AND MESSMER, K. (1966) Elektrolytveranderungen in tiefer Hypothermie. 11. Beziehungen zur klinischen und biologischen uberlebenzeit. Pfliigers Archiv, 228, 220-238.

COLLEWIJN, H. AND S C H A DJ.~ ,P. (1962) Cerebral Impedance Changes in Hypothermia. Arch. Intern. Physiol. Biochem., 70, 20&2 10.

-, (1964) Chloride, Potassium and Water Content of Apical Dendrites and Their Changes after Circulatory Arrest at Body Temperatures from 37°C to 20°C. Arch. Intern. Physiol. Biochem., 72, 194-2 10.

-, (1964) Conductivity of the Cerebral Cortex after Circulatory Arrest from 37°C to 18°C. Arch. intern. Physiol. Biochem., 72, 181-193.

DAVSON,H. AND SPAZIANI, E. ( I 962) Effect of Hypothermia on Certain Aspects of the Cerebrospinal Fluid. Expt. Neurol., 6, 118-129. DRASKOCI, M. FELDBERG, W.,FLEISCHAUER, K. AND HARANATH, P. S. R. (1960) Absorption of Histamine into the Blood Stream on Perfusion of the Cerebral Ventricles and its Uptake by Brain Tissue. J. Physiol., 150, 50-72. KLATZO,I.. MIGUEL,J., FERRIS,P. J., PROKOP,J. 0.AND SMITH, D. E. (1964) Observations on the Passage of the Fluorescein Labeled Serum Proteins from the Cerebrospinal Fluid. J. Neuropathol. Exptl. Neurol., 23, 18-35. KRISTIANSEN, K., KROG.J. AND LUND,1. (1960) Experiences with Selective Cooling of the Brain. Acta Chirurg. Scand. Suppl., 253, 151-161.

LAJTHA,A. (1962) Amino Acid Transport in the Brain. In: Properties of Membranes and Diseases of the Nervous System. M. D. Yahr (Ed.). Springer Publishing Co., New York, pp. 43-54. LEVIN, E., NOGUEIRA, G. J. AND GARCIA ARGIZ,C. A. (1966) Ventriculo-Cisternal Perfusion of Amino Acids in Cat Brain. J. Neurochem., 13, 761-767. LI, C. L. (1955) Action and Resting Potentials of Cortical Neurones. J . Physiol., 130, 96-108. -, (1958) Effect of Cooling on Neuromuscular Transmission in the Rat. Amer. J. Physiol., 194, 200-206.

-, (1959) Cortical Intracellular Potentials and Their Response to Strychnine. J. Neurophysiol., 22, 436-450.

LI, C. L., BAK,A. F. A N D PARKER, L. 0. (1966) Some Electrophysiological Changes in the Brain under Hypothermia. (In preparation). Lr, C. L. AND GOURAS,P. (1958) Effect of Cooling on Neuromuscular Transmission in the Frog. Amer. J. Physiol., 192, 464-470. Lr, C. L. AND MCILWAIN, H. (1957) Maintenance of Resting Membrane Potentials in Slices of Mammalian Cerebral Cortex and Other Tissues I n Vitro. J . Physiol., 139, 178-190. OHTA,T., PARKER, L. 0. AND Lr, C. L. (1966) The Perfusion Pressure in Hemicooling of the Brain. (In preparation). C. F. (1962) Extracellular Space of Brain as Determined by RALL,D. P., OPPELT,W. W. AND PATLAK, Diffusion of Inulin from the Ventricular System. Life Sci., 2, 43-48. REULEN, H. J., AIGNER,P., BRENDEL, W., AND MESSMER, K. (1966) Elektrolytenveranderungen in tiefer Hypothermie. I. Die Wirkung akuter Auskuhlung bis 0°C und Wiedererwarmung. Pfliigers Arch., 228, 197-219.

STEINWALL, 0. AND KLATZO,I. (1966) Selective Vulnerability of the Blood-Brain Barrier in Chemically Induced Lesions. J. Neuropathol. Exptl. Neurol., 25, 542-559. VANHARREVELD, A. (1957) Changes in Volume of Cortical Neuronal Elements During Asphyxiation. Amer. J. Physiol., 191, 233-242. VAN HARREVELD, A., CROWELL, J. AND MALHOTRA, S. K. (1965) A study of Extracellular Space in Central Nervous Tissue by Freeze-Substitution. J . Cell Eiol., 25, 117-137. VAN HARREVELD, A., AND OCHSS. (1956) Cerebral Impedance Changes after Circulatory Arrest. Amer. J. Physiol., 189, 180-192.

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DISCUSSION C. CRONE: I was very impressed by Dr. Klatzo’s contribution, and I have a few questions: I did not understand your argument when you said that the constancy of the membrane potential, particularly at decreasing temperatures, signified that the cells did not increase in size. If you accept the Goldnian equation, as describing the relevant factors which determine the membrane potential, I don’t see how you get from that to the size of the cell.

The other comment relates to the impedance studies you mentioned. If the impedance increases in a damaged brain, this may be due to a decrease in the extracellular space. One would expect an intercellular distance below 2008, under these conditions. As this seems not to be the case, the logical consequence is eidier that artefacts invariably occur when preparing brain tissue for electron microscopy, leaving an intercellular slit of 200 A, whatever the condition was in situ, or that the impedance studies do not inform us about the extracellular space. 1. KLATZO:With regard to my statement that the constancy of the membrane potential, particularly at decreasing temperature, signified lack of cellular swelling; what I had in mind was the fact that the swelling is usually related to intracellular penetration of sodium accompanied by water. In such swelling, as it occurs, e.g., in asphyxia, the translocation of sodium influences the resting membrane potential. It is true that in some instances, such as reported by Pappius in osmotically induced swelling of the brain in uremic dogs subjected to hemodialysis, the Na/K remains unchanged and intracellular swelling may occur without inflow of sodium. I would like to emphasize, however, that gross observations in our material were definitely against presence of swelling. The hypothermic brains appeared smaller and firmer to touch than normal controls. With regard to your second comment, I am wholeheartedly in agreement that in the living brain the intercellular spaces must be larger than 150-200 8, as suggested by some electronmicroscopists. Presently, there is data forthcoming indicating that this assumption may be wrong and may be related to artefacts of tissue processing. Physiological experiments, such as, e.g., Van Harreveld’s, elegantly demonstrate variations in extracellular compartment, whereas in many electronmicroscopic observations the distance between the cell membranes appears to be rigidly always the same, even in drastic conditions such as acute asphyxia or edema.

R. KATZMAN:I would like to raise for the record the question as to the interpretation of the impedance measurements. If indeed one is dealing (as was originally done on the red cell membranes) with a group of spherical cells with very high membrane resistances, suspended in an aqueous medium, then indeed one can use the impedance measurements to directly determine the size of the surrounding fluid compartment. When you deal with a very complex tissue, it becomes much more difficult. And if you use the analysis of Dr. Ranck (1963, Exprl. Neurol., 7: 153-174), who took into account the various components of this complex tissue, one comes up with quite a different value for the extracellular space than Van Harreveld estimates. Ranck calculated that the spaces were from 5 to 12% instead of Van Harreveld’s 25;& using essentially the same data with a different conceptual interpretation. There is one final point that I think ought to be emphasized, and that is that the cell membrane by itself does not have a low impedance in the dead state; in fact it has a very high impedance. A biomolecular lipid layer without any protein in it has an impedance of 10” Ohm/cm2, compared to the lo? of the neuron, and perhaps 100hm/cm2of the glial cell. In order to maintain the low impedance of the glial cell and of the neuron, vital processes are probably necessary. And one of the effects of fixation may be to destroy some of the mechanisms that underly the usual lower impedance of some cell membranes. J . DOBEING: It will be interesting to know people’s views on where Dr. Rall’s inulin is, if it is not in an extracellular space, and the inulin fills 12 to 15% of the total space.

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D I S C U S S I O N O N T H E P E R M E A B I L I T Y OF T H E B L O O D - B R A I N B A R R I E R

The field of pathology can furnish material that permits under certain conditions, the study of problems related to the permeability of the blood-brain barrier. Two examples are given here: ( I ) the results obtained studying the changes in the composition of the central nervous system during cerebral edema, and (2) the results showing possible relationships between the free amino acid pools of blood, or cerebrospinal fluid, and urine in certain congenital disturbances of metabolism. 1. In cerebral edema there is an increase in water content of the nervous tissue and an increase in sodium; in addition, there is an increase of a rapidly moving protein fraction on the electropherogram. These proteins migrate on agar gel to the level of albumin or fast a-globulins (Fig. I), and can

Fig. 1. Agar gel electropherogram of water-soluble proteins from edematous cerebral tissue. Note the presence of a significant rapidly migrating fraction.

be isolated after passing through columns of Sephadex and DEAE cellulose. The protein responsible for the increase in content of rapidly migrating material could be shown (Karcher and Lowenthal) to be albumin, although it differed slightly from serum albumin with respect to molecular weight and sensitivity to the action of tryptic digestion, and also in its immunological characteristics. 11. The investigation of changes in the concentration of free amino acids in serum, cerebrospinal fluid, and urine in patients with congenital disturbances of amino acid metabolism shows that the changes are not parallel in all three pools. For example, in phenylketonurics on a phenylalaninedeficient diet, the drop in phenylalanine concentration is not parallel in all fluids (Fig. 2). The variations in phenylalanine concentrations (Mardens and collaborators) are inversely proportional to variations in the concentrations of other amino acids in the serum (Fig. 3) while this is not the case for cerebrospinal fluid.

Len ... Guy

E 8 300c v1

-

s t i200-

al

'\ \

Mois

Fig. 2. Changes in serum and CSF phenylalanine concentrations in a phenylketonuric on a phenylalanine-deficient diet. Note that the changes in concentration are not parallel in serum and CSF.

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399

'f

R&mc

Guy Len

c .PHE SERIQUE p m o l e s / l O O m l *--a AMINOACIDEMIE, PHE non compr lse + + PHE L c R pm oles /10 0 ml x-

- -x

AMINOACIDORACHIE, PHE non comprise

Fig. 3. Concentrations of phenylalanine and other amino acids in the serum of phenylketonurics on a phenylalanine-deficient diet. Note that the concentration of amino acids increases as the phenylalanine concentration decreases during the course of treatment, and vice-versa.

These data would point to the usefulness of human material in observing the permeability of the blood-brain barrier. A. Lowenthal

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40 I

Changes in Brain Accumulation of Amino Acids and Adenine Associated with Changes in the Physiologic State DONALD H. F O R D

State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York (U.S.A.)

The observations by Ehrlich (1885, 1887) that certain aniline dyes stained all tissues rapidly when injected into the blood stream, except the brain, was undoubtedly the first step in the creation of a concept of a blood-brain barrier. Confirmation of this concept has been provided by many investigators (Bakay, 1956; Broman, 1955; Goldman, 1913; Lewandowsky, 1900; Roux and Borrel, 1898) using a wide number of techniques. Attempts to localize the site of the barrier to a specific anatomical structure (Barrnett, 1963; Dempsey and Wislocki, 1955; De Robertis and Gerschenfeld, 1961; Donahue et al., 1961 ; Farquahar and Hartman, 1957; Hess, 1953, 1955; Maynard et a]., 1957; Rodriguez, 1955; Tschirgi, 1962; Wislocki and Leduc, 1952) have finally led to the conclusion that every membrane existing between the blood stream and neuronal cytoplasm may possibly contribute. The absence of perivascular and perineuronalspaces (De Robertis, 1962; Maynard et al., 1957) as well as the particular interdigitated organization of brain capillary endothelium (Maynard et al., 1957) may also be important in influencing rates of exchange of solutes between the blood and brain parenchyma. Reasons for exclusion of materials from the brain have included binding to plasma proteins, molecular charge, lipid solubility, aqueous solubility and the electrical charge of a molecule (Garoutte and Aird, 1955; Goldworthy et al., 1954; Krogh, 1946; Robbins and Rall, 1960; Review by Bakay, 1956). The restriction on penetration of metabolic substrates into the nervous system was at one time considered as being so stringent that it was suggested that the only amino acid capable of reaching the neurons was glutamine. Glucose, oxygen, carbon dioxide, lipid soluble materials and water appeared to move freely into brain parenchyma, while inorganic ions and other highly dissociated ions equilibrated between brain and blood more slowly (Tschirgi, 1962). More recently, the investigations of Waelsch and Lajtha (1961), Lajtha and Toth (1963), Gaitonde and Richter (1955), Guroff and Udenfriend (1962), Chirigos et al. (1960), Ford et al. (1965), as well as many others, have demonstrated that most amino acids move freely into the brain. However, the rapid influx of amino acids seems well balanced by a rapid efflux, and both depend upon specific transport systems (Lajtha, 1964; Levi and Lajtha, 1965a and b; Blasberg and Lajtha, 1965). Considering these rapid transport mediated movements of amino acids in and out of brain parenchyma, Rc,ferences p. 411413

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D. H. F O R D

one might readily suppose that the entrapment of amino acids into neurons might be influenced by the metabolic activity within the neuron and that the over-all metabolic turnover rates within neurons might be influenced by the over-all metabolic activity of the body. This suggestion that uptake of amino acids into neurons might be influenced by the metabolic demands can in part at least be attributed to Dobbing (1961). If increases in amino acid uptake into neurons can be associated with the metabolic activity of neurons, it would appear that the membranes which constitute what is considered the blood-brain barrier may be facilitative as well as restrictive in function. Uptake of triiodothyronine in brain compared with muscle Before proceeding to changes in internal or external environmental states which may influence brain metabolism in such a way as to appear to influence neuronal amino acid accumulation, I would like to point out that CNS accumulation of some compounds, while exceeded by many organs, is paralleled by a comparable uptake in muscle (Fig. I). Such a similarity in uptake between brain and muscle (Ford, 1965)

Fig. 1. A comparison of the percent of an injected doseof 131 I present per gram of tissue in the pineal, plasma, cerebral grey matter, striated muscle and adrenal gland at various time intervals after the I.V. injection of 1311-labeledtriiodothyronine (0.5 ,ug/kg) into normal male rats (Ford, 1965).

is well illustrated by triiodothyronine (T3). In this instance a dose of 0.5 ,ug/kg of ['3lI]T3 was given intravenously and animals killed at varying times after injection. The amount of the injected dose of 1311 present in the tissue was calculated on the basis of the percent of the injected dose present/g. Most of the tissue radioactivity was shown to be triiodothyronine by paper chromatography (Ford, 1965). It is clear, that while pineal and adrenal uptakes and plasma levels of radioactivity are higher than that seen in cerebral grey matter, muscle levels of radioactivity were essentially the

CHANGES IN BRAIN ACCUMULATION

403

.

Fig. 2 Autoradiograms of ventral horn neurons from the cervical spinal cord of a normal rat (right) and a rat exercised in an activity wheel for 2 h (25 feet/min) and injected during exercise with [3H]leucine (Courtesy of Altman, 1964).

same as those demonstrated in the cerebral grey matter. This similarity in accumulation might mean that there is a blood-muscle barrier to triiodothyronine, which seems unlikely. One may add parenthetically that the percent of an injected dose of radioR[~i~re.nw p.s411-413

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D. H. F O R D

activity present in the brain and muscle after intravenous injection of labeled lysine, tyrosine and glutamine is also quite comparable. Effect of exercise on the accumulation of [3H]leucine by neurons In 1964,Altman noted that rats run in an exercise wheel at a rate of 25 feet/min had an increased amount of radioactivity in neurons and surrounding neuropil if the labeled amino acid was injected during the exercise period. The animals were killed after an exchange period of one hour and the uptake determined by radioautography (Fig. 2). The increases in the amount of radioactivity present occurred in a wide variety of neurons as well as in the neuropil of the exercising rats, which Altman felt was more marked in such of the motor regions as the motor cortex, cervical cord neuropil, and ventral horn cells and in the visual cortex than in some non-motor areas, such as the hippocampus, cochlear nucleus and choroid plexus. Injections of labeled amino acid after the exercise were associated with increased uptakes of radioactivity which were much less marked and in which there were no distinctive differences between “motor” and “non-motor” neurons. The radioautographic illustration also demonstrates that the neuronal uptake of amino acid as compared with the uptake in the surrounding tissue was quite high. Since the volume of grey matter occupied by neurons is relatively small (Brizzee er al., 1964; SchadC, et al., 1964), it is apparent that studies on brain accumulation of labeled materials using whole tissue slices or blocks will be likely to provide data suggesting that over-all incorporation is low due to the very low amount of the labeled materials present in the tissue termed neuropil. This heterogeneity of uptake complicates interpretation of blood-brain barrier influences. Effect of neuron regeneration Complicated changes in neuronal protein and RNA are known to occur following nerve section or crush, as determined by biochemical methods (Brattgard et al., 1957). These changes can also be demonstrated radioautographically (Rhodes et a/., 1964). In a large series of rats the hypoglossal nerve was sectioned. Two weeks later the animals received a dose of [3H]lysine (593 pg/kg) intravenously. A half hour later the animals were killed by intracardiac perfusion and the tissues processed for radioautographic analysis. The uptake of radioactivity was compared in the normal and regenerating hypoglossal neurons in the same animal by grain counting procedures (Table 1). Ascending paper chromatographic analysis of brains from other animals injected with lysine demonstrated that the main labeled compound present in the brain was lysine. Inasmuch as the presence of labeled amino acid in cells following the usual histological procedures utilized with radioautographic studies is believed to be directly related to protein synthesis (Leblond, 1965), the increased grain count over the regenerating neurons suggests an accelerated protein synthesis in these cells (Column A). This was also apparent in animals which were either hypo- or hyperthyroid in metabolic activity. Cell size was also appreciably increased in regenerating

405

CHANGES IN B R A I N ACCUMULATION

TABLE I C O M P A R I S O N OF U P T A K E O F [:'H]LYSlNE OF T H E HYPOGLOSSAL

IN NORMAL A N D REGENERATING NERVE CELLS

NUCLEUS AS AFFECTED

Euthvro id A. Grain counts/cell Regenerating Normal B. Cell size (mg of paper/cell outline, reflecting cell area) Regenerating Normal C. Grain count/mg of paper (reflects cell area) Regenerating Normal

BY V A R Y I N G T H Y R O I D

Hypothyroid

+

32.210 4.592" 19.095 & 1.714

50.098 -+ 3.914bC 26.102 1.886

28.464 1.984d 18.883 I 1.502

28.205 16.924

1.110 *0.037 1.034 i 0.097

STATES

Hyperthyroid

36.337 f 2.035b 21.711 f 2.139

+ 1.208e + 1.718

26.683 f 1.090e 17.781 f 1.296

1.7758 f 0.1131 1.5769 & 0.096'

1.3680 f0.0759 1.2154 & 0.059

Significantly greater than normal cells ( p , : 0.02, 0.05). Significantly greater than non-regenerating cells ( p < 0.001). c Significantly greater than euthyroid cells ( p < 0.02, 'i 0.01). d Significantly larger than non-regenerating cells ( p > 0.001, < 0.01). e Significantly larger than non-regenerating cells ( p i 0.001). Significantly greater than euthyroid and hyperthyroid cells ( p > 0.001, < 0.01). g Significantly greater than normal and regenerating euthyroid cells ( p > 0.001, < 0.01). (From Rhodes el a/., 1964.) a

K :

b

neurons (Column B). The net result of the increases in grain count and in cell size was that the concentration of labeled lysine in regenerating cells remained close to what it was in the normal cells of euthyroid animals (Column C ) . However, to maintain this normal concentration in the regenerating enlarged hypoglossal neurons, a net increase in total amount of labeled amino acid present was necessary. Both changes in thyroid state (Column C) increased amino acid uptake in regenerating cells in a manner comparable to that seen in euthyroid rats. Moreover, both changes in thyroid state were associated with increased neuronal accumulation of labeled material which was not relative to regeneration. While it seems likely that the increased incorporation of labeled amino acid in neurons from hypo- and hyperthyroid states may be caused by dissimilar reasons, an explanation for this observation is not clear at this time. It may be pointed out, however, that a change in the metabolic requirements of the neuron, in this instance a need for increased amounts of amino acid for the synthesis of new protein in a regenerating cell, was associated with an increased incorporation of labeled material. The extent to which the labeled amino acid was accumulated by either normal or regenerating neurons also seemed to be influenced by the thyroid state of the animal. A similar radioautographic experiment was performed with regenerating hypoglossal neurons, using 13Hladenine as the test material. It seemed that it might be useful to use this purine for what was virtually the same experiment because so much of a dose of exogenously provided adenine becomes associated with AMP, ADP and ATP (Pakkenberg et al., 1965). The effect of dysthyroidal states was also included in Refcrcnrrs p. 411-413

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D. H. F O R D

TABLE I1 C O M P A R I S O N OF U P T A K E O F [ 3 H ] A D E N l N E I N N O R M A L A N D R E G E N E R A T I N G N E R V E C E L L S O F T H E H Y P O G L O S S A L N U C L E U S AS A F F E C T E D B Y V A R Y I N G T H Y R O I D S T A T E S

Grain Counts Neuropil areas equivalent to neurons

Control (11)* Hypothyroid (1 1)8 Hyperthyroid (1 3p

130 & 12.70+ 326 & 52.68*+ 174 & 13.48*+

Nerve cells, Normal Xllth Nucleus

**

53 14.16 73 9.94 73 i 12.61

Nerve cells, regenerating Xllth Nucleus 33 f 12.33 52 & 18.62 60 10.65

*

Significantly different from neuropil counts in euthyroid rats ( p < 0.02). Significantly higher than the count from normal and regenerating hypoglossal nucleus cells (on an equivalent area basis) ( p < 0.001). * Number of animals studied. (From Pakkenberg et al., 1965). +

this investigation on uptake of intravenously injected adenine by normal and regenerating neurons. The dose of adenine given was 104.0 pg/kg, and the animals were killed by intracardiac perfusion one-half hour after injection. In this study, the regenerating cells had a lower radioautographic level of activity than did the control cells of the adjacent nucleus (Table 2). Again, both dysthyroidal states influenced cell accumulation of radioactivity and in the same direction with comparable effects being seen in both normal and regenerating cells. The comparison of cell-neuropil levels of radioactivity following r3HIadenine injection was the opposite from what was observed following [3H]lysine injection (Table 1). Thus, with the injection of adenine, neuropil was observed to have higher levels of activity than did the neurons 1/2 h after injection. This neuropil associated radioactivity was significantly higher than that of the normal neurons for both the euthyroid and dysthyroidal groups, and the level of radioactivity in the neuropil around regenerating neurons was, like the activity of the neurons, lower than on the control side. Thus, it appears thatuptake of adeninein the neuropil and neurons in the regenerating hypoglossal nucleus is depressed by the processes associated with regeneration. However, this may be an illusion, since the adenine taken up into neural tissue appears primarily associated with the synthesis of the adenine nucleotides, which may be more rapidly expended in the synthesis of new protein and tissue respiration in regenerating than in normal cells. The high concentration of radioactivity in the neuropil after adenine injection is maintained for only a short period (Fig. 3), for when one examines the levels of radioactivity in neuropil and a variety of neurons at later time intervals after injection by radioautography, neuronal uptake clearly exceeded that of the neuropil,

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8ol

xl\L . t,

Fig. 3. A grain count analysis of radioautograms of the amount of 3H accumulated in various neuron types at various intervals after I.V. injection of [3H]adenine. Grain count is based on total counts from areas encompassed by 20 neurons for each neuron type and compared with the grain count from an equivalent area of neuropil. Data for the f hour period was available for only hypoglossal (XII) motor neurons and neuropil, which is continued at subsequent periods with data from ventral horn motor neurons and neuropil. The dose of adenine given was 969 pg/kg (314 /tC/kg).

Amino acid uptake into grey matter and neurons as influenced by thyroid state Theeffect ofdysthyroidal states on uptake of amino acids into brain grey matter and ventral horn motor neurons following intravenous injection of labeled amino acids may also be analyzed with liquid scintillation counting techniques (Ford et al., 1965) utilizing a Nuclear Chicago liquid scintillation counting system. In this study a dose of 590 to 600 pg of [ 3 H ] ~ ~ - l y s iwas n e injected intravenously/kg into male rats, which were then killed by intracardiac perfusion with citrated saline at various time intervals after the injection. Accumulation of radioactivity was determined on the basis of the percent of the injected dose present/g of tissue, which was then plotted against time. Ventral horn motor neurons and spinal cord grey matter will be the only types of neuronal tissue considered. The spinal cord grey samples were prepared by removing the white matter from the region to be analyzed. Ventral horn motor neurons were dissected with fine needles from a comparable region free hand, using a dissecting microscope with a magnification of 130 x . For details of the procedure see Ford et al. (1965). In the spinal cord grey, the amount of radioactivity in euthyroid rats was intermediate between that seen in the hypo- and hyperthyroid animals (Fig. 4). The attainment of a maximal concentration occurred earlier in the hyperthyroid group and latest in the hypothyroid group, which suggests that turnover in the hyperthyroid group is somewhat faster. Such a faster turnover of the labeled amino acid contained in a larger non-labeled pool of lysine could readily account for the lower amount of labeled material present in tissue samples taken from the hyperthyroid group, while a slower Rrfermces p . 411-413

408

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0

6.0[

U C

0 0.3:: c

-:

.-

I'

Ventral horn neurones

1: ;

:

I

Spinal cord grey /

O . hypo

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Fig. 4. A comparison of the amount of 3Hpresent in ventral horn motor neurons and spinal cord grey matter in euthyroid and dysthyroidal male rats at various time intervals aftes I.V. injection of [3H]DL-lysine (Ford et a/., 1965).

turnover of labeled lysine in the hypothyroid rats could reasonably explain the higher accumulations which occur in this group. The amount of radioactivity in the ventral horn motor neurons was significantly higher than that of the spinal cord grey for all three groups. Furthermore, ventral horn cell radioactivity on a percent dose per gram basis exceeded even that of the liver. There was again a tendency for the radioactivity in the neurons from hyperthyroid rats to be lower than normal, while that entering the neurons from hypothyroid rats was above normal. The pattern of the distribution of radioactivity at different time intervals after injections was not one, however, which would permit one to arrive at the same conclusions regarding turnover rates as were made for the spinal cord grey, which illustrates in part the difficulties inherent in metabolic studies with tissues as heterogeneous in nature as brain grey matter. A similar investigation on the effect of dysthyroidal states on accumulation of labeled amino acids was done with [14C]~-glutaminein which a dose of 1.44 mg/kg of glutamine was given intravenously (Ford and Rhines, 1967). The amount of radioactivity present in ventral horn motor neurons and spinal cord grey were compared (Fig. 5 ) in the same manner as after injection with [3H]lysine. Again the ventral horn cell levels of radioactive material were appreciably higher than those seen in the spinal cord grey, and again both dysthyroidal states appeared to have some influence on the levels of radioactivity present in both tissues. This was most noticeable in the hyper-

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Fig. 5. A comparison of the amount of I4C present in ventral horn motor neurons and spinal cord grey matter in euthyroid and dysthyroidal male rats at various time intervals after I.V. injection of [14C]glutamine(Ford and Rhines, 1967).

thyroid neurons which not only contained a higher level of radioactivity, but attained maximal concentration during the experimental period. (Chromatographic analysis indicated that most of the 14C in the brain was associated with glutamine or glutamic acid.) A final example wherein CNS and neuronal uptake of labeled amino acid may be influenced by a change in the environmental state of an animal may be observed in animals subjected to a condition in which they are respiring pure oxygen at a pressure of 3 atmospheres (Fig. 6). [3H]Lysine was given intravenously at a dose level of 190.0 pg/kg and the animals were killed by intracardiac perfiision at various time intervals after iiijection. The amount of radioactivity present in the various tissue samples was expressed in terms of the percent of the injected dose present/g. Again, the level of radioactivity in the ventral horn cells was significantly higher than that determined in spinal cord grey matter. The effect of respiring pure oxygen at a pressure of 3 atmospheres was to significantly depress activity levels in both the ventral horn neurons and the spinal cord grey, on the basis of group analysis. This depression in uptake may be related to interference in the activity of various enzymes (Chance et al., 1965; Dickens, 1962; Jacobsen et al., 1964; Kety and Schmidt, 1948; Thomas et al., 1963; Wood et al., 1963, 1964) or to the ischemia known to occur in the CNS under such conditions. It might be noted parenthetically that the changes in uptake of amino acid occur much sooner than do the convulsions which are induced by high pressure oxygen, and which require subjecting the animals to higher pressures. Riferences p. 411413

410

D. H. F O R D

1

0 High

Control

pressure oxygen

0.7E

/-----Ventral horn cells O ,

,

.-I

, -

Spinal cord grey

n

z

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10

20

30

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Fig. 6. A comparison of the amount of 3H present in ventral horn motor neurons and spinal cord grey matter in male rats at normal atmospheric pressure and in rats subjected to hyperbaric oxygen (3 atmospheres) after I.V. injection of [3H]lysine (Ford and Rhines, 1967).

CONCLUSION

That some mechanism exists in the brain which appears to restrict entry of many compounds, as compared with other tissues, has been accepted for many years. A variety of anatomical, physiological and biochemical explanations have been introduced to describe this phenomenon. However, this barrier phenomenon does not appear to hold true in all instances. Certainly, if one compares the amount of labeled triiodothyronine or amino acid in brain and muscle after intravenous injection, one finds that the amount of labeled material present in the two tissues is rather comparable, as is the turnover. If one then compares the distribution of various labeled amino acids and adenine between neurons and the surrounding neuropil radioautographically (Altman, 1964; Rhodes et al., 1964) (Figs. 2 and 5), one finds that neuronal uptake is considerably higher than that of neuropil. In other words, the low concentration of a physiologic substrate incorporated into a large mass of neuropil may mask the relatively high neuronal levels when one does total tissue slice or block analysis. The high amino acid accumulation by neurons as compared to neuropil was then confirmed by the application of liquid scintillation counting techniques to determine the extent to which accumulation occurs in neurons as compared with blocks of tissue containing both neurons and neuropil. An increase in neuronal physiologic activity induced by exercise and regeneration of neurons were both shown to increase incorporation of labeled amino acid in neurons. Adenine uptake was depressed in regenerating cells, possibly because of an increased utilization rate of adenine associated with the ATP utilized during the increased protein synthesis which occurs in neuron regeneration. Changes in thyroid

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41 1

function (hypo- or hyperthyroidism) were both associated with changes in accumulation of amino acid or adenine into normal or regenerating neurons or into neuropil. Finally, an increase in the amount of oxygen in the respired gas in association with an increase in pressure to three atmospheres significantly depressed amino acid accumulation by neural tissues. Thus, the uptake or incorporation of amino acids or adenine into neural grey matter or into neurons appears to be easily and readily altered by internal or external environmental factors, which would appear more likely to exert their influences on transport systems than on particular anatomical subunits existing between the blood stream and the neuronal cytoplasm. The term “barrier” seems somewhat inappropriate in relation to amino acid uptake into neurons, since the uptake appears to occur easily and to be readily influenced by changes in cellular environment in a dynamic fashion. ACKNOWLEDGEMENTS

Supported in part by a U.S.P.H.S. Research Grant (NB-04568-03) from the National Institute of Neurological Diseases and Blindness, Public Health Service, and in part by a Research Grant from the Physiology Branch (NONR 4018(00)), Office of Naval Research. The author wishes to express his appreciation for the assistance provided by Mr. Ralph Rhines, Mrs. M. Buschke and Mrs. Gloria Cohan.

REFERENCES ALTMAN, J. ( I 964) The use of fine-resolution autoradiography in neurological and psychobiological research. Response ofthe Nervous Systeni to Ionizinr Radiation, 2nd Ed. T. J. Haley and R . S. Snider (Eds.). Boston, Little, Brown and Co., (p. 336). BAKAY, L. (1956) The Bloo(l-Brain Barrier: With Special Regard to the Use of Radioactive Isotopes. Springfield, Illinois, Thomas. BARRNETT, R. J. (1963) Fine structure and function of the neurone. Trans. Anier. Neurol. Ass., pp. 123-126. BLASBERG, R. A N D LAJTHA,A. (1965) Substrate specificity of steady-state amino acid transport on mouse brain slices. Arch. Biocheni. Biophys., 112. 361-377. BRATTGARD, S.-O., EDSTROM, J.-E. A N D HYDEN,H. (1957) The chemical changes in regenerating neurons. J. Neurocheni., I, 3 16-325. BRIZZEE, K. R., VOGT,J. A N D KHARETSCHKO, X. (1964) Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat. Growth and Maturation of the Brain, D. P. Purpura and J. P. SchadC (Eds.). Progress in Brain Research, vol. 4, Amsterdam, Elsevier, (p. 136). BROMAN, T. (1955) On basic aspects of the blood-brain barrier. Acta Psychiat. Scand., 30, 115-124. CHANCE,B., JAMiEsoN, D AND COLES,H (1965) Energy-linked pyridine nucleotide reduction. Inhibitory effects of hyperbaric oxygen in vitro ‘and in vivo. Nature, 206, 257-263 CHwmos, M A , GREENGARD, P A N D UDENFRIEND, S (1960) Uptake of tyrosine by rat brain in vivo. J. B i d . Cheni., 235, 2075-2079. DEMPSEY, E. W. A N D WisLocKi. G. B. (1955) An electron microscopic study of the blood-brain barrier in the rat, employing silver nitrate as a vital stain. J. Biophys. Biocheni. Cytol., 1, 245-256. DE ROBERTIS, E. (1962) 1. Some old and new concepts of brain structure. World Neiirol., 3, 98-1 11.

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DE ROBERTIS, E. AND GERSCHENFELD, H. M. (1961) Submicroscopic morphology and function of glial cells. Intern. Rev. Neurobiol., 3, 1-65. DICKENS, F. (1962) The toxic effects of oxygen in nervous tissue. Neurochemistry, 2nd. ed., Elliott, K. A. C., Page, I. H., Quastel, 5. H. Editors, Springfield, Illinois, C. C. Thomas (p. 851). DOBBING, J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. DONAHUE, S. AND PAPPAS, G. D. (1961) The fine structure of capillaries in the cerebral cortex of the rat at various stages of development. Amer. J. Anaf., 108, 331-348. EHRLICH, P. (1885) Das Sauerstoff-Bediirfnisdes Organismus. Eine Farbenanalytische Studie, Berlin, A. Hirschwald, (p. 69-72). -, ( I 887) Zur Therapeutischen Bedeutung der Substituerenden Schwefelsaure-Gruppe. Therap. Monatsh., 1, 88-90. FARQUAHAR, M. G. AND HARTMAN, J. F. (1957) Neuroglial structure and relationships as revealed by electron microscopy. J. Neuropafhol. Exptl. Neurol., 16, 1 8-39. FORD,D. H. (1965) Uptake of 1131-labeled triiodothyronine in the pineal body as compared with the cerebral grey and other tissues of the rat. Structure and Function of the Epiphysis Cerebri, J. A. Kappers and J. P. Schadk (Eds.). Progress in Brain Research, vol. 10, Amsterdam, Elsfvier (p. 530). FORD,D. H. AND RHINES,R. (1967) Uptake of C14 into the brain and other tissues of normal and dysthyroidal male rats after injection of C14-~-glutamine.Acta Neurol. Scand., 43, 3 3 4 7 . FORD,D. H., WINES,R., HARTSTEIN, M. AND RHODES, A. (1965) The uptake of DL-Lysine-H3 into the nervous system as compared with other tissues in euthyroid and dysthyroidal male rats. Acfa Neurol. Scand., 41, 21 5-232. GAITONDE, M. K. AND RICHTER, D. (1955) The metabolic activity of the proteins of the brain. Proc. Roy. SOC.Brit., 145, 83-99. GAROULTE, B. AND AIRD,R.B. (1955) Diffusion of sodium ions from cerebral tissue in vifro. Science, 122, 333-334.

GOLDMAN, E. E. (191 3) Vitalfarbung am Zentral Nervensystem. Beitrag zur Physiologie der Plexus Choroideus und der Hirnhaute. Berlin, G. Eimer. R. A. (1954) The blood-brain barrier: The effect of GOLDWORTHY, P. D., AIRD,R. B. AND BECKER, acid dissociation constant on the permeability of certain sulfonamides in the brain. J. Cell. Comp. Physiol., 44, 519-526. GUROFF, G. AND UDENFRIEND, S. (1962) Studies on aromatic amino acid uptake by rat brain in vivo. J . Biol. Chem., 237, 803-806. HESS,A. (1953) The ground substance of the central nervous system revealed by histochemical staining. J. Comp. Neurol., 98, 69-92. -, (1955) The relation of the ground substance of the central nervous system to the blood-brain barrier. Nature (Lond.), 175, 387-388. JACOBSON, I., HARPER, A. M. AND MCDOWALL, D. G. (1964) The effects of oxygen at 1 and 2 atmospheres on the blood flowand oxygen uptake of the cerebral cortex. Surg. Cynecol. Ohstet., 119, 7 3 7-742.

KETY,S . S. AND SCHMIDT, C. F. (1948) The effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young men. J . Clin. Invest., 27, 484-492. KROGH,A. (1946) The active and passive exchange of inorganic ions through the surfaces of living cells and through living membranes generally. Proc. Roy. SOC.(London), B., 133, 140-200. LAJTHA, A. (1964) Protein metabolism of the nervous system. Infer. Rev, Neurobiol., 6, 1-97. LAJTHA, A. AND TOTH,J. (1963) The Brain-Barrier System. V. Stereospecificityof amino acid uptake, exchange and efflux. J . Neurochem., 10, 909-920. LEBLOND, C. P. (1965) What radioautography has added to protein lore. The Use of Radioautography in Investigating Protein Synthesis. C. P. Leblond and K. B. Warren, Editors, New York, Academic Press, (p. 321). LEVI,G. AND LAJTHA,A. (1965a) Cerebral amino acid transport in vitro. 11. Regional differences in amino acid uptake by slices from the central nervous system of the rat. J. Neurochem., 12,639-648. LEVI,G., CHERAYIL, A. AND LAJTHA, A. (196513) Cerebral amino acid transport in vitro. 111. Heterogenecity of exit. J . Neurochem., 12, 757-770. LEWANDOWSKY, M. (1900) Zur Lehre von der Cerebrospinal-Flussigkeit.Z . Klin. Med., 40,480494. MAYNARD, E., SCHULTZ, R. L. A N D PEASE, D. C. (1957) Electron microscopy of the vascular bed of rat cerebral cortex. Amer. J . Anat., 100, 409434.

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PAKKENRERG, H., FORD,D. H., RHINES, R. AND ISRAELY, R. A. (1965) Adenine H3-uptake in nervous tissue including regenerating nerve cells, as compared with other tissues in euthyroid, hypo and hyperthyroid male rats. Actu Nerirol. Scand., 41, 497-512. RHODES, A., FORD,D. H. A N D RHINES, R. (1964) Comparative uptake of ~L-Lysine-H~ by normal and regenerative hypoglossal nerve cells in euthyroid, hypothyroid and hyperthyroid male rats. Exprl. Neurol., 10, 251-263. ROBEINS, J. AND RAI.L,J. E. (1960) Proteins associated with the thyroid hormone. Physiol. Rev., 40, 41 5 4 8 9 . RODRIGUEZ, L. A. (1955) Experiments on the histologic locus of the hematoencephalic barrier. J. Conip. Ncrirol., 102, 2145. Roux, E. AND BORKtL, A. (1898) Tetanos cerebral et immunite contre le tetanos. Ann. Inst. Pusteur (Paris), 12, 225-239. SCHADE, J. P., VAN BACHER, H. A N D COLON, E. (1964) Quantitative analysisofneuronalparameters in maturing cerebral cortex. Gron~tliand Mcitrirarion ofthe Brain, D. P. Purpura and J. P Schade (Eds.). Progress in Brain Research, vol. 4, Amsterdam, Elsevier (p. 150). TtioMAs, J. J. JR., NEPTUNE, E. M. JR. AND SUDDUTH,H. C. (1963) Toxic effects of oxygen at high pressure in the metabolism of D-glucose by dispersions of rat brain. Biochem. J., 88, 31-45. TSCHIRGI, R. D. (1962) Blood-brain barrier; fact or fancy? Fed. Proc., 21, 665-671. WAELSCH, H. A N D LAJTHA,A. (1961) Protein metabolism in the nervous system. Pltysiol. Rev., 41, 709-736. WISLOCKI, G. B. A N D LEDUC,E. H. (1952) Vital staining of the hematoencephalic barrier by nitrate and trypan blue, and cytological comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. Comp. Neurol., 96, 3 7 1 4 5 . WOOD,J. D. AND WATSON,W. J. (1964) The effect of oxygen on glutamic acid decarboxylase and gamma-amino butyric acid-alpha-ketoglutaric acid transaminase activities in rat brain homogenates. Canad. J. Pliysiol. Pharmzcol., 42, 277-279. WOOD,J. D., WATSON,W. J. AND CLYDESDALE, F. M. (1963) Gamma-amino butyric acid and oxygen poisoning. J. Neiirochern., 10, 625-633.

DISCUSSION P. MANUEL:I should like to make some general remarks. The first one concerns the incorporation of amino acids into tissue using radioautography. When you look for the incorporation of amino acids into proteins in vifro and you precipitate the proteins with TCA, you may have a high degree of incorporation. However, to determine if there has been real incorporation of amino acids into proteins, one must treat the preparation with TCA at 90°C because when you provide exogenous basic or acidic amino acids they may simply become attached to another amino acid in a protein chainzwithout actually becoming incorporated into the proteins. I think that many mistakes might result from this kind of phenomena in experiments with radioautography. During perfusion you have a free exchange between free and bound amino acids, because you are doing two kinds of experiments. One is called a “chase-experiment”. When this is done at 10°C there can still be an exchange with a peptide chain. With water perfusion there may also be an exchange with the amino acids which are attached to the peptide chains and which are not incorporated in the-chains. Thus, the problem remains as to whether the incorporation of amino acid represents a protein synthesis or not. I think that the control experiments which you did with the scintillation counting after the radioautography are very useful, but it seems to me that it will also be useful to do this after treatment by TCA, before the counting. The second remark concerns the nucleotide pool. We have observed an increase of nucleotide synthesis following diffcrcnt forms of stimulation. There was namely an increase in the turnover of ATP or in ATP-synthesis. If one looks for RNA synthesis and finds an increase in the specific radioactivity of a purine or pyrimidine base in RNA, it might be difficult to determine if R N A synthesis occurred because of an increase in the specific activity of the precursor pool of bases.

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D. H. FORD: This might be true for RNA or ATP. P. MANDEL:It is believed that there is an increase of the RNA synthesis in stimulated neurons. As far as the ATP-synthesis-increase is concerned, we have experiments with amphetamine, wherein examination of the y-phosphate or the u-phosphate demonstrates a good correlation between the increase of functional activity and nucleotide synthesis. The last point concerns the different salts which one uses. We found some years ago that there are really differences after injection of a precursor. There are differences in the radioactivity of the free nucleotides in different salts, which agrees well with what you found.

D. H. FORD:The problem of interpreting radioautography is very complex, since most people rarely determine the chemical nature of the labeled material producing the radioautograph. In our findings with the dissected neuron preparations from perfused animals, there may be activity in the cells which are being monitored, which is not associated with protein, but which is associated with a carrier or some other marker, or which may be a lipoprotein. When [3H]lysine was injected, the major labeled material in the neurons was lysine as determined chromatographically. With adenine injected rats, tissue samples were hydrolyzed with KOH which was followed by perchloric acid precipitation. This provides quite different results than those obtained by acid hydrolysis and precipitating with the base. The labeled molecules are not completely split t o liberate the adenine base itself. Thus, one ends up with literally all of the possible compounds that adenine can be associated with, including the break-down products of adenine (representing perhaps about 30% of the total pool). Most of these were found in the hydrolyzed brain preparations. K. D. NEAME: I would like to bring up a point about the terminology. Dr. Ford used the word upl(ikP, implying it with reference to incorporation of amino acids into protein. “Uptake” is also used to refer to the movement of free amino acids. I think one must definitely distinguish between these two types of movement, one: incorporation into protein, and the other: movement into the cell, but not into the protein. D. H. FORD:Actually, if one could determine the total amount of labeled material taken up into the cell, the values would probably be higher. However, with the process used, these small amino acids, free bases and perhaps peptide units as well are by and large removed. So in our preparation one is literally left with only what has been incorporated or bound in some fashion within the cell if not actually into protein. However, you are quite right in cautioning against the rather loose use of the term “uptake”. P. G. SCHOLEFIELD: Did I understand you, Dr. Ford, to say that most of the tritiated adenine went into the nucleotides when you measured incorporation? D. H. FORD:The greatest fraction of activity which we have found is in nuclcotides. There were other smaller fractions, which we have not been able to identify.

P. G. SCHOLEFIELD: It seems to me that in other studies with tritiated adenine, certainly in in vitro studies, it disappears within a matter of minutes. As 1 understand it, previous irr vivo work on incorporation of adenine indicates that it disappears very quickly, but in some of your slides you showed that adenine was going into the cell, and being incorporated into nucleotides even after 48 hours. How do you explain this?

D. H. FORD : We have performed chromatographic analysis on animals killed 24 hours or 48 hours after the injection with labeled adenine. ADP and AMP could be seen as identifiable compounds, plus a very small amount of adenosine, and traces of activity which migrated chromatographically like adenine. It would seem, therefore, that most of the adenine has disappeared and become incorporated into nucleotides. Of course, if one hydrolyzes with perchloric acid in the usual fashion, over 60% of the activity is present as ade nine.

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P. G . SCHOLEFIELD: Yes, that is normal, because if you hydrolyze it you liberate the purines. Could you hydrolyze the R N A for example with KOH? D. H . FORD:We have done some preliminary salt extractions of brain for RNA, and the total amount of activity taken up into the RNA fraction as compared to the rest of the activity in the sample is so much lower, that I distrust the figures. In liver, I have more confidence in the results, since the extracted fraction has a U V absorption comparable to that of RNA. Therefore, the radioactivity present in this fraction would seem to be, in part at least, RNA. However, in brain the technique which we used does not seem to be adequate. P. G. ScHoLEwLD: Just one more inquiry, if I may. Did you identify this ATP? Would not you have thought that the alkali would have hydrolyzed most of the ATP? P. MANDEL:In the normal hydrolysis you need barium hydroxide at loo". You cannot hydrolyze in the same condition as you hydrolyze RNA. A. LAJTHA: The changes in the label in the tissue may be caused either by changes in the transport of the labeled precursor or by changes in the rate of incorporation of this precursor into an acidinsoluble product. I wonder which one you think is affected by hypothyroidism. If I remember well, Sokoloff showed an increase in protein formation in in virro systems.

D. H. FORD:In young animals? A. LAJTHA: Only in young animals.

D. H. FORD:We have been interested in this, because we think, looking at our data from adult animals, that a point of maximal concentration of activity occurs earlier in hyperthyroid rats than normally observed. This suggests that there is actually a slight acceleration of incorporation of labeled material into the protein in the hyperthyroid state. We observed while doing amino-nitrogen determinations, that the amino-nitrogen levels of normal and the hyperthyroid rats was essentially the same. Thus, if the total amount of amino nitrogen is relatively constant, but there is an acceleration of turn-over of protein in neurons, one might logically expect the time needed for attaining maximal concentration of an amino acid might be altered, reflecting the change in metabolic rate, which might well occur without changing the amount of label present in a protein at any given time. H. KOENIG: You referred to Dr. Altnian's work in which he demonstrated an increased incorporation in the protein of ['JCIleucine in the nerve cell in exercised animals. He also observed an increase of a similar magnitude into the choroidal plexus, which he did not discuss. So one is left with the question: what was the meaning of this increased incorporation? The problem of radioautography is that while it tells you where the incorporated label is located, it tells you nothing about penetration since the acid soluble precursor pool is eluted out. In studies of this kind, it would be well to obtain measurements on the acid soluble pool from fresh tissue adjacent to that being radioautographed to gain some idea of penetration as well as incorporation.

D. H. FORD:We also have information on the distribution of lysine in the various pools which, as you have indicated, is very helpful in understanding the radioautograph. P. MANDEL:It is quite sure that in 30 minutes after injection there is very little incorporation of adenine into RNA: it is mainly present in the acid soluble pool.

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417

The Development of the Blood-Brain Barrier JOHN DOBBING Department of Physiology, London Hospital Medical College, London (England)

Classical views on the blood-brain barrier have usually included the idea that it is not present in early life, but develops gradually as the organism develops, to become fully operative only when the animal achieves maturity. I n the first decades of this century, during the period when the “barrier” was considered exclusively in terms of dyestuffs or other histologically identifiable substances, it was widely claimed and believed that trypan blue entered the foetal and newborn brain readily from the blood stream, and was only excluded in the matureindividual (Behnsen, 1927; Stern and Peyrot, 1927). When radioactive isotopes became available for biological research, it was quickly found that such non-specific markers as 32P appeared to be less restricted in their entry into neonatal brain than into adult (Fries e t a / . , 1940; Bakay, 1953), and this was naturally interpreted as a further manifestation of the “developing blood-brain barrier”. Since this time, the concept has been further extended. Analagous to the alleged behaviour of dyestuffs, it has been held to account for the phenomenon of kernicterus, a condition confined to the neonatal period in humans in which unconjugated bilirubin from the plasma enters certain areas of the brains of jaundiced babies. Also, as an extension of the early findings with 32P many other types of metabolic materials have been found to enter the immature brain more readily than the adult, in accordance with the much greater activity of many metabolic processes at this time. One of the more recent examples was reported for inulin (Woodbury, 1967). In many quarters the belief dies hard that trypan blue enters immature brain freely. Nevertheless, for several years now, experimentalists in the field have known that however immature the brain, dyestuffs like trypan blue do not enter it any more readily from the blood than in the adult state (Millen and Hess, 1958). More recently it has been shown that bilirubin will not enter the normal newborn brain (Lucey, Hibbard, Behrman, de Gallardo and Windle, 1964) unless it is present in sufficient quantity to have exceeded the capacity of the plasma albumin to bind it (Diamond and Schmid, 1966). The extreme rarity of an unconjugated hyperbilirubinaemia of this magnitude in adults accounts for kernicterus being confined to the neonatal period in humans. However, in the Gunn strain of rats in whom there can be a high plasma level of such pigment due to a conjugation defect, kernicterus does occur in adults provided the form and physical state of the plasma bilirubin is suitable. The operative factor is in the plasma, not in the maturity of the brain. RpJ’fiwnci.rp. 424-425

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For those whose concept of the blood-brain barrier is limited to the behaviour of dyestuffs across the blood-brain interface, the idea of a developing barrier of increasing impermeability with age, must therefore be abandoned. Most experimenters, however, are concerned with the blood-brain relationships of other classes of substances, and it must be agreed that something like this older concept still seems to be valid for many metabolic materials as well as in cases like that of inulin mentioned above. The best methods of investigation of the blood-brain barrier involve direct measurements of the rates of entry of substances from blood to brain. In the developing brhin these methods can perhaps be supplemented by observations on the rate of accumulation of brain constituents during the normal growth period of the organ. With certain reservations these rates of accumulation must represent their rates of arrival (or those of some of their precursors) within the brain from the blood. In most species the brain undergoes a period of maximum growth (measured in increments of weight) earlier than the corresponding period for the rest of the body. During this “growth spurt” it is to be presumed that the rate of entry of many substances must be greater than at any other time. The brain “growth spurts’’ of several species are portrayed in Fig. 1 which illustrates two points: that there are important species

\ Human---Months Rat ---.Days ’ Days -ma PI9 d-Weeks Dog 1’ I- Weeks Pig

-35 -30 -25 -20 -15 -10 - 5 Age

4

Birth

5

10

15

20 25

30

Age

Fig. 1 . The timing of brain growth in different species in relation to birth (Davison and Dobbing 1966). Curves of the rate of brain growth in different species are expressed as weight increments (percentage of adult wet weight of brain) per unit period of time. Brain-weight data are taken from the following sources: man (Spector, 1956); rat (Dobbing, unpublished); dog (Himwich and Petersen, 1959); pig (Dickerson and Dobbing, 1967); guinea pig (Dobbing, 1968). The time scale in each case has been adjusted according to the life span of the species, on the quite arbitrary assumption that brain development may be related to life span (Donaldson, 191 1).

differences in the timing of the brain “growth spurt” in relation to birth; and that the periods of maximum rate of accumulation of brain substance is preceded as well as succeeded by a time of slow, or even negligible rate of accumulation. Because of the species differences it is evident that to speak of the neonatal brain, or even of the foetal brain without a knowledge of the particular species intended, can be misleading.

THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

419

The brain of the guinea pig foetus three-quarters of the way through gestation is of a similar state of maturity to that of a rat towards the end of its second postnatal weeks, and can presumably be expected to have a similar metabolic performance or “bloodbrain barrier”. Is it possible that our impression of a more permeable blood-brain barrier to metabolic substances in early life, may simply be a reflection of the enhanced activity associated with the “growth spurt”? If so, it should be possible, by selecting the correct timing for a given species to demonstrate the “pre-growth-spurt” period of slow entry into the brain (as well as the “growth spurt” itself and its subsequent decline) by direct experiment. We would still be left with the almost philosophical question of whether changing “blood-brain barrier” permeability dictates the changing growth rate, or whether the rate of entry is merely a reflection of the characteristics of brain growth; but at least we should be faced with a developing “blood-brain barrier” whose characteristics were not simply those of gradually increasing permeability with age.

-Wejght --DNA-P

_-.. Cholesterol

L.:..,> -10

. ,. . -5

.

:. t

‘Irth

*

L IL .

.

5

.

10 Age weeks,

.

.

.

.

.

15

.

.

I

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2021

Fig. 2. Rate curves of the increase in fresh weight and amounts of cholesterol and DNA-P in the whole brain of the developing pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature value. (Dickerson and Dobbing, 1967).

Fig. 2 represents the rate of accumulation of DNA-P and cholesterol in the brain of the developing pig. The velocity curve of increasing weight is occupied first by a period of rapidly increasing numbers of cells, followed by a period of lipid deposition. These broadly represent the two phases of myelination: a phase of oligodendroglial proliferation followed by the manufacture of lipid-containing myelin sheaths by the established oligodendroglia. It is known that both these substances are mainly concerned with brain structures of very slow (if any) turnover (Davison and Dobbing, 1961). Is i t valid to equate the rate of access of a brain constituent or its precursors to the growing brain with the observed rate of accumulation of the product? Provided that the constituent has a sufficiently slow turnover once incorporated into the brain, it is probably reasonable to assume that the two observations are related. That is to say if a brain constituent, on incorporation into the developing brain structure, is subsequently metabolically inert, or nearly so, then its rate of accumulation is likely to References p. 424-425

420

J. D O B B I N G

resemble its rate of entry. In practice most brain constituents are synthesized in situ, and therefore it is the rate of access of their precursors which is likely to be represented by the velocity curve of accumulation of inert product. In order to demonstrate this association directly, the entry rate of labelled cholesterol into developing rat brain has been compared with the velocity curve of its normal accumulation during growth (Dobbing, 1963b). Although most brain cholesterol is synthesized within the developing tissue, a measurable proportion is derived as the preformed molecule from the blood and this can be demonstrated by injecting [4-“C]cholesterol intraperitoneally and finding some of it unchanged in the brain. Fig. 3 showed the result of such an experiment, from which it can be seen that the relative specific activity a short time after injection into rats of different ages closely follows the velocity curve of its accumulation. In other words the peak rate of entry is preceded as well as succeeded by conditions in which the rate of entry is slow. It may well be asked which of the two factors normal growth or “blood-brain barrier” - controls or limits the other?

Body weight (g)

Fig. 3. Pattern of entry of [4-14C]cholesterol into rat brain at different body weights compared with a relative specific activity of brain cholesterol rate curve of brain cholesterol accumulation. 0-0 1 day after injection. 0-0 increments in whole brain cholesterol for each 5 g gain in body weight. (Dobbing, 1963b).

Fig. 4 shows velocity curves for DNA-P, weight and cholesterol in rats, and it can again be seen how the one for DNA-P is declining rapidly at a time when that for cholesterol is rising fast. Any “developing blood-brain barrier” hypothesis must therefore include a different timing for each substance as well as the “stop-go-stop” characteristics outlined earlier. Fig. 5 shows a similar pattern of events in the developing guinea pig brain. The use of 32P as a label for investigating the “blood-brain barrier” has many critics on account of the variety of metabolic events which must influence its rate of entry. A great deal of the phosphate entering the brain is concerned, particularly during myelination with phospholipid metabolism, and a peak period of incorporation has often been demonstrated, particularly into myelin lipids at the time of

THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

42 1

14 -

DNA- P -- Brain weight ---Cholesterol

,

----- - - _ _ _ _ _ _ _ _ _ _ _ \

-5

5 'Irth

10 Days

15

20* 25 Weaning

30

.

,

35

.

40

.

45

I

50

Days

Fig. 4. Rate curves of the increase in fresh weight and amounts of cholesterol and DNA-P in the whole brain of the developing rat, plotted as increments per 2 day interval. All values calculated as a percentage of the mature value. -DNA-P; -.-.-.-. brain weight; -------- cholesterol. (Dobbing unpublished).

Fig. 5. Rate curves of the increase in fresh weight and amounts of cholesterol and DNA-P in the whole brain of the developing guinea pig plotted as increments per one day interval. All values calculated as a percentage of the mature value. -brain weight; whole brain cholesterol; DNA-P. (Dobbing, 1968).

____--_-

References p . 424425

-.-.-.-.

422

J. D O B B I N G

Days

Birth

Fig. 6. Rate curve of the increase. in the amount of cholesterol in the whole brain of the developing rabbit, plotted as increments per 5 day interval. All values calculated as a percentage of the mature value. (calculated from Davison and Wajda, 1959).

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Age (weeks)

Fig 7. Rate curve of the growth of the whole brain, spinal cord and cerebellum in the pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature weight. ------whole brain; -cord; cerebellum. (Dickerson and Dobbing, 1967).

-.-.-.-.

myelination (McMurray, 1964). Most of the evidence has naturally come from in vitro experiments, or following intracerebral injection, techniques which are not applicable to the direct investigation of the “blood-brain barrier” in its usual sense. Recently it has been claimed on the basis of in vivo experiments, that the newborn rabbit brain admits 32P no more readily than the adult, when proper regard is had to different conditions such as vascularity (Grontoft, 1965). Fig. 6 shows the rate curve for the accumulation of cholesterol into rabbit brain of different ages. This closely follows the entry characteristics of the other lipids, particularly the myelin lipids, and the same features of a transient period of increased entry can be seen. Is it possible, in selecting the two ages (newborn and adult), that this intermediate period has been missed?

T H E DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

“it

423

s4

90

-J

2s

~

Fig. 8. Rate curves of the increase in amounts of DNA-P in the forebrain, cerebellum and spinal cord of the developing pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature value. ___ cerebellum; .. .. . .. forebrain; ------ cord (Dickerson and Dobbing, 1967).

.

Fig. 9. Rate curves of the increase in amounts of cholesterol in the forebrain, cerebellum and spinal cord of the developing pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature value. ___ cerebellum; ...... forebrain; ------ cord (Dickerson and Dobbing, 1967).

.

.

One of the findings in this very careful study by Grontoft was that the newborn cerebellum took up 32P more readily than the adult. The reverse was true of the cerebrum, and in the brain stem uptake was equal at both ages. It is, of course, well known that there are regional differences in the metabolic behaviour of the brain. In the same way different regions develop at different relative rates, and the cerebellum develops relatively earlier and more quickly than other parts (Dickerson and Dobbing, 1966). As far as is known no figures are available for the rabbit, but in the human and in the Re-fcrences p. 424-425

424

J. D O B B I N G

pig, it is documented and there is no reason to believe this is not a common mammalian phenomenon. Fig. 7 shows velocity curves of weight increase in cerebellum, whole brain and cord. In each case these can be divided into an earlier period of cellular proliferation (Fig. 8) and a later one of lipid deposition (Fig. 9). It would seem, therefore, that the findings reported by Grontoft could well correspond with the characteristics of normal regional growth. It has often been suggested (Dobbing, 1961, 1963a) that the “blood-brain barrier” for metabolic substances can be accounted for on the basis of known metabolic behaviour. In the developing brain it does indeed still seem that the so-called barrier may in large measure be simply a reflection of some of the characteristics of brain growth. ACKNOWLEDGEMENTS

I wish to thank the Multiple Sclerosis Society (of Great Britain and Northern Ireland) and the Pig Industry Development Authority for supporting this work.

REFERENCES BAKAY,L. (1953) Studies on blood-brain barrier with radioactive phosphorus. 111. Embryonic development of the barrier. Amer. Med. Ass. Arch. Neurol. Psychiat., 70, 30-39. BEHNSEN, G. (1927) Zellforsch., 4, 515. DAVISON, A. N. AND DOBEING, J. (1961) Metabolic stability of body constituents. Nature, 191, 844848.

-, (1966) Myelination as a vulnerable period in brain development. Brit. Med. Bull., 22, 40-44. DAVISON,A. N. AND WAJDA,M. (1959) Metabolism of myelin lipids: Estimation and separation of brain lipids in the developing rabbit. J. Neurochem., 4, 353-359. DIAMOND, I. AND SCHMID,R. (1966) Experimental bilirubin encephalopathy. The mode of entry of bilirubin - 14C into the cextral nervous system. J . Clin. Invest., 45, 678-689. DICKERSON, J. W. T. AND DOBEING, J. (1966) Peculiarities of Cerebellar Growth. Proc. Roy. SOC. Med., 59, 1088. -, (1967) Prenatal and postnatal growth and development of the central nervous system of the pig. Proc. Roy. Ser. B, 166: 384-395. DOEBING, J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. -, (1963a) The blo3d-brain barrier: some recent developments. Guy’s Hosp. Repts., 112, 267-286. -, (1963b) The entry of cholesterol into rat brain during development. J. Neurochem., 10, 739-742. -, (1968) Prenatal and postnatal growth and development of the central nervous system of the guinea pig (in preparation). DONALDSON, H. H. (1908) A comparison of the albino rat with man in respect to the growth of the brain and of the spinal cord. J. comp. Neurol. Psycho/., 18, 345-389. FRIES, B. A., CHANGUS, G. W. AND CHAIKOFF, I. L. (1940) The influence of age o n the phospholipid metabolism of various parts of the central nervous system of the rat. J. Biol. Chem., 132, 23. GRONTOFT, 0. (1965) The permeability to P32 in different regions of the brain of new-born and adult rabbits. Acta pathol. microbiol. Scand., 63, 481-492. HIMWICH, W. A. AND PETERSEN. J. C. (1959) In: Messerman, J. H. edition Biological Psychiatry, p. 2. New York. Grune and Stratton. LUCEY, J. F., HIBBARD, E., BEHRMAN, R. E., ESQUIVEL DE GALLARDO, F. 0.AND WINDLE, w.F. (1964) Kernicterus in asphyxiated newborn rhesus monkeys. Exptl. Neurol., 9,43-58. MCMURRAY, W.C. (1964) Metabolism of phosphatides in developing rat brain - 1. Incorporation of radioactive precursors. J. Neurochem., 11, 287-299,

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MILLEN,J. W. AND HESS,A. (1958)The blood-brain barrier: an experimental study with vital dyes. Brain, 81, 248-257. SPECTOR, W. S. (1956) Hatidbook of Biological Data. Philadelphia. Saunders. STERN,L. A N D PEYROT,R. (1927) Le fonctionnement de la barriere hematoenckphalique aux divers stades de development chez les diversesesptces animales. C.R. SPances SOC.Biol. Fil., 96, 1124. WOODBURY, D. M. (1967) This volume pp. 297-314.

DISCUSSION L. BAKAY:I wanted to ask you one question, and I want to make a small comment. There must be a species difference as far as the immature or infantile barrier is concerned. Previously, the ground substance, which was supposed to be a polysaccharide, was thought to fill an extensive area. Now it is delegated to be nothing more than the capillary wall. I understand that in guinea pig it is already present at birth, while in other animals it is not. This region was supposed to be an area where the barrier is already well established at the moment of birth as far as the guinea pig is concerned, while in other animals it is still undergoing “maturation.” As far as bilirubin is concerned, injecting large amounts of bilirubin in some species of animals could produce bilirubin staining of the brain (Rozdilsky and Olzewski). The same amount injected into other animals (dog, cat or rabbit), appears very different under the same experimental circumstances. The question I wanted to ask you, pertains to Barlow’s [35S]-studiesand why it is that we think that there is an increased permeability of the barrier at the time of birth which provides for the uptake of large amounts of sulphate. Is the inorganic sulphate space an extracellular space and is it larger in the newborn and does it become smaller as the animal grows older? What do you think about this? J. DOBBING: I don’t know. However, it should not be difficult to investigate the rates of entry in vivo, although determining the in vivo entry rates is more difficult than it would first appear. If results were obtained which represented an entry rate, then it might be considerably influenced by the rate of sulphatide accumulation during this period. Now to refer to the point that you made about experimental kernicterus in different species, there are very definite species differences in the accumulation of all of these things which we have measured.

T. Z. C S ~ K YWith : regard to the suggestion that unconjugated bilirubin actually saturates all the protein binding receptors, do you recall what was the technique used for this study? Should this really occur, it would be very interesting to determine if phenylbutazone will cause kernicterus. Phenylbutazone is one of those very rare substances which can saturate with great avidity all the protein receptors, and can actually displace other drugs after they have been bound to proteins. If this occurs, then a large dose of phenylbutazone could cause kern-icterus in many animals, even in adults. Phenylbutazone will also dislodge gantrisin and cause a larger extracellular distribution. J. DOBBING: I think the fact that this will occur with gantrisin and produce kernicterus in babies at a very low level of plasma bilirubin puts the etiology of kernicterus in the plasma rather more than on the age of the brain. J. FOLCH-PI:The difficulty with which we are dealing is that the developing brain is the same organ as the adult brain in name only. The rapid changes which occur in the cell population and the shift in the position and perhaps the size of the spaces means that one cannot really study steady states, except in a very limited period of time. This is further complicated by the increases in weight as cells grow and fibers become myelinated. Since the brain may increase in weight as much as 10% in one day, it is very difficult to really draw valuable comparisons unless you do a longitudinal study, a

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complete profile of the brain of one animal. Once you have that profile, which has to be completed with a full awareness of the whole anatomy and physiology and biochemistry of the brain, it is only then that you are in a position to compare between one species and the other. Also, we place too much value on a new point of view, which may permit the entrance of the uninformed into a field, which has happened many times in science. This has often been a source of completely absurd experiments. Now I think that we neurochemists are very well aware that we are finally getting data and the man who educated us was Flexner. He was a man who demonstrated a beautiful multidisciplinary approach to science which is classic. V. TENNYSON: I just want to say that as far as spaces are concerned, there is a very little more space in the embryo than we find in the adult. The only difference that I can see in the embryo in the blood vessels is that the basement membrane is not as well developed. J. FOLCH-PI:On the other hand, Cr. Tennyson, in in vivo chemical spaces, such as the sodium and chloride spaces, there are very marked and well documented differences which really illustrate how cautious one has to be in correlating or trying to make equivalents between in vivo anatomical spaces and in vivo chemical compounds. V. TENNYSON: But as one sees in the electronmicroscope there seem to benolargespacesin theembryo.

D. B. TOWER: Could I ask you to tell us in some sort of relative terms what is the difference between “somewhat bigger” and “not very much bigger”? V. TENNYSON: 1 really would hesitate to say that there is any. Once in a while one sees cell processes rounding off, and you get a more triangular space rather than a space between perfectly parallel walls of cells.

D. B. TOWER: We are not really talking about inches or feet. A few A difference may make quite a lot of difference volumetrically. 0. STEINWALL: Dr. Woodbury, a short question about inulin: Was it possible that it entered the brain by first entering the cerebrospinal fluid? D. M. WCODEURY:Our data, based on calculation of the concentrations in the two compartments, indicate that inulin en ters the brain first, then the cerebrospinal fluid next and not vice versa. A. LAJTHA: Let me mention amino acids here, although one can’t generalize from the behavior of one type of metabolite to that of another. In measuring the exchange of amino acids between plasma and brain, we found a very high rate for most amino acids, with a half-life in brain pools in minutes. This rate of exchange is probably unrelated to the rate of metabolism of the amino acids and it certainly is at least one order of magnitude larger than metabolic rates. Cholesterol may be an entirely different case, where the influx of cholesterol increases when the highest rate of deposition of this compound occurs, which may be a turning-on and off kind of transport phenomenon. An alternate explanation would be that there is a constant rate of exchange of cholesterol, but you find significant counts in the brain only when significant amounts are deposited.

DOBEING: It is of course, a problem of semantics to argue whether it is a change in the permeability of the system which is controlling growth, or that it is reflected by changes in what we have called the blood-brain barrier. I would not claim to know what controls growth and I think we ought to maintain an open mind about what is found in the literature of our colleagues on the subject.

J.

There may be a small free cholesterol pool in constant equilibrium with plasma which A. LAJTHA: may be too small to detect. This pool. however, can contribute significant labeling to the myelin during myelination. If this is the case, the label will measure not the transport but the incorporation of the molecule into myelin.

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421

J. DOBBING: We happen to have evidence with the materials we use, that the very molecules which get into the brain stay for a considerable period after they have arrived there, in some cases for several years.

P. MANDEL:Of course it has not been proven that there is not a free cholesterol pool, J. DOBBING: I am sorry, no, it has not indeed, and my data for rates of entry are very inferential

...

I agree.

P. MANDEL:I think that the experiments concerning growth are still significant if we keep in mind that any kind of cell has its own rate of uptake and synthesis. This is all that we can say, and in this way it is useful. J. FOLCH-PI:I have not said it is not useful, but I have always maintained that a single isolated point may mean very little. You need a whole thesis-point, you need a time sequence, a longitudinal study in the way the physiologists are already working in behavior. J. DOBBING: For meaningful time curves, you need a great many observations. P. MANDEL:It depends on the kind of cells with which one is working. H . M . ADAM:How many points did in fact attribute to your growth rate curves? J. DOBBING: In the case of the pigs, there were animals at 40 different ages from the last 50 or 60 days of gestation to the first two or three months of life. The animals were not equally spaced in age and tended to be further apart at the older ages. With that number, it was a relatively simple analysis with these substances, but we have done more complicated things. With the relatively simple substances used, it is possible without any very great exercise of judgement to draw these kinds of rate curves.

H. M . ADAM:Would there be any objection against using a logarithmic scale for the time sequence?

J . DOBBING: No, the original intention of the work was to test the hypothesis that at the peak period of activity this might be a particularly vulnerable period to such restricted influences as malnutrition or anoxic conditions.

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Intrinsic* Amino Acid Levels and the Blood - Brain Barrier C L A U D E F. BAXTER Neurochemistry Laboratories, Veterans Administration Hospital, Sepulveda, California; Department of Biochemistry City, of Hope Medical Center, Duarte, California; and Department of Physiology, UCLA School of Medicine, Los Angeles, California.

The instantaneous substrate level of a compound at a given site in the nervous system reflects the interaction of at least five dynamic processes: synthesis, degradation, uptake, extrusion and exchange. Since the latter three processes define the kinetic interrelationship between the blood and the brain, it is apparent that substrate levels not only are affected by but will themselves affect this interrelationship. A. I N T R I N S I C A N D E X T R I N S I C AMINO A C I D P O O L S

In recent years several laboratories have studied the effect of changes in the substrate levels of amino acids upon uptake, extrusion and exchange phenomena in nervous tissues, both in vitro and in vivo (Abadom and Scholefield, 1962; Blasberg and Lajtha, 1965; Lajtha et al., 1963; Lajtha, 1964; Levi et al., 1966; Levi et al., 1965; Nakamura and Nagayama, 1966; Neame and Smith, 1965; Tsukada et al., 1963; Wiechert, 1963). Substrate level equilibria were altered by utilizing extrinsically added amino acids. In many experiments the movement of a specific, isotopically labeled amino acid was charted. Results from these studies fail to distinguish whether an amino acid is taken up, extruded or exchanged from only one specific compartment in the tissue, or if the movement of the amino acid reflects an involvement of the total tissue pool. All available evidence shows that an extrinsically added amino acid, such as y-aminobutyric acid (GABA), even when added to a subcellular fraction of brain tissue in vitro, equilibrates within a reasonable period of time with only a part of the intra-particulate pool (Weinstein et al., 1965). In vivo studies have indicated a similar situation (Roberts et al., 1958). So-called “free” amino acids in brain tissue appear to be contained in several compartments, and labeled amino acids, injected into the brain, may become distributed rapidly in only a few of the many compartments of the total substrate pool. Berl, Lajtha and Waelsch (I96 I ) found that [U-l4C]glutamate injected intracerebrally gave rise to glutamine with a higher specific activity than that of glutamate. This result, which has been confirmed repeatedly (Berl et al., 1962; Baxter, 1963; Gaitonde, 1965; Berl and Pur-

* The term intrinsic has been used to designate “located within”. The evidence contained in this paper suggests that most oft he intrinsic amino acids in brain also must beendogenous“originating internally”. Rrfcrrnces p. 441-444

C. F. B A X T E R

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TABLE I C O M P A R T M E N T A L I Z A T I O N OF G L U T A M I C A C I D A N D G L U T A M I N E I N R A T B R A I N

Time after Compound injected

injection (niin)

[ I -14C]glutamic acid [U-14C]glutamic acid [I -14C]y-aminobutyric acid [U-14C]glucose

Activity Ctutamine Glutamic acid (cts/niin/100 mg brain tissue)

Ratio of relative specific activity*

GMEIGA*Z

40 40

6270 3840

4600 1997

2.7 3.8

40 32

3827 380

2230 1025

3.4 0.7

* Based upon a glutamine pool which is f the size of the glutamate pool.

**

GME = glutamine: GA

=

glutamic acid.

One to two microcuriesof W-labelled metabolite was injected stereotaxically into the lateral ven'ricle of fed female Sprague Dawley rats weighing 200 to 220 g. The injected dose was contained in 1OA of a neutral solution. Rats were lightly anesthetized with Nembutal (8 to 9 mg/200 g rat) prior to placement into the stereotaxic instrument. Glutamic acid and glutamine were isolated from tissue extracts by two dimensional high voltage electrophoresis. Radioactivity was determined as described by Baxter and Senoner (1964).

pura, 1966), is the same irrespective ofwhether [l-"C]- or [U-"T]glutamic acid is injected into the ventricle of the rat brain (see Table 1). The widely accepted interpretation of these results holds that the 14C-labeled glutamic acid did not become distributed evenly in all of theglutamic acid pool of brain tissue, but only in a specific compartment which happens to be in rapid equilibrium with the major portion of the glutamine pool. Thus, there must be at least two glutamate compartments in brain. In more recent experiments [I-WIGABA was injected intracerebrally and again it was found that, within minutes, the glutamine had acquired a higher specific activity than the glutamic acid. The relationship, 40 minutes after the injection, is recorded in Table 1. Since GABA cannot be carboxylated to glutamic acid, all 14Clabeling from GABA must have passed through the intermediates of the tricarboxylic acid cycle before reaching glutamate or glutamine. The results suggest therefore that all of the tricarboxylic acid cycle intermediates derived from [1-l4C]GABA were in metabolic pools which did not equilibrate with a major compartment of the glutamic acid pool, within the time period of the experiment. A similar conclusion can be drawn from the experiments of Roberts and Morelos (1965), who found that glutamine acquired a higher specific activity than glutamic acid, after [U-"C]leucine had been injected intravenously into rats. Even the intracerebral administration of [I-"C]- or [2-W]-acetateto mice resulted in a higher specific activity in glutamine (Van den Berg et al., 1966). By contrast, the injection of 1%-labeled glucose has consistently led to a higher specific activity in glutamate than in glutamine (see ratio GME/GA Table I), a result which is in agreement with those published by Cremer (1964); Gaitonde et al. (1965); Van den Berg et al. (1966); and by Lindsay and Bachelard (1966). Presumably,

I N T R I N S I C A M I N O A C I D LEVELS

43 1

these experiments show that [“W]glucose can label a glutamate pool in brain tissue which was not labeled by the direct intracerebral injection of [14C]glutamate, [“TIGABA, or [14C]acetate. It is not within the scope of this paper to speculate about the interpretation of these results. The data are presented only to illustrate that amino acids added extrinsically to a biological system are not necessarily equivalent kinetically to an elevation of intrinsic levels of the same amino acids. In the past, the effects of changed amino acid levels upon their uptake, extrusion or exchange across brain barriers have been investigated almost exclusively through the addition of extrinsic amino acids to biological systems. It seemed desirable, therefore, to study these phenomena also with a system in which intrinsic levels of amino acids were changed. Intrinsic levels of “free” amino acids in brain tissue are extremely stable under a variety of physiological conditions (Roberts and Simonsen, 1962). We know of only some acute vitamin deficiency studies and one nutritional study in which a few select amino acid levels were altered by a synthetic diet (Roberts, 1963). No normal physiological conditions have been described which have altered, dramatically, overall amino acid levels in the central nervous system. The object of this paper is to describe a vertebrate system in which intrinsic levels of amino acids of brain tissue are changed drastically in response to a normal physiological stress and to explore in a preliminary way the possible involvement of brain barriers in these changes. B. ENVIRONMENTAL EFFECTS ON AMINO ACID LEVELS IN EEL AND TOAD BRAINS

Aquatic species found in or near an euryhaline environment can be divided into two groups : osmoregulators and osmoconformers. Whereas the former group can maintain a sizable osmotic gradient between the external environment and the internal tissue environment, the latter group adapts to brackish or ocean water by establishing an iso-osmotic equilibrium between external salt concentrations and the body tissues. Many marine invertebrates are osmoconformers and it is well established that amino acids play a role in their internal osmotic regulation (Florkin and Schoffeniels, 1965). Similar changes in free amino acids which parallel alterations in environmental salinity have been described in muscle tissue and plasma of some amphibians (Gordon, 1965; Tercafs and Schoffeniels, 1962), and in teleosts such as the flounder and the three-spined stickleback (Lange and Fugelli, 1965). The regulation of intracellular amino acid concentrations appears to be the only mechanism employed by the sipunculid marine worm (Golfingia gouldii) to counteract a decrease in external salinity (Virkar, 1966). Changes in free amino acids in the central nervous system in response to osmotic stress have not been described previously. Studies were conducted with two species comparing GABA levels in brain tissues of individuals found in fresh water with those adapted to an euryhaline environment.The first study used a teleost, the silver eel (Anguilla anguilla) which during its life cycle migrates from ocean water to fresh RrJivenres p. 441444

432

C. F. B A X T E R

water and back to ocean water. Fresh water eels were collected in the Zuider Zee in the Netherlands and salt water eels in the Atlantic Ocean off the Netherlands coast. For the second study, the Western toad (Bufo boreas) was chosen because it is a dweller in mud caves near fresh and brackish water. These toads were adapted in the laboratory to a brackish environment. GABA was chosen as a representative amino acid for the central nervous system since, in vertebrates, it is found almost exclusively in this organ system. TABLE I1 Y - A M I N O B U T Y R I C A C I D I N B R A I N T I S S U E S O F S I L V E R E E L (AnEuilla anguilla) A N D W E S T E R N T O A D (Bufo boreas) I N F R E S H A N D O C E A N W A T E R E N V I R O N M E N T

Environment

Fresh water Ocean water

Change in GABA level

Silver Eel* GABA (pnoleslg)

Westerit Toad* * GABA (pnoleslg)

3.8 ( & 0.7)8 4.2 (rt 0.5)b 3.3 (f0.8)c 4.5 (f0.6)d

25.8 (3 0.9)

N.S.

-I-32 %

33.8 ( * 1.0)

* Eels caught in fresh water were predominantly females, those from ocean water were predominantly males. Each group value represents a n average of 6 t o 12 eel brains. Standard deviations are shown in parenthesis. Eels were designated as: (a) immature, (b) mature, (c) 39 to 41 cm long, and (d) larger than 50 cm in length. ** Western toads ranged in weight from 25 to 108 g, with an average body weight in each group of 44g. Each group consisted of 24 toads. Four brains were pooled for every GABA determination. Ocean water toads were adapted for 2 days to a n environment containing 20% ocean water and 2 days t o 40% ocean water. Toads were kept in aquaria inclined at a n angle so that f the floor area was covered by aqueous phase while the other half remained dry. Results in Table I1 show that in the eel, which is an osmoregulator, no statistically significant difference in levels of GABA in the brain of ocean water and fresh water specimens could be detected. This finding is in agreement with the observations by Boucher-Firly (1935), that in the silver eel the osmolality of body fluids increased by less than 15 % when these fish migrated from fresh to ocean water. Large female eels in fresh water or salt water showed no significant differences at all in the osmolality of their body fluids. Under similar conditions, only small changes in Naf concentration of eel serum have been reported by Sharrett, Jones and Bellamy (1964), whose investigations essentially confirm the earlier osmolality measurements. By contrast, the toad (Bufo boreas) is an osmoadapter and in the laboratory adjusts readily to an environment containing up to 50 % ocean water and 50 % fresh water. A significant increase in levels of GABA in brain tissue was found when these amphibians were placed for two days in a mixture consisting of 20 % ocean water and 80 % fresh water and two days in 40 % ocean water and 60 % fresh water. The results, shown in Table 11, were the same for males, females and hermaphrodites (for details see Baxter and Ortiz, 1966). Amino acid levels in the brains of toads from any single

433

INTRINSIC AMINO ACID LEVELS

shipment were quite uniform, as was the biochemical response to ocean water adaptation. There appeared to be, however, seasonal variations, both in normal levels of amino acids and i n the amount of change induced by the brackish water environment. Thus, levels of GABA in osmotically stressed toads were elevated in some experiments by only 30 while in others the increase was as high as 80 %. Seasonal variations in metabolism and metabolites of amphibians have been observed by other investigators (Mizell, 1965).

x,

T A B L E I11 MAJOR C O M P O S I T I O N A L C H A N G E S I N N I T R O G E N O U S C O N S T I T U E N T S O F T O A D B R A I N A S A RESULT O F A D A P T A T I O N T O BRACKISH WATER

(From Baxter and Ortiz, 1966) Atiiino Acid

Aspartic acid Alanine Glycine Glutamic acid y-Aniinobutyric acid Glutarnine $- asparagine Urea Ethanolamine

Fresh water 40u/, Ocean water (pnioleslg tissue, wet weight)

0.94 0.37 0.87 5.5

2.7 3.5 15.8 1.7

2.4 0.87 1.6 9.1 4.0 4.8

31.8 0.8

Change ( %I

4-149 +135 84 65 48 37 139 - 55

+ + + + +

Alcoholic tissue extracts were prepared and processed as previously described (Baxter, 1961). Ninhydrin positive compounds were measured using a Technicon amino acid analyzer system connected to a Gilford Model 2000 spectrophotometer and recorder. Results obtained by column separation were verified for glutarnic acid, glycine, y-aminobutyric acid and urea wing established enzymatic assay techniques (Graham and Aprison, 1966; Aprison and Werman, 1965; Baxter, 1961; Bernt and Bergrneyer, 1963).

In Table I l l are shown the changes of some amino acids found in brain tissue which were observed when the toads were adapted to 40 % ocean water. Although aspartic acid showed the greatest increase on a percentage basis, the change in glutamic acid level was quantitatively the most significant. On a molar basis, increases in aspartic acid and GABA were the same. The increase in urea concentration by 22 pmoles per gram of brain tissue (wet weight) was the largest change found for any nitrogenous compound in brackish water toads. This change was reflected also in muscle and plasma and doubtlessly helped to maintain osmotic equilibrium. The very high levels of urea in the tissues of Bufo boreas are noteworthy. In a fresh water environment, urea levels in the brain of this species were higher than those reported for any other vertebrate known to us and three times higher than the levels in frog brain as measured by Buniatian and Davitian (1966). These results fit the correlation which Schoffeniels* has made between the ability of animals to adapt to an euryhaline environment and the level of urea in their tissues.

*

Schofleniels, E., personal communication.

Rcfcrmws p. 4 4 1-444

433

C. F. BAXTER

It is apparent from the results in Table 111 that osmotic stress primarily increased the concentration of those amino acids in toad brain which are closely related metabolically to the tricarboxylic acid cycle. Only minor elevations in the concentration of threonine, valine, tyrosine, phosphoethanolamine and phosphoserine were observed. No significant changes in levels of creatine, taurine and ammonia were detected. Of the other amino acids found in brain extracts, leucine increased and lysine decreased in concentration, but the molar changes were extremely small (see Table VII for leucine). It is probable that minor elevations in amino acid concentrations, expressed as a function of wet tissue weight, reflect only the dehydration of brain tissue in osmotically stressed toads. It was found that under our experimental conditions the water content of toad brain, usually about 85 % of the wet weight, decreased by a maximum of 1.8 % corresponding to a maximum increase in dry weight of 12 % over control values. Therefore it is clear that the major changes shown in Table 111 are not the result of dehydration. C. MECHANISMS O F A M I N O A C I D A C C U M U L A T I O N I N T O A D B R A I N

There is some evidence to suggest that the elevated levels of urea and amino acids in muscle tissue of euryhaline adapted amphibians can be accounted for, in part, by an enhanced degradation of larger molecules (Tercafs and Schoffeniels, 1962). A decrease in the rate at which amino acids are metabolized has also been postulated (Florkin et al., 1964). However, the detailed mechanisms by which intracellular amino acid levels are altered in response to a brackish environment are not clearly understood. Studies by Schoffeniels and Gilles (1963), on glutamic dehydrogenase in aquatic invertebrates suggest activation of this enzyme system by cationic constituents whereas Chaplin et al. (1965) favor a mechanism involving anions. Activation of other

Fig. I . Toad brain amino acids. All salt solutions and sucrose were prepared to represent an osmotic concentration which was equivalent to a brackish water mixture containing 40% ocean water and 60% fresh water. These solutions were 410 10 mOs. Toads were adapted to salt solutions and sucrose for 2 days at f strength and 2 days at full strength in inclined aquaria as described in Legend for Table 11. Control animals were maintained in salt free water.

435

INTRINSIC AMINO A C I D LEVELS

enzyme systems related to amino acid and energy metabolism by ionic constituents is well documented and could play a role in modifying amino acid levels in the central nervous sytem of Bilfo boreas. When toads were adapted to environments containing specific salt solutions, equivalent in osmolality to 40 % ocean water, changes in amino acids of brain tissue were observed. Results for glutamic acid and GABA shown in Fig. 1 confirm preliminary data (Baxter, 1966). Solutions of sodiumsalts proved to be the most effective environment for elevating glutamic acid and GABA levels in brain. They exceeded in effectiveness ocean water of equivalent osmolality without causing any additional water loss from the brain tissue. Potassium chloride had no effect on levels of amino acids in brain tissue, despite the fact that it was the most toxic environment used. Presumably these effe,nts of sucrose, brackish water and sodium salt solutions are mediated through ionic changes in the internal environment of the toad. The plasma i n fresh water (control) toads ranged from 235 to 265 milliosmolal, but increased by 60 to 70 percent when these amphibians were adapted to brackish water or equivalent sodium salt solutions (Table V). Invariably the elevated levels of amino acids in brain tissue of these toads were accompanied by raised levels of Na+ both in brain tissues and blood plasma. Preliminary data indicate, that the Na+/K+ ratio in brain tissue was also increased (Table IV). T A B L E 1V SODIUM A N D POTASSIUM ION C O N C E N T R A T I O N IN T O A D B R A I N E X T R A C T S -.

Brairi coriiposiriori ~~

B i virorinicnf

Water ( 1/,)

.

Naf

Kf

Nal K

(rwquiv.lkg)

(niequiv./kg)

ratio

85 98 114 107 85

0.69 0.81 0.86 0.67 0.82

Fresh Water

83.6

40 :‘c Ocean Waler

82.0

59 80

NaCl KCI Sucrose

81.6 83.1 81.9

72 70

98

~~~

Sodium and potassium were determined by flame photometry. For details of salt concentrations and environmental conditions, see Legends of Table I 1 and Fig. I .

Toads in the potassium chloride and sucrose solutions lost weight rapidly because of dehydration. However, dehydration of brain tissue in these animals was no greater than that observed in toads living in a brackish or salt water environment. All of these results indicate a well-developed mechanism i n toad brain by which solute concentrations are regulated so as to minimize hydration changes in the cerebral tissues. The effect of a saline environment upon plasma osmolality, Na+ plus K+ concentration and the molarity of glutamic acid and urea, is shown in Table V. Our results for changes in cationic constituents of plasma in brackish water toads are in overall agreement with those reported by Gordon (1965). Heferencrs p, 441-444

436

C. F. BAXTER

TABLE V EFFECT O F E X T E R N A L S A L I N E E N V I R O N M E N T U P O N SOME T O A D P L A S M C O N S T I T U E N T S

Eiivirotinient

Fresh water 40;( Ocean water NaCl Solution (420 mOs)

Na+ plirs K+ (iiieyuiv.lliter) *

Clutaiiiic Acid

(niOs/kg)

256 422

127 173

0.04 0.20

19.8 42.1

435

184

0.21

48.9

Osinolality

Urea

(liiiioles/iiil plasma)

* The K t concentration of plasma was extremely low and the increase observed was due primarily to Na+. In order to eliminate the addition of Na+ with the anticoagulant, ammonium citrate or citric acid was added to those aliquots of blood in whichNa+and K ' xere determined by flame photometry. Plasma for amino acid and urea determinations was obtained from heparinized blood. All results are based upon pooled samples. Measurements were made on blood obtained by heart puncture as described in text. Red cells were removed by centrifugal precipitation in cold, within 30 min after collection of sample. Levels of urea and amino acids were elevated not only in brain tissue, but also in the plasma of toads in a sodium salt environment. Since amino acid levels in toad plasma are normally very low, the proportionally large changes observed after osmotic stress represent only an insignificant fraction of the total osmolal effect in the plasma. In the case of urea, the degree of elevation is similar in both brain and plasma and represents, on a molar basis, the largest change of all of the nitrogenous substances. Although many of the enzymes of the urea cycle have been found in brain tissues (see Kemp and Woodbury, 1965 for review), one of the essential ones, carbamyl phosphate synthetase, appears to be present only at rather low levels (Jones et al., 1955). In the liver, on the other hand, carbamyl phosphate synthetase is quite plentiful. Recent experiments have shown that the activity of this enzyme in the liver of the dogfish can be considerably increased by placing this species into dilute ocean water (Watts and Watts, 1966). Since the blood-brain barrier does not exclude the net entry of urea from the blood (Kleeman et al., 1962), all of the above evidence suggests that a major portion of the urea found in brain tissue may have originated in other organs and was carried into the brain via the plasma. D . B L O O D - B R A I N B A R R I E R A N D I N T R I N S I C A M I N O A C I D LEVELS

The foregoing results have shown that the levels of nitrogenous solutes in brain and plasma of the toad (Bufo boreas) are elevated when this species is placed in an aqueous environment containing a sodium salt concentration which is equivalent in osmolality to that of 40 % ocean water. Changes in Na+ concentration and gradients between blood and brain also have been observed. The origin of the amino acids which accumulate in the brain tissue of osmotically stressed toads is a matter for conjecture. Since there is restricted net transport of most amino acids from blood into the adult brain of vertebrates, both mammalian and non mammalian (Wiechert and Knaape, 1963; Lajtha et al., 1963; Guroff and Udenfriend,

INTRINSIC AMINO A C I D L E V E L S

43 7

1962; Kuttner et al., 1961 ; Neame, 1961 ; Dingman and Sporn, 1959; Lajtha et a]., 1957; Schwerin et a / . , 1950), the most likely sources for many of the amino acids in brain are the metabolic cycles within the brain tissue itself. Vidaver (1964a, 1964b) has shown that the net direction of a mediated transport for glycine in the pigeon erythrocyte depends upon the direction of the Na+ gradient between the exterior and interior of the cell. Experiments by Christensen's group and by others (Wheeler e t a / . , 1965; Finerman and Rosenberg, 1966; Fox et a / . , 1964) confirm that the involvement of Na+ in transport mechanisms for amino acids is a widespread phenomenon. In a situation of osmotic stress with altered Na+ gradients, it might be possible, therefore, that some amino acids would transfer more readily between plasma and brain tissue. Under the same conditions it is equally possible that the blood-brain barrier is altered as a function of changed intrinsic amino acid levels. The experimental results recorded below represent a preliminary effort to test the feasibility of the latter two possibilities. Evidence for a change in the blood-brain barrier was sought by measuring the uptake of 1Clabeled amino acids from plasma into the brain of toads kept in a fresh water environment and toads adapted to an aqueous environment containing a salt concentration of 420 milliosmolal NaCI. Toads were adaoted to their environment as previously described (Baxter and Ortiz, 1966).

Fig. 2. The heart was exposed by making a longitudinal incision through the skin and pectoral girdle with a pair of scissors. The incision was made along the mid-ventral line extending approximately from 1.5 cm below to 1.5 crn ahove the xiphisternurn. A segment of the xiphisternum was removed and a small hole cut into the pcricardial sack. By applying slight pressure to the abdomen of the toad the heart was pushed through the opening. Isotope injections were made with a size k'27 needleand blood was withdrawn using a size ,# 18 needle and a heparinized syringe. References p . 4 4 1 4 4 4

438

C.

F. B A X T E R

Intravenous injections or blood sampling proved to be a problem. However, by externalizing the toad's heart (Fig. 2) it was possible to make all injections into, and to withdraw blood from, the ventricles of this organ. Animals with externalized hearts appeared normal, hopped around, ate and survived for many days without loss of weight. Death occurred only when the exposed pericardium became infected. In preliminary experiments, plasma space in brain was measured in toads both from fresh water and saline water environments. Two microcuries of 125I-labeIed albumin were injected and after 5 min blood was collected and the toads sacrificed. Brains were excised rapidly, blotted lightly and weighed. Both 0.1 ml plasma aliquots and brain were counted directly in a well counter. [125I]albumin is retained for considerable periods of time in the circulation without penetrating appreciably into tissue spaces (Von Lombard0 and Tamburino, 1963; Love, O'Meallie, Lemmon and Burch, 1962). Assuming that five minutes is sufficient time to permit complete mixing of injected albumin with the total plasma pool of toads at 21"C, the ratio of specific TABLE V l C H A N G E S I N [L"I]ALBUMIN

Brain

Environment

Fresh water control NaCl Solution (410 mOs)

(crslmitilg)

SPACE OF TOAD BRAIN

Pla.srna

(ct.s/niiti/ni/)

x 10-1

x 10-1

445 913

46 650 51 500

Plarrna space (ndlg) 0.0095 0.01 77

For details of environmental conditions, see Legends of Table I1 and Figure I

activity in brain tissue versus plasma is proportional to the plasma space in the brain tissue. Results are shown in Table VI. Plasma space, expressed as ml plasma/g of brain tissue, is calculated from the ratio: counts/min/g of brain tissue counts/min/ml blood plasma The plasma space of brain almost doubled in toads adapted to a saline environment. Corrections for differences in plasma space were made in TablesVII and VIII. I t can be seen in Table VII that the correction factors did not significantly influence the results in which [I-"W]I-leucine uptake by the toad brain was measured in vivo. The distribution of [ I-l4C]leucine between plasma and brain tissue was determined 30 and 60 minutes after the isotope was injected into the blood stream. The results show that the brain/plasma ratio of isotope concentration in toads adapted to the NaCl environment was twice as great as that found in controls. Expressed as a function of plasma concentration, the level of [ I-14C]leucineretained in brain tissue was doubled by osmotic stress. At the same time, however, the total leucine pool was more than doubled. It seems probable that both of these phenomena involve intracellular as well as extracellular spaces in the brain tissue.

+ z

TABLE VII

%

c1

2 P

UPTAKE OF

[I-“c]/-LEUCINE

9

2

9

2

Group

1

2 3 4

Owironnient

H2O Control NaCl Solution HzO Control NaCl Solution

Time after 1-1~CCJ’leuririe itijectiori ( m i t i ) .r

30 30 60 60

BY TOAD BRAIN

Brain Leircitie

(.rcl,,oles/gl 0.067 0.137 0.067 0.137

Plasma

Toral cis Plast,ia space Net crs (cts/niiri/g wet 10) ,: 10-2 734 1301 532 1323

30 45 18

40

704 1256 514 1283

10-2

Ruiio brain/ plasma

3171 2525 1877 2236

0.22 0.50 0.27 0.57

(ct”t’lit’/‘’il) *\

NaCl adapted toads were kept in an aqueous environment containing salt at the level of 400 mOs/liter. Each group represents four or more toads. 0.75 pc of [l-14C]leucine,together with 0.015 pnoles of I-leucine carrier, was injected in a 0.1 ml neutral soli*tiondirectly into the left ventriclc of control and saline adapted toads. The dose injected into each toad was the same, but as the weight of individual toads differed the counts/min/ml of plasma also differed considerably. Ratios of counts brainiplasma were, however, extremely consistent for each group. Results above are the average for each group.

P

w

\o

440

C. F. B A X T E R

Lajtha and Mela (1961) have shown that exchange rates of isotopically labeled lysine between plasma and brain of the rat are increased if extrinsic [12C]lysine is injected subarachnoidally into cerebral tissues. Thus, the results in Table VII could be interpreted as showing that an increase in the size of the intrinsic leucine pool in toad brain affected ["Tlleucine uptake and exchange from the plasma in a manner analogous to the one described by Lajtha and Mela (1961) with extrinsic lysine in the rat. As long as the source of elevated intrinsic leucine in toad brain remains unknown, it is possible only to conclude that under conditions of osmotic stress a new equilibrium ratio between the pools of leucine in plasma and brain is obtained. In the case of a-aminoisobutyric acid (Table VIII) no known pool of this amino acid exists in brain or plasma. It has been used extensively as a model amino acid to study uptake phenomena in various systems (Christensen and Riggs, 1956; Akedo and Christensen, 1962). When [ I-"T]a-aminoisobutyric acid was injected into the circulation of toads at room temperature (21"C), the rate of uptake by brain tissue was extremely slow. As in the case of [I-"C]leucine, the brains of osmotically stressed toads accumulated [ 1 -14C]a-aminoisobutyric acid more rapidly than the brains of controls. TABLE VIII U P T A K E O F [I-'4C]U-AMtNOtSOBUTYRtC A C I D B Y T O A D B R A I N

Group

Environment

Time after injection (min)

Brain (cts/nrin/g*)

Plasnia (cts/min/tnl)

Ratio brain/ p,asr,la

965

21 130

0.046

1 2

HzO control NaCI** solution

60 60

1563

12 900

0.121

3 4

HzO control NaCI** solution

90

1058

20 880

0.05I

90

1479

10 760

0.139

* corrected for "Plasma Space" in brain. ** 400 mOs/l NaCl for 2 days.

Approximately 0.5 pc and 3.1 pmoles of carrier u-aminoisobutyric acid were injected as a dose of0.2 rnl directly into the heart of each control and saline adapted toad. Each group represents4or more oads. Results above represent averages.

The brain/plasma ratio of isotope concentration was almost three times greater. These data, too, must be interpreted with caution. The transport of a-aminoisobutyric acid in and out of the brain tissues appears to be mediated through carrier mechanisms which are used also for the transport of naturally occurring, neutral amino acids such as alanine, glycine and serine. Competition for the carrier mechanisms may affect both rates of entry and exit in brain slices in vitro (Levi, Blasberg and Lajtha, 1966; Blasberg and Lajtha, 1965; Blasberg and Lajtha, 1966). As can be seen in Table 111, the pool of both alanine and glycine in brain approximately doubled when toads were adapted to a saline environment. It is possible, therefore, that the effect recorded

INTRINSIC A M I N O A C I D LEVELS

44 1

in Table VlII does not reflect an alteration in the blood-brain barrier but is once again an expression of the increased amino acid pool in the brains of osmotically stressed toads. SUMMARY

The influence of intrinsic amino acid levels in brain tissue upon amino acid movements from blood to brain has been studied. 1 . Isotopically labeled amino acids, administered to rats intracerebrally, had ready access to only a portion of the amino acid pools of brain tissues. Thus, there is a kinetic difference in brain tissues, between intrinsic amino acids and those applied extrinsically. 2 . The intrinsic levels of many amino acids and of urea in brain tissue of the toad (Bufo boreas) were elevated dramatically when these animals were exposed to an environment containing 40 percent ocean water or osmotically equivalent salt solutions containing Naf. Levels of aspartic acid, alanine and urea were raised by more than 100 percent and those ofglutamic acid, glycine and GABA by 49 percent or more. Osmotically equivalent solutions of sucrose had a lesser effect and of K+ had no effect. 3. All elevations in amino acid levels of brain were correlated with increased levels of Na+ in blood plasma and an elevated Na+/K+ ratio in brain tissue extracts. 4. There is limited evidence to suggest that the unusually high levels of urea in toad brain originate, in part, outside of the brain. 5 . The relationship of blood amino acids to brain aniino acids was altered as toads became adapted to a saline environment. This was shown by an increased uptake by the brain of isotopically labeled amino acids from the blood. These findings are not necessarily indications of a changed blood-brain barrier, but may be the result of the enlarged intrinsic amino acid pools in brain. ACKNOWLEDGEMENTS

This research was supported by Grant #NB-03743 from the National Institutes of Health, Institute for Neurological Diseases and Blindness, U.S. Public Health Service. I am greatly indebted to Dr. C. L. Deelder and his colleagues at the Rijksinstituut voor Visserij Onderzoek in IJmuiden, The Netherlands, who made this study feasible by collecting eels from fresh and ocean water and sending the weighed brains to me, pickled in alcohol for analysis in California.

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442

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BAXTER,C. F. (1961) Estimation of y-aminobutyric acid. Methods in Medical Research, 9, J. H . Quastel (Ed.). Chicago, Year Book Medical Publishers Inc. (p. 192-195). BAXTER, C. F. (1966) Changes in “free ”amino acid composition of central nervous system in response to osmotic stress. Fed. Proc., 25, 713. BAXTER, C. F. AND ORTIZ,C. L. (1966) Amino acids and the maintenance of osmotic equilibrium in brain tissue. Li/e Sci., 5, 2321-2329. BAXTER, C. F. AND SENONER, I. (1964) Liquid scintillation counting of C14 labeled amino acids on paper, using trinitrobenzene-I-sulfonicacid and an improved combustion apparatus. Anal. Biochem., 7.55-61. [For more details see same authors and title (1965) in Advances in Tracer Methodology, 2, S. Rothchild, Editor. New York, Plenum Press (p. 97-105).] BERL,S., LAJTHA, A. AND WAELSCH, H. (1961) Amino acid pool and protein metabolism. IV. Cerebral compartments of glutamic acid metabolism. J. Neurochem., 7, 186-197. BERL,S. AND PURPURA, D. P. (1966) Regional development of glutamic acid compartmentation in immature brain. J. Neurochem., 13, 293-304. BERL,S., TAKAGAKI, G., CLARKE, D. D. AND WAELSCH, H. (1962) Metabolic Compartments in vivo. Ammonia and Glutamic acid metabolism in brain and liver. J. Biol. Client., 237, 2562-2569. BERNT,E. AND BERGMEYER, H. U. (1963) Urea. Methods of Enzymatic Analysis, H. U. Bergmeyer (Ed.). New York and London, Academic Press (p. 401406). BLASBERG, R. A N D LAJTHA,A. (1965) Substrate specificity of steady-state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. -(1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Res., 1, 86-104. BOUCHER-FIRLY, S. (1 935) Recherches biochimiques sur les Teleosteens Apodes. Ann. I n s t . Ocianogr. Monaco, 15, 2 19-227. BUNIATIAN, H. CH. A N D DAVITIAN, M. A. (1966) Urea synthesis in brain. J. Nerrrochem., 13, 743. CHAPLIN, A. E., HUGGINS, A. K. AND MUNDAY, K. A. (1965) Ionic effects on glutamate dehydrogenase activity from beef liver, lobster muscle and crab muscle. Comp. Biochem. Physiol., 16, 49-62. CHRISTENSEN, H. N. ANDRIGGS, T. R. (1956) Structural evidence for chelation and SchiR”s base formation in amino acid transfer into cells. J. Biol. Chem., 220, 265-278. CREMER, J. E. (1964) Amino acid metabolism in rat brain studied with I4C labelled glucose. J. Neurochem., 11, 165-185. DINGMAN, W. AND SPORN,M. B. (1959) The penetration of proline and proline derivatives into brain. J. Neurochent., 4, 148-153. FINERMAN, G. A. M. AND ROSENBERG, L. E. (1966) Amino acid transport in bone. Evidence for separate transport systems for neutral amino and imino acids. J. Biol. Chem., 214, 1487-1493. FLORKIN, M., DUCHATEAU-BOSSON, CH.,JEUNIAUX, CH.AND SCHOFFENIELS, E. (1964) Sur le mkcanisme de la regulation de la concentration intracellulaire en acides aminks libres chez Eriocheir Sinensis, au cours de I’adaptation osmotique. Arch. Intern. Physiol. Biochim., 72, 892-906. FLORKIN, M. AND SCHOFFENIELS, E. (1965) Euryhalinity and the concept of physiological radiation. Studies in Comparative Biochemistry, Munday, K. A. (Ed.). International Series of monographs on pure and applied biology, 23, Oxford, London, Edinburgh, New York, Paris, Frankfurt, Pergamon Press (p. 6-40). FOX,M., THIER,S., ROSENBERG, L. A N D SEGAL,S. (1964) Ionic requirements for amino acid transport in the rat kidney cortex slice. 1. Influence of extracellular ions. Biochim. Biophys. Acta, 79, 167-1 76. GAITONDE, M. K. (1965) Rate of utilization of glucose and “compartmentation” of u-oxoglutarate and glutamate in rat brain. Biochem. J., 95, 803-810. GAITONDE, M. K., DAHL,D. R. AND ELLIOTT,K. A. C. (1965) Entry of glucose carbon into amino labeled glucose. Biochem. J., 94, acids of rat brain and liver in vivo after injection of uniformly 345-352. GORDON,M. S. (1965) lntracellular osmoregulation in skeletal muscle during salinity adaptation in two species of toads. Biol. Bull., 12, 218-229. GRAHAM, L. T. JR.A N D APRISON,M. H. (1966) Fluorornetric determination of aspartate, glutamate and 1,-aminobutyrate in nerve tissue using enzymic methods. Anal. Biochem., 15, 487497. GUROFF, G. AND UDENFRIEND, S. (1962) Studies on aromatic amino acid uptake by rat brain in vivo. J. Biol. Chem., 237, 803-806. JONES,M. E., SPECTOR,L. A N D LIPMA”, F. (1955) Carbamyl phosphate, the carbamyl donor in enzymatic citrulline synthesis. J. Amer. Cheni. SOC.,77, 819-820.

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KEMP,J. W. AND WOODBURY, D. M. (1965) Synthesis of urea-cycle intermediates from citrulline in brain. Eiochim. Eiophvs. Acta, 111, 23-31. KLEEMAN, C. R., DAVSON, H. AND LEVIN,E. (1962) Urea transport in the central nervous system. Artier. J. Physiol., 203, 739-747. KUTTNER, R., SIMS,J. A. AND GORDON, M. W. (1961) The uptake of a metabolically inert amino acid by brain and other organs. J. Neitrocheni., 6, 31 1-317. LAJTHA.A. (1964) The uptake of amino acids by the brain in various species. Coniparative Neitrocheniistry. D. Richter (Ed.). Proceedings of the 5th International Neurochemistry Symposium. Oxford, London, New York and Paris, Pergamon Press (p. 193-204). LAJTHA, A., FURST,S., GERSTEIN, A. A N D WAELSCH, H. (1957) Amino-acid and protein metabolism of the brain. 1. Turnover of free and protein-bound lysine in brain and other organs. J. Neurochern., 1, 289-300. LAJTHA, A., LAHIRI, S. A N D TOTH,J. (1963) The brain barrier system. IV. Cerebral amino acid uptake in different classes. J. Neurochern., 10, 765-773. LAJTHA,A. A N D MELA,P. (1961) The Brain Barrier System. I. The exchange of free amino acids between plasma and brain. J. Neitrocheni., 7 , 210-217. LANCE.R. AND FUGELLI, K. (1965) The osmotic adjustment in the euryhaline teleosts, the flounder fleitroriectes flesits and the three-spined stickleback, Gasterosteits aculeatus. Conip. Biocheni. Physiol., 15, 283-292. LEVI,G., BLASRERC, R . AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biocheni. Biophys., 114, 339-351. LEVI,G., CHERAYIL, A. AND LAJTHA.A. (1965) Cerebral amino acid transport iri vitro. 111. Heterogeneity of exit. J. Neitrochetn., 12, 757-770. LINDSAY, T. R . A N D BACHELARD, H. S. (1966) Incorporation of 14C from glucose into a-keto acids and amino acids in rat brain and liver iti vivo. Biochetii. fharriiacol., 15, 1045-1052. LOVE,W. D., O'MEALLIE,L. P., LEMMON, W. W., AND BURCH,G. E. (1962) Evaluation of a new method for measurement of cerebral and other regional circulation times. J. Lab. Cliti. Med., 60, 478-49 I . MIZELL,S. (1965) Seasonal changes in energy reserves in the common frog Raiia pipiens. J. Cell. Conip. fhysiol., 66, 251-258. N AKAMURA, R. AND NAGAYAMA, M. (1966) Amino acid transport by slices from various regions of the brain. J. Neiirocherii., 13, 305-313. NEAME, K. D. (1961) Phenylalanine as inhibitor of transport of amino acids in brain. Nature, 192, 173-174. NEAME, K. D. AND SMITH,S. E. (1965) Uptake of D and L alanine by rat brain slices. J. Neurochetii., 12, 87-9 I . RnRERTs, E., ROTHSTEIN, M. AND BAXTER, c. F. (1958) Some metabolic studies of y-aminobutyric acid. Proc. SOC.Exptl. Eiol. Med., 97, 796-802. RnBERTs, E. AND SIMONSEN, D. G. (1962) Free amino acids in animal tissue. In: Aniirio Acid Pools, J. T. Holden (Ed.) Amsterdam, London and New Ycrk, Elsevier Publishing Company (p. 284-349). ROBERTS, S. (1963) Regulation of cerebral metabolism of amino acids. 11. Influence of phenylalanine deficiency on free and protein-bound amino acids in rat cerebral cortex: Relationship to plasma levels. J. Neiirocherii., 10, 931-940. ROBERTS, S. AND MORELOS, B. S. (1965) Regulation of cerebral metabolism of amino acids. 1V. Influence of amino acid levels on leucine uptake, utilization and incorporation into protein iri vivo. J. Neitrocherri., 12, 373-387. SCHOFFENIELS, E. A N D GILLES,R. (1963) Effect of cations on the activity of I-glutamic acid dehydrogenase. L$e Sci., 1, 834-839. SHARRATT, B. M., JnhEs, 1. C. A N D BELLAMY, D. (1964) Water and electrolyte composition of the body and renal function of the eel (Arigitilla atiguilla). Coriip. Biocheni. fhysiol., 11, 9-18. SCHWERIN, P., BESSMAN, S. P. AND WAELSCH, H. (1950) Uptake of glutamic acid and glutamine by brain and other tissues of the rat and mouse. J. Eiol. Cheni., 184, 37-44. TERCAFS, R. R. A N D SCHOFFENIELS, E. (1962) Adaptation of amphibians to salt water. Life Sci., 1, 19-23. TSUKADA, Y., NAGATA, Y., HIRANO, S. A N D MATSUTANI, T. (1963) Active transport of amino acid into cerebral cortex slices. J. Neitrocheiri., 10. 241-256. V A NDEN BERG,C. J., MELA,P. A N D WAELSCH, H. (1966) On the contribution of the tricarboxylic acid

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cycle to thesynthesis of glutamate, glutamineand aspartate in brain. Biochenr. Biophys. Res. Conrrn., 23,479-484. VIDAVER, G. A. (1964a) Transport of glycine by pigeon red cells. Biochein., 3, 662-667. -(1964b) Some tests of the hypothesis that the sodium-ion gradient furnishes the energy for glycineactive transport by pigeon red cells. Biochein., 3, 803-808. VIRKAR, R. A. (1966) The role of free amino acids and the adaptation to reduced salinity in the sipunculid Golfingia gouldii. Comp. Biochein. Physiol., 18,617-625. VON LOMBARDO, G. AND TAMBURINO, G.(1963); Das Studium der Blutliquorschranke mittels radiojodierten Serumalbumins. Untersuchungen am normalen und am experimentell meningitischen Hund. 2. Naturfursch., 18b, 716-777. WATTS, D. C. AND WATTS,R. L. (1966) Carbamyl phosphate synthetase in the elasmobranchii; osmoregulatory function and evolutionary implications. Conip. Biochenr. Pliysiol., 17, 785-798. WEINSTEIN, H., VARON,S., MUHLEMAN, D. R. AND ROBERTS,E. (1965) A carrier-mediated transfer model for the accumulation of 14C y-aminobutyric acid by subcellular brzin particles. Biochem. Pharmacol., 14,273-288. WHEELER, K. P., INUI,Y., HOLLENBERG, P. F., EAVENSON, E. AND CHRISTENSEN, H. N. (1965) Relation of amino acid transport to sodium-ion concentration. Biuchem. Biophys. Acta, 109, 620-622. WIECHERT, P. (1963) Uber die Permeabilitat der Blut-Liquor-Schranke fur einige Aminosauren. Acta Biol. Med. Germ., 10, 305-310. WIECHERT, P. AND KNAAPE, H. H. (1963) Uber der Einfluss experimentell ausgeloster cereberaler Krampfanfalle auf die Permeabilitat der Blut-Liquor-Schranke fiir einige Aminosauren. Acta Biol. Med. Germ., 11, 494-501.

DISCUSSION R. KATZMAN: I just want to ask Dr. Baxter if he can give the average osmolarity of the total blood in the normal state and in the sodium-chloride stressed state.

C. F. BAXTER:In the normal state it is around 230 milli-osmols; in the stressed state it is slightly above that of the environment, that is about 420 milli-osmols.

I should like to make two comments. First of all I don’t agree that a substrate may have a P. MANDEL: regulatory influence on the metabolism of a cell, unless its amount is limiting. The most important fact we know in molecular biology is that the syntheses are dominated by some form of RNAsynthesis which depends on DNA or some inducers, repressors or depressors, but not on the substrate. The main experiment performed by Kornberg was that he got a primer-like D N A with several different pools of free nucleotides. It is the same for RNA, so 1 think that first of all we should admit that the substrate has not a very important role in the metabolism of a cell rrnless there is a limiting factor. The second comment is that you have a tremendous increase of glutamic acid over the glutamine. D o you have some data concerning the turn-over of the proteins in relation to the high pool ot glutamic acid or glutamine? C. F. BAXTER: To discuss the first question: I think I would have to disagree with you that substrate levels are not important in regulating the metabolism of the cell. I don’t think very many enzymologists hold the view that we have complete saturation of most enzymes with substrate in a normal metabolic system. I don’t know either, for that matter, if in any one transport system the carrier is fully saturated at any one time. If you are talking about adaptation and adaptive enzyme synthesis, this is a different matter altogether. But in the case of just normal metabolism, I believe substrate levels often are rate limiting to an enzyme system. Your second question concerned the protein turn-over. I do not have any data concerning this. You may have noticed the fact that the urea content in the saline adapted toad brain was extremely high, and this may indicate that there is quite a bit of protein break-down. There are also some data for invertebrates which ‘‘seem” to indicate that during osmotic stress there may be an increased break-down of protein contributing to the amino acids in muscle tissue.

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G . Ltvi: I think that the higher exchange rates of labeled amino acids that you find has an explanation in the fact that thc cerebral concentration of these amino acids in the brain is increased. Higher exchange rates of amino acids have also been shown by Dr. Lajtha when the amino acid level in the brain is artificially increased. I agree with you for leucine, but for ci-amino iso-butyric acid there is no pool. C. F. BAXTER: G . LEVI:But there is a pool of amino acids of similar structure and charge (such as alanine), with which (1-amino isobutyric acid could easily exchange.

K. A. C. ELLIOTT: Could Dr. Baxter speculate on the teleology of these changes, or in more orthodox terms: what is the survival value of thesechanges? C. F. BAXTER: Well, 1 think it is fairly obvious that if these toads could not adapt to their normal environment they would not survive for very long. K. A . C. ELLIOTT: But the nature of the changes, how d o they contribute to this survival? By changing the osniolarity of the tissue; at least that is all we know of to date. C. F. BAXTER:

K. A. C. ELLIOTT: So it is sufficient to make an appreciably large contribution to the new osmolarity? C. F. BAXTER:Yes, I think the nitrogenous compounds which we have measured so far make a ontribution of about 37 milli-osmols. K. A. C. ELLIOTT: And you have got to make up about 150? C. F. BAXTER: This is correct. I would agree with you that it is only a portion of the total, but 37 milliosnioles is a noteworthy contribution - about 20 plus percent of the total requirement. R. CUTLER: In your last slide you showed a tissue plasma ratio about twice as high for the sea water animals. But the plasma counts were about half those of the control animals. 1 wondered if therc was a difference in dose, or how you could account for the difference.

C. F. BAXTER: We injected the same dose into both groups of animals. You were asking why plasma counts are so much lower in the sodium chloride stressed toads? Presumably in these animals, the labelcd (1-aniinoisobutyric acid has also penetrated more rapidly into tissues other than brain, and therefore the plasma counts would be lower than those in fresh water toad plasma after a very short period of time. R. CUTLER: Wouldn't this be sufficient to account for your higher ratio in the brain? C. F. BAXTER: Yes, it probably would be. However, the brain is in contact directly or indirectly with the plasma, and not with other tissues. Therefore, the comparison we have to make is hetween plasma level and brain level. So I think thc significance of the ratio should still apply. D. M. WOODBURY: Is there any evidence that the salt stress increased in some way the number of polypeptide or smaller molecules in the cell and thereby increased osmolality by increasing the total number of particles in the cell? C. F. BAXTER: I have no such evidence. D. M. WOODBURY: And one other question. Were there any pH changes produced which could increase the total charge on the protein molecules and thereby allow an increased number of osmotic particles to exist in the cells? C. F. BAXTER: There is a change in plasma pH. Normal heparinized toad plasma has a pH of abou 7.9 which is about 0.3 pH units higher than the plasma of toads in 40% ocean water.

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P. G. SCHOLEFIELD: Dr. Baxter, I just wonder how long you have to incubate your toads in saline to get this effect? I remember that toads produce a steroid analogue, and 1 thought about ouabain being a steroid analogue. In this respect I wondered how much you are altering the control of the sodium movement, e.g., through the change in production of steroids, and this led me to wonder just how the toads produced these effects. C. F. BAXTER:We can get this change without any difficulty by placing the toads into 40”/, ocean water for one day. However, in practice we have adapted them more gradually to their saline environment. We have not done a systematic study with steroids.

GENERAL DISCUSSION

D. B. TOWER:Many substances have been discussed here in the last few days, for most of which someone has suggested a transport system in one way or the other. However, inulin seems to stand alone in being excluded by many membrane systems. 1 just wonder how we are going to consider inulin in the context of this meeting. D. M. WOODBURY: I think I can explain the distribution of inulin, at least its movement out of the CSF, and its low levels in the CSF and brain, merely by flow in the CSF-sink, coupled with slow entrance across the endothelial barrier between the brain capillaries and the interstitial space of the brain. lnulin follows the same sort of behavior as radioiodinated serum albumin or any other large molecule. As Dr. Rall and others including Dr. Reed and 1 have shown, all such non transported molecules leave the CSF at the same rate by bulk flow. D. B. TOWER:Yes, but why is it excluded from corning in?

D. M. WOODBURY: It isn’t excluded, it just enters more slowly than most substances. The rate of entrance of such water soluble substances across the endothelial barrier appears to be merely a matter of their size. Thus, sucrose enters more rapidly than inulin, but at a lower rate than the smaller molecule. mannitol. D. B. TOWER: I am just asking this for the sake of clarification. D. M. WOODBURY: I don’t think there is any question that there is a barrier between the endotheliuni and the interstitial space of the brain for inulin, and related substances since their rate of entrance is extremely slow.

A. LAJTHA: We have some information on inulin flux in brain slices, and may 1 mention that brain slices are, in some respects, an altered system as compared to the living brain. If the influx of inulin is measured, there is a definite break. One of the possibilities, as mentioned before, would be that inulin penetrates slowly into damaged ctlls.

D. M. WOODBURY: May I discuss that? The only experience I have had in this respect is with some studies on the thyroid gland. We were interested in measuring the extracellular space of the thyroid to see if the inulin entered just the stromal space or whether it also penetrated into the luminal space. Dr. Chow and 1, therefore, performed some radioautographic studies with tritiated inulin. The inulin was found only in the stromal (interstitial) space outside the follicle. We also measurtd the inulin space of the thyroid by detrrmination of the uptake curve and compared the spaces at various times with radioautographs determined at one and 24 hours after administration of inulin. During the initial phase (1 hour) when the inulin was equilibrated, it was only in the stromal (interstitial) space by radioautography and nowhere else in the thyroid. After 24 hours the inulin space of the thyroid had increased to quite an extent; at this time the radioautographs showed that it was still in thc interstitial space and had not penetrated the cells or into the lumen, but that it was now beginning t o accumulate in clumps in the interstitial space, probably due to phagocytic activity by macrophages. The increase in these “clumps” corresponded to the increase in the space. Thus, during the early uptake phase, inulin measures the extracellular space of the thyroid, but after that, due to the accumulation in macrophages it no longer measures the volume of this compartment.

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A number of years agoCotlove also showed that inulin penetration into muscle involved two phases: a rapid phase and a very slow phase that took 24 hours for equilibration. He showed that the slow phase involved penetration into the connective tissue of the muscle. This also is probably due to accumulation in macrophages. Thus, the inulin space, after the initial rapid phase of equilibration, is not a valid measure of extracellular space in skeletal muscle. It is evident, therefore, that in both these experiments inulin remains in the extracellular space although secondary accumulation occurs. Our data for the inulin space of the brain in young rats also strongly suggest an extracellular distribution only. A. LAJTHA: Was this measured after several days?

No, only up to 24 hours after injection. D. M.WOODBURY: K . A. C. ELLIOTT: Is it in order to go back to Dr. Ford’s paper? If you give an animal radioactive glutamate, a lot of it will be rapidly metabolized and the radioactivity will be distributed all over the place. However, some of the labeled material will presumably be incorporated into protein. If you give the animal radioactive lysine which is not rapidly metabolized, some goes into protein. Now I am wondering: will the protein which incorporates the lysine be the same in kind and amount as the protein that is formed from the glutamate. In fact would it be possible to do the same experiment with glutamate as with lysine, by eliminating the metabolites and studying only the substances incorporated into protein? If the protein does get labeled in this way, this is partial evidence of turnover, then there should be a disappearance of the label from these neurons after a certain length of time. I wonder how soon this disappearance of the label occurs. 1 seem to be asking two questions: Does glutamate give you a similar degree or rate of labeling of protein as lysine, even though glutamate is being metabolized in another way? Does labeling of the protein in either way demonstrate that it is being turned over?

D. H. FORD:I think Dr. Lajtha has more information actually on this than I do. As 1 recall he has inforniation which suggests that there is a family of proteins present in differing amounts which would lead one to think that the turn-over rates or half-lives of any amino acid that could go into different proteins could have difference half-lives. He has one experiment in particular with young animals which 1 think is pertinent here. A. LAJTHA:May I mention a pertinent point here form the results of experiments that we recently published, where we fed mice with food containing labeled lysine with constant specific activity. The labeled diet was fed before the animals became pregnant, and the same labeled diet was continned during pregnancy and during subsequent growth until adult age. With this procedure we were sure that every protein molecule in the brain was labeled, since throughout the development the animal had only labeled lysine with constant specific activity available. When the animals were adult, we withdrew the labeled food and substituted unlabeled food. Within five months most of the label disappeared, showing that at least 95 per cent of the cerebral protein in adults is in a dynamic state. Then we analyzed lysine itself, and this was, of course, metabolized. In the long time experiment we found that about half of the label in brain proteins was in compounds other than lysine; one third was in glutamic acid. The incorporation of glutamic acid as compared to lysine was fairly similar, although we did not measure this very carefully. May 1 also mention that the average rate of turnover, calculated from this experiment, was rather similar to what we found when we calculated it from initial rates of incorporation. In mice, the average half-life was about 14 days. I don’t think there is very good evidence at the present time showing that there is an exchange of a single amino acid in cerebral proteins without a complete turnover of the whole protein molecule. Not many people measured this, and so, although we don’t have too much evidence against it, we certainly have no evidence that is convincing for it.

C. F. BAXTER:I wonder whether it would be in order to go back to Dr. Tower’s question to Dr. Quadbeck about the effect ofvitamin Bti and its analogues on the blood-brain barrier, or penetration rate: The similarity in structure between pyrithioxine and vitamin Bti has already been pointed out. However, Dr. Quadbeck stated that vitamin B6 had no effect upon the blood-brain barrier criteria which he has measured.

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Preliminary observations in our laboratory might lead to an opposite conclusion and we would suggest that, under certain conditions, vitamin Bemight play a role in blood-brain barrier mechanisms. Some years ago we were engaged in experiments with rats, in which thiosemicarbazide (TSC) was injected intraperitoneally and convulsions prevented, by the administration of pyridoxine o r pyridoxal some time later (Baxter and Roberts, 1962). Measurements were made of glutamic acid decarboxylase (GAD), GABA lcvels and y-aminobutyric-a-keto-glutaric transaminase (GABA-T) in brain tissues of the treated rats. G A D and yABA-T activities were measured in virro without the addition of pyridoxal phosphate. While TSC administered in vivo decreased lcvels of GABA in brain and inhibited GAD, it had no apparent effect on GABA-T. When TSC treated animals were injected with pyridoxal, the levels of GABA in the brain continued to decline while G A D activity appeared reactivated and GABA-T remained unchanged. There were various ways to interpret these results. It had been reported in the literature (Gammon er al., 1960) that the blood-brain barrier to GABA was altered in rats injected with methoxypyridoxine. It seemed possible that in our experiment a similar alteration in the bloodbrain barrier had occurred as a result of TSC plus pyridoxal treatment. The continued decrease in cerebral levels of GABA at a time when GABA synthesis was accelerated, would then be explainLd by a leakage of GABA out of brain tissues into the rest of the body. This hypothesis was tested in a preliminary way using 3 groups of 4 adult male Swiss mice. At zero time of the experiment, groups 2 and 3 received TSC intraperitoneally at a level of 12 nig/kg body weight. At I5 minutes, group 3 micl received an anticonvulsant dose of pyridoxal HCI (40 mg/kg). At 30 minutes, 101of2 [I4C]-GABA(containing about 2pC) was injected into the left brain ventricle of each mouse. Fifteen minutes after the isotope injection (and before the TSC treattd animals could convulsr) all mice were quick-frozen in liquid nitrogen. Whole body sections were prepared and radioautographs made using the techniques pioneered by Ullberg and his colleagues (Ullberg, 1954).

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Although the number of animals tested precludes a conclusion and quantitative data are yet to be obtained, these preliminary experiments suggest that the administration of pyridoxal to TSC treated mice increaspd the outflow of isotopically labeled material from the ventricle or brain tissue into other tissues of the body. This increased flux then might reflect a change in permeability of the brainblood barrier i n response to vitamin BS administration. BAXTER, C. F. A N D ROBERTS, E. (1962)Effect of 4-MethoxyniethylpyridoxineandCarbonyl-Trapping Agents on Amino Acid Content of Mammalian Brain and Other Tissues. In Amino Acid Pools, J. T. Holden (Ed.), pp. 499-508. Elsevier, Amsterdam, London and New York. GAMMON, G. D., GUMPUIT, P. rt a/. (1960)The Effect of Convulsant Doses of Analeptic Agents upon The Concentration of Amino Acids in Brain Tissue. In Irihibifiotrin the Nervous System and Gamma Arniiiohrityrir Acid, E. Roberts e f a/.(Ed.), pp. 328-330, Pergamon Press. U L L H t w , S. (1959)Autoradiographic Studies on the Distribution of Labeled Drugs in the Body. Progress if1 Nuclear Energy - Series 6, Vol. 22 - Biological Sciences, pp. 29-35, Pergamon Press, London, Oxford, New York and Paris. G. QUADBECK: I don't think that this has anything to d o with pyrithioxine, because pyrithioxine has no vitamin Be like-effect. It is also ineffective in the intoxication with thiosemicarbazide compound. P. MANDLL: I should like to come back to the problem of substrate metabolism. We agree that the level of substrate has an influence on the level of hydrolytic enzymes; for example, if we inject more arginine, more argenase is found in the liver. I agree that there is a feed-back control for the synthesis of amino acids. But when we deal with the uptake of amino acids, we are concerned mainly with protein synthesis. I don't believe that there is any evidence that change in the conccntration of amino acids (unless an amino acid is limiting), has any influence on the protein synthesis. Moreover, if we admit that the pool of amino acids could have any influence on the protein synthesis in a cell and that the genetic control of protein synthesis is not constant, we should know what to expect; because as far as the distribution of amino acids in the system is concerned, the kind of synthesis which depends on the RNA which is present in the cell or on the DNA is always the same. Therefore, the problem of the substrate level of amino acids in view of protein synthesis cannot play a role unless it is a limiting factor. A. LAJTHA: The effect of changes in amino acid pool on cerebral metabolism is very important, but unfortunately only a few experiments have investigated this point. Dr. Sidney Roberts did some vcry interesting experiments in which he found that omitting phenylalanine from the diet changes cerebral amino acid pools. When he measured the transport and the metabolism of free leucine and the incorporation of leucine into proteins in the brain, he found that all threc of these processes were significantly altered, showing that a change in the pool may affect both amino acid and protein metabolism.

P. MANDEL: That is entirely different. He removed from the medium an amino acid which is limiting, because it is an essential amino acid, without which protein synthesis is not possible. Under these conditions you have protein break-down. A. LAJTHA: No. Phenylalanine did not disappear from the brain; however, the altered diet changed, among other things, the level of leucine in the brain. I don't know what all the changes were; perhaps they were manifold. One of the possibilities would be that changes in the ribosomal structure caused by alterations in the pool are then reflected in the synthetic activity of the ribosomes. P. MANDEL: But if you remove phenylalanine from the diet the synthesis of proteins is disturbed. A. LAJTHA:No, the changes were in the structure of the ribosomes, because of the diet. But somehow the ribosomes reacted to the pool size and their synthetic avidity was altered. I can't believe that the structure of the ribosomes change when you remove an amino acid. P. MANDEL: The evidence which we have was that even removing whole proteins from the diet did not alter the pattern of proteins in the ribosomes. So, what occurs is that there are fewer ribosomes and there is a break-down of proteins in liver and muscle providing amino acids to produce the proteins. The kind

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of proteins YOU produce depends on the information which comes from DNA or messenger-RNA. You cannot do anything to change it. If vou remove an essential amino acid from the diet, what you produce is a decrease of the synthesis of messenger-RNA by a phenomenon which would take too long to explain. Thus, in a deficient diet there would be fewer polysomes which would decrease the synthesis of protein and the incorporation of [14C]lysine. The synthesis of protein depends only on the genetic information and not on the available substrate.

C. F. BAXTER: There is plenty of evidence in the literature that ions can stimulate the rate of an existing enzyme. And in the particular case of the osmotically stressed toads we have ionic changes, so we may have changes in rate of enzyme activity, because of the ion stimulation. Would you accept that? P. MANDEL: Yes, but the synthesis of protein does not depend on the substrate concentration. It depends on whether you induce a new enzyme, e.g. Under these conditions there is a synthesis of an enzyme from the pool of amino acids. However, I think it would be difficult to obtain any evidence that if you stimulate the synthesis of an enzyme, that it depends (again, it is the same problem) on the substrate level, unless there is an amino acid which is limiting. What we can say is that glutaniic acid in brain is not a limiting factor. C. F. BAXTER:But I don’t think we have to stipulate that there is an increase in enzyme synthesis under the conditions that I showed. In an increased level of amino acids in the brains of those animals, 1 don’t think that is necessary.

D. P. RALL:May I change the subject? It occurs to me along the same lines of your paper, Dr. Baxter, that there is another thing that you can d o to these toads. You can functionally dehydrate them the way seawater does, by barring their access to water, either fresh water or sea water. If you have done this, what happened? C. F. BAXTER: We have done this, and it has no appreciable effect. D. P. RALL:Do toads drink sea water? C. F. BAXTER: In the case of Bufo boreas, I do not know, but they well might do so. We have increased internal osmotic concentrations in these toads by keeping them on moist blotting paper. In this way they cannot excrete any of their urine, and the urea level in the plasma goes way up. You can also dehydrate them. The brain is the last organ that will dehydrate to any significant degree.

N. M. VANGELDER: What is the inhibition of blotting paper? C. F. BAXTER: I don’t know. That is the way Dr. Malcolm Gordon at UCLA found he could prevent toads from urinating.

Spaces in the CNS

Introduction to Session on Brain Spaces K. A. C . ELLIOTT Departnient of Biocheniistry and Montreal Neurological Institute, McGiN University, Montreal (Canada)

I was very glad to be invited to this interesting symposium but I was embarrassed by the fact that for the last few years 1 have been primarily an office worker and steadily getting out of date. So, I invited myself to be chairman of this session. In this position, and as probably the oldest member of the symposium, I felt I could sermonize a little without greatly hindering the progress of knowledge. I have been interested in cerebral edema and in brain spaces since I was put on to the study of cerebral edema with Herbert Jasper in relation to war surgery when I first went to the Montreal Neurological Institute towards the end of the last war. This interest was accentuated by my observation of the way brain tissue slices swell when immersed in media resembling cerebrospinal fluid. My early work on this subject seems to have more or less missed the point, as has been shown largely by later work by Dr. Pappius. Perhaps my missing the point has a moral. Perhaps I am not the only one who must be prepared to find his work lead in different directions from what he originally expected. In the work with Dr. Jasper (Elliott and Jasper, 1949) we were watching’and trying to measure cerebral edema in rabbits. One of the most striking observations in rabbits with their brains exposed was the sporadic, quite sudden, bulging of the brain. The literature showed that this occurred on unaccountable occassions in various animals and, in fact, it is a known trouble in human brain surgery. The only people I could find who seemed to have considered it experimentally were Obrador and Pi-Suner (1943) in Mexico, who described sudden inflation of exposed brain on production of lesions near the fourth ventricle. We found that the bulge collapsed when the animal was decapitated and we could find no evidence of extra fluid in the tissue. So we called the phenomenon “inflation” and blamed it on vascular dilatation and perhaps extra cerebrospinal fluid production. Perhaps increased intracranial pressure due to a tendency to “inflation” is a part of the trouble when symptoms ascribed to “edema” are observed. Cannon had postulated a metabolic-osmotic mechanism of brain swelling and Jasper and I produced real brain swelling, or shrinkage, by hypoosmotic or hyper-osmotic (25 per cent glucose) intravenous infusions. Dr. Pappius, however, has indicated that osmotic swelling is not the common feature of cerebral edema though osmotic swelling may occur during drastic changes in plasma osmolarity, as during use of the artificial kidney (Pappius ef al., 1967). The water content of the brain as a whole, grey and white matter, can be reduced osmotically by hypertonic iieferences p . 454

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urea injections but the edema near lesions is not affected (Pappius and Dayes, 1965). The problem of the swelling of slices has been energetically worked out by Dr. Pappius and finally, through collaboration with Dr. Klatzo, she has proved that the main swelling in slices in vitro is in a space that is extracellular, in free communication with the surrounding medium, and which occurs appreciably only af the edges of slices where there is the mechanical damage of cutting (Pappius et af., 1962). So it is very doubtful that this kind of swelling is the main swelling of cerebral edema. In fact it is almost certainly not, since Dr. Pappius has shown, as surgeons know from experience, that cerebral edema occurs in white matter, scarcely at all in grey (Pappius and Gulati, 1963), whereas the swelling of slices due to the damage caused in their preparation occurs with both grey and white slices and it was slices of grey matterthat we studied particularly. Still this swelling at the site of damage does indicate the existence of a potential space. Evidently certain structures normally keep brain tissue properly compressed and when these are damaged the tissue can swell up like a sponge. I don’t think we should forget this potential space though it is not a normal space. In fact it seems that a space of this kind, the fluid in which seems to be extracellular and in communication with plasma, opens up under conditions that produce cerebral edema and is actually the main space of cerebral edema. But why this space opens up in white matter, and not in grey matter, remains to be figured out. Histological considerations might explain this. The neuropil feltwork of grey matter may bind this tissue so tightly that spaces cannot open unless there is mechanical damage to the feltwork as in cut slices. Our early studies did show that there is also the possibility, under certain abnormal conditions, of a change in an infra-cellular space. This occurs, for instance, during hypoxia or in the presence of high extracellular glutamate or potassium. This may be the glial swelling that has been observed with the electron microscope in swollen slices. Such intracellular changes seem too small to account for the volume of cerebral edema but they do require us to think carefully in other directions. For instance, there was the discovery by Krebs and his coworkers that, when glutamate is present in the surrounding medium, brain slices take up both extra glutamate and potassium. But consideration of intracellular space changes show that there is no concentration of potassium (Pappius and Elliott, 1956). Actually the potassium concentration in the increased intracellular space is not higher than normal. In Table I, I have summarized our thinking on this subject. Theconsideration of spaces in the brain, or in any tissue, is really rather complicated. You start with three apparently obvious spaces: the blood vessels, an extracellular space and an intracellular space. But then these, at least the last two, are not so obvious. We have a flat contradiction between the quite large extracellular space in brain if this is equated with sodium and chloride spaces or, at least in slices, with thiocyanate or sucrose spaces, and the smaller spaces apparently seen by the electron microscope. But the electron microscope space agrees approximately with the chemically determined inulin or protein spaces of Dr. Pappius (Pappius et al., 1962). But what do we mean by intra- and extra-cellular spaces? Does the extracellular space extend into the innerds of the cell via the endoplasmic reticulum? And if so, how

B R A I N SPACES

453

TABLE I T H E S E S CONCERNING C E R E B R A L E D E M A

I . “Inflation” can be confused with edema. 2. Brain swelling (and shrinkage) can be produced experimentally by osmotic action. but 3a. Traumatic edema is not osmotic swelling. b. Traumatic edema is rnnstly an uptake of fluid into an extracellular space. This may be only a potential space in normal tissue. (The fluid apparently enters this space from defective blood vessels) c. Traumatic edema occurs in white matter, very little in grey matter. 4. Trauma nevertheless produces some generalized change in grey and white matter though only the white swells grossly. 5. Direct damage to brain, as at edges of cuttissue,allows local uptake of fluid into a potential space in grey or white tissue. 6. Unfavourable metabolic conditions can produce mine intracellular swelling, at least in vifro. 7. Osmotic swelling of white and grey matter can occur when there is rapid change of concentration of a plasma solute as in the use of the artificial kidney. (Osmotic swelling is, of course, intracellular) 8. Osmotic reduction of intracranial pressure can be achieved with hypertonic urea (or other substances). This involves general reduction of brain volume, not reduction of edema.

f a t will proteins, metabolites and electrolytes diffuse in and out of this tortuous space? That is, though it may be connected with the really external space, will it behave as extracellular space for all solutes? Once we concede that we are in the intracellular space, we find we are merely reaching further complexity. We have spaces : intramitochondrial, intra-lysosomal, intraGolgi apparatus, intra-nucleus, intra-nucleolus and intra-subdivisions of these. And presumably we have a little fluid that is plain intra-cellular, bathing all these organelles and interacting with extracellular fluid osmotically, through simple diffusion and through various kinds of pumps and active transport systems. Most probably such pumps and systems also apply to interactions between the various subcellular spaces. But even the commonly used term “space” is ambiguous. We have an example in our own experience. GABA exists in brain in at least three different states (see e.g. Elliott 1965; Varon et al., 1965). One is in free solution, one is occluded in particles that are obtained in sucrose suspension and is completely separated from free GABA. Labelled free GABA does not mix with it. The third exists when sodium is present, and its presence is inhibited by cold. It is completely exchangeable with free GABA. Now the free and the occluded GABA may be regarded as being in two particular spaces. But what about the third state? It is presumably adsorbed in some way and not occupying a “space” as we commonly mean the word. How often do we talk of a “space” which isn’t really a space? The substance whose space we are determining may not all be in ordinary solution but may be partly absorbed or adsorbed to a solid surface or to a substance in solution, as in enzyme-substrate, antigen-antibody, lipidlipid, or electrostatic combinations, or in micellar or clathrate occlusions. And even when we speak of a space, meaning a volume of solution containing the substance in question, are we sure we know what we mean? The solvent may in many cases not be water as we know it in bulk. Water in narrow spaces, the walls of which Rrferrncrs p . 454

454

K . A. C. ELLIOTT

are full of ionized groups and hydrogen bonding potentialities, is probably structuredice-like according to Szent-Gyorgyi. We could hardly expect solutes to behave in such matter as though they were in free solution. I am inclined to think that this kind of condition applies not only in intracellular spaces but also to some of the narrowly confined extracellular space. So we could have to consider several different kinds of extracellular fluid spaces according to the precise location and state of the fluid. There would be, for instance, the truly external fluid and perhaps fluid within connecting endoplasmic reticular tubules, and there would be the normal water and the structured water. Finally, we should remember that these structures and fluids are not all necessarily stationary. We should keep in mind old observations on protoplasmic streaming and the more recent evidence of axoplasmic flow in neurons and the pulsations and gyrations of cultured glial cells that we used to see in the movies of Dr. Pomerat. I have now, I believe thoroughly scrambled the issue and I shall hope that our main speakers and the general discussion will straighten things out. Dr. Pappius has shown that tangible observations can be made in spite of all these complexities. I call on her to speak now.

REFERENCES

ELLIOTT, K. A. C. (1965) y-Aminobutyric acid and other inhibitory substances. Br. Med. Bull., 21, 70-75; see also Strasberg, P. and Elliott, K.A.C. (1967) Can. J. Biochem., 45, 1795-1807. ELLiOTT, K. A. C. AND JASPER, H. H. (1949) Measurement of experimentally induced brain swelling and shrinkage. Amer. J. Physiol., 177, 122-129. OBRADOR, S. AND PI-SUNER, J. (1943) Experimental swelling of the brain. Arch. Neurol. Psychiat., 49, 826-830.

PAPPIUS, H. M. AND DAYES, L. A. (1965) Hypertonic urea. Its effect on the distribution of water and electrolytes in normal and edematous brain tissue. Arch. Neurol., 13, 395402. PAPPIUS, H. M. AND ELLIOTT, K. A. C. (1956) Factors affecting the potassium content of incubated brain slices. Canad. J. Biochem. Physiol., 34, 1053-1067. PAPPIUS, H. M. A ~ GULATI. D R. (1963) Water andelectrolytecontent ofcerebral tissues in experimentally induced edema. Acta Neuropathol., 2,451460. PAPPIUS, H. M., KLATZO, I. AND ELLIOTT, K. A. C. (1962) Further studies on swelling of brain slices. Canad. J. Biochem. Physiol., 40,885-898. PAPPIUS, H. M., OH, J. H. AND DOSSETOR, J. B. (1967) Effect of rapid hemodialysis on brain tissues and cerebrospinal fluid of dogs. Canad. J. Physiol. Pharmacol., In press. VARON,S., WEINSTEIN, H., KAKEFUDA, T., AND ROBERTS, E. (1965) Sodium-dependent binding of y-aminobutyric acid by*.morphologically characterized subcellular brain particles. Biochenr. Pharmacol., 14, 1213-1224.

455

Spaces in Brain Tissue in vdro and in viva H A N N A M. PAPPIUS The Dorurer Lahoratory of Experinletrial Neurocheniisiry. Montreal Neurological Insiiiuie and the Deparinrerri of Neurology and Neurosurgery, McCill Uiriversiiy, Montreal (Canada)

Several years ago, Dr. Elliott and I studied in detail the distribution in incubated cerebral cortex of substances thought to equilibrate with extracellular tissue water. We used thiocyanate, sucrose, inulin and labeled protein (Pappius and Elliott, 1956; Pappius et a/., 1962; Pappius, 1965). In our experiments, rat cerebral cortex slices about 0.5 mm thick were weighed and incubated in Ringer-bicarbonate-glucose medium containing one of the ‘markers’. After incubation, the slices were drained, reweighed and suitably extracted for chemical analysis of the ‘marker’ substance under study. The ‘marker’ space in the tissue in each case was taken as equal to that volume of the medium which would contain the same amount of the ‘marker’ as was found in the slice. I would like to stress that when discussing spaces I am referring to the volume of distribution of a particular substance when steady-state conditions exist. Such conditions are fairly easily established in vifro. In vivo serious problems are encountered in attaining equilibration of the ‘marker’ substance between the various compartments available to it. FLUID COMPARTMENTS I N CEREBRAL CORTEX INCUBATED AEROBICALLY FOR 60 M I N .

c

0

20

60 80 100 Milligrams o r Microliters

40

Dry weight Tissue water not equilibrated with Inulin, Protein, Sucrose and Thiocyanate

I 120

140

Tissue water equilibrated with Sucrose and Thiocyanate, but not with lnulin and Protein Tissue water equilibrated with Inulin, Protein, Sucrose and Thiocyanate

0

Fig. 1. Fluid compartments in cerebral cortex incubated aerobically for 60 min. Composite summary of data. For details see text.

Fig. 1 is a composite summary of data obtained in many experiments in which the ‘marker’ spaces were determined separately. The results are expressed on the basis of 100 mg or microliters of the original tissue. The total length of the bar represents the average weight or volume of the slice after incubation, the portion to the right of the Ri~feriwcap. 460

456

H. M. P A P P I U S

broken vertical line representing the extent of swelling. The solid portion represents dry weight. We found that all the ‘markers’ equilibrated with the fluid taken up on incubation, about 30 pl in a 100-mg slice. Inulin and protein equilibrated also with about 20 pl of the original tissue water, giving a total inulin-protein space in the swollen slice of about 50 pl. Sucrose and thiocyanate equilibrated with a significantly greater portion oftheoriginaltissue water-about 55pI-so that the total sucrose-thiocyanate space in the swollen slice was 85 pl. These results show that three distinct fluid compartments exist in incubated cerebral cortex slices : ( I ) the non-thiocyanate, non-sucrose space, (2) the inulin-protein space and (3) the compartment which is penetrated by sucrose and thiocyanate but not by inulin and protein. With the help of Dr. Klatzo we were able to visualize microscopically the tissue water compartment equilibrated with protein labeled with a fluorescent dye (Pappius et al., 1962). The bulk of the protein could be seen in a zone near the edge of the slice which consists of tissue damaged during its preparation. On the basis of this finding, we were able to conclude that protein and, by inference, inulin equilibrate with the fluid in damaged regions of the slice where the bulk of the in vitro swelling has occurred. On the basis of other experiments (Pappius et al., 1962; Pappius, 1965) which I have no time to describe, we know that under our standard aerobic experimental conditions intracellular swelling, associated mainly with anaerobic conditions, has been kept at a minimum. Thus the 2 0 4 of original tissue water which equilibrates with inulin in vitro must include the damaged areas of the slice and therefore represents an overestimate of the true extracellular space of brain. On the other hand, the non-inulin space of the slice can be regarded as the intracellular space of grossly undamaged cells. We interpret the fact that thiocyanate and sucrose equilibrate with a significant fraction of the non-inulin compartment of tissue water as evidence that they have someintracellular distribution and we have postulated that it may be intraglial (Pappius et al., 1962; Pappius, 1965). In fact, the distribution of sucrose in incubated tissue is not quite the same as that of thiocyanate. In recent experiments cat cerebral cortex slices were incubated in media containing thiocyanate and trace amounts of either [14C]sucrose or [“Tlinulin. Thus two spaces were determined simultaneously in each tissue sample. The results of these experiments are summarized in Fig. 2. It will be seen that under aerobic conditions the thiocyanate space was on the average 7 pl greater than the sucrose space in the same slice. This difference was quite consistent and statistically highly significant (p < 0.01). When sucrose and thiocyanate spaces were measured in the presence of 1 % sucrose, the highest concentration of sucrose in our original experiments on sucrose distribution (Pappius and Elliott, 1956), both the non-sucrose and non-thiocyanate spaces were diminished t o about the same extent, presumably due to osmotic dehydration. The agreement between the non-thiocyanate space in one case and the non-sucrose space in the other is thus coincidental. Since there is experimental evidence that thiocyanate, like chloride, crosses neuronal membranes (Coombs et a/., 1955) and thus must be distributed approximately according to the Nernst equation, the small dis-

SPACES IN B R A I N TISSUE

in VitrO A N D in

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Distribution of Inulin, Sucrose and Thiocyonote in Ctrebral Cortex Slices of Cat Incubated for 60 min. Aerobic

,

Aerobic; 1% sucrose in medium

I

(4) 1

d

20

40

60

80

100

120

140

Id0

Milligrams or Microliters

Fig. 2. Distribution of inulin, sucrose and thiocyanate in cerebral cortex slices of cat incubated for 60 min. Number of experiments in brackets. Short markers indicate standard deviation when six experiments were averaged and range of results in case of four experiments.

crepancy between sucrose and thiocyanate distribution does not necessarily invalidate our suggestion that both penetrate the glial space (see Discussion, Pappius and Elliott, 1956). The question arose whether the apparent intracellular distribution of sucrose and thiocyanate is associated with the in vitro situation and reflects partial damage, or whether, in fact, certain cellular elements in cerebral tissue are permeable to thiocyanate and sucrose. In an effort to answer thisquestion, thiocyanate distribution in cerebral tissues of cat was studied in vivo under conditions where equilibrium between the blood, the brain tissues and the cerebrospinal fluid, if not fully achieved was at least closely approached. In these experiments twice isotonic (2.5 %) sodium thiocyanate solution was slowly infused intravenously into cats over 30 to 40 min to give final blood concentration of about 10 mM. This relatively high concentration of thiocyanate was chosen in the hope of saturating the mechanisms which are known to actively transport thiocyanate from the cerebrospinal fluid to blood (Pollay and Davson, 1963). The animals, anesthetized with Nembutal, did not show any obvious signs of respiratory difficulties or other toxic effects of thiocyanate. Their blood remained well oxygenated with spontaneous breathing. The thiocyanate content of the blood was found to remainconstant throughout the experimental period. The animals were killed 2,4, 6 or 10 h after the infusion and the water and thiocyanate contents of the cerebral tissues and of the cerebrospinal fluid were determined. In vitro dialysis experiments showed that at 10 mM thiocyanate concentration in the blood 83 % of the thiocyanate was in free solution. In Fig. 3 the thiocyanate contents of the tissue water and of the cerebrospinal fluid are expressed as percent of the free thiocyanate concentration in the blood. Each point represents the average of results from three animals except at 10 h where five animals were used. The range of results is also indicated. With reference to the paper presented by Dr. French, at two hours after infusion there was indeed a suggestion of slight dehydration of the brain. The dry weight of both cerebral tissues was found to be at the higher border of the normal range. At later time intervals the dry weight was within normal limits (see Table I). Rrfercncrs p . 460

H. M. P A P P I U S

458

TABLE I EFFECT O F I N F U S I O N O F T H I O C Y A N A T E O N T H E D R Y W E I G H T O F C E R E B R A L TISSUES O F CAT

Time in hours after start of insusion of thiocvanatel

Dry weight mg % fresh weight of tissue Cerebral cortex Subcortical white matter

Number of atiinials

-

19.2 40.7 20.5 19.3 19.1 19.1 0.4

2 4 6 10

31.8 f 1.2 33.7 32.5 31.2 32.05 k 1.5

Averages -fr standard deviation where 5 or more animals used. 1 2.504 sodium thiocyanate was infused slowly over 30 to 40 min. Final concentration of thiocyanate in the plasma 11.3 3: 2.4 mM (14 animals).

I

0

I

‘

2

I

hrs.

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I

Ib

.

A W.T O U hrrmu I1 4 , I I Mill] W. ha 0C Er*d Y cam, 0 u n p c d .ku Mtfl(

Fig. 3. Percent of cerebral tissue water and cerebrospinal fluid water equilibrated with free thiocyanate in blood. For details see text.

Results presented in Fig. 3 show that the thiocyanate content of the CSF rose slowly and even 10 h after the thiocyanate infusion it had not come to an equilibrium with the free thiocyanate in the blood. At 10 h the average concentration ofthiocyanate in the CSF was 75 % of that in blood. The apparent thiocyanate concentration in the water of cerebral cortex and white matter increased slowly up to 6 h at which time it reached a plateau. At 10 h the concentration of thiocyanate in the water of both cerebral tissues amounted to 35 % of that in blood. When expressing the thiocyanate content of cerebral tissues in vivo in terms of thiocyanate space, it can be assumed either that the tissue thiocyanate has come to an equilibrium with thiocyanate in the blood or that it has equilibrated with the thiocyanate in the CSF. Since it is difficult to decide which of the two assumptions is the

SPACES I N B R A I N TISSUE

in vitro

AND

in vivo

459

Thioqanate Spate in Cerebral Tissurs of Cat in Vivo lllod l b l q l w ( ~11.3 f 2.4 mM 110I CEREBRAL CORTEX

Equilibrated with

2 hrs.

BLOOD

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SUBCORTICAL WHITE MATTER 2 hrr.

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(Sf

1

0

10

40

b0

80

I 100

Milligrams or Microliters

Fig. 4. Thiocyanate space in cerebral tissues of cat in v i v a Number of animals in brackets. Short markers indicate range of results. For other details see text.

correct one, in Fig. 4 thiocyanate space in the upper portion of each bar was calculated assuming equilibration with the blood and in the lower portion assuming equilibration with the CSF. At 2 h after infusion when steady state conditions have not been achieved, it is clear that expressing the thiocyanate space in terms of the concentration of thiocyanate in the blood gives an underestimate of the total space in vivo available to thiocyanate while in terms of thiocyanate in the CSF on overestimate of the tissue space is obtained. By 10 h when the difference between the blood and the CSF thiocyanate content has greatly diminished, the two estimates of thiocyanate space were considerably closer. It can be concluded that the thiocyanate space in cerebral cortex of cat in vivo when equilibrium between all water compartments has been achieved must be within the limits of 29-38 %, probably closer to the former figure. This, by the way, agrees fairly well with the figure of 30% for iodide space under conditions when iodide transport out of the CSF was inhibited, as reported at this conference by Dr. Woodbury. Fig. 5 is a comparison of the distribution of inulin and thiocyanate in vivo and in vitro. For the purpose of this discussion, I have assumed an in vivo inulin space of 10 3%, basing this estimate on the published inulin space figures of Rall and his colleagues (Rall et al., 1962). This is not too far from the figure of 13.5%inulin space mentioned here by Dr. Woodbury and 15 % sulfate space referred to by Dr. Cutler. Dr. Tower has reported considerably higher values for inulin space in vivo (Bourke et al., 1965), but it is unlikely that under his conditions even very careful experimentation can overcome the basic problem that equilibration between the various water compartments was not established. While both in vivo and in vitro existence of three

H. M. P A P P I U S

460

Fluid Conportmtntr in Cerebrol Cortex Tissue in Vivo ond i n Vitro Blood Thiocyanate 9.8 m M . 10 hrs. after lhiocvanats inlurion.

60 rnin. aerobic incubation

I

"

0

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Fig. 5 . Fluid compartments in cerebral cortex tissue in vivoand in vitro. Number of animals in brackets. Short markers indicate range of results for in vivo experiments and standard deviation for in vitro experiments.

fluid compartments can be demonstrated in terms of the distribution of inulin and thiocyanate, it is quite clear that quantitative relationships are rather different under the two sets of conditions. We know that the increased inulin space in vitro is associated with swelling in the damaged areas of theslice. However, thiocyanatedistribution relative to inulin distribution is also increased in v i m . Thiocyanate equilibrated with approximately 30 % of the non-inulin space in vivo and nearly 60 % of the non-inulin space in vitro. Further experiments will show, I am sure, whether this increase in the space available for thiocyanate distribution is an artefact of damage involved in the in vitro preparation or whether it is another demonstration of the restricted permeability into cerebral tissues in vivo, the so-called blood-brain barrier.

REFERENCES E. S. AND TOWER, D. B. (1965) Variation of cerebral cortex fluid spaces BOURKE, R. S., GREENBERG, in vivo as a function of species brain size. Amer. J. Physiol., 208, 682-692. COOMBS, J. S., ECCLES, J. C. A N D FAW,P. (1955) The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential. J . Physiol., 130, 326-373. PAPPIUS, H. M. (1965) The distribution of water in brain tissues swollen in vitro and in vivo. Biology of Neiiroglia. E. D. P. De Robertis and R. Carrea (Eds.), Progress in Brain Research, vol. 15 (p. 135-154). PAPPIUS, H. M. AND ELLIOW,K. A. C. (1956) Water distribution in incubated slices of brain and other tissues. Canad. J. Biochern. Physiol., 34, 1007-1022. H. M., KLATZO, I. AND ELLIOTT, K. A. C. (1962) Further studies on swelling of brain slices. PAPPIUS, Canad. J. Biochem. Physiol., 40, 885-898. POLLAY, M. AND DAVSON, H. (1963) The passage of certain substances out of the cerebrospinal fluid. Brain, 86, 137-1 50. C. S. (1962) Extracellular space of brain as determined by RALL,D. P., OPPELT,W. W. A N D PATLAK, diffusion of inulin from the ventricular system. LifeSci., 2, 43-48.

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VitVO A N D

in ViVO

46 1

DISCUSSION D. B. TOWER:I would like to say that the areas of agreement between Dr. Pappius and myself are vast compared to the areas of disagreement, and I would like to support what Dr. Pappius has said. In other tissues (e.g., liver, kidney, etc.), not only is there a disproportion between the space accessible to inulin and that accessible to thiocyanate, but also to that accessible to sucrose. Sucrose occupies almost the same-sized spaces as thiocyanate or chloride in the liver and kidney. In the white matter there is a real problem. I think Dr. Pappius must be much more skillful than I am at cutting slices of white matter, because I could not get slices to work, and finally resorted to a preparation of what might be called “hemi” corpus callosum, which in the cat, fortunately, is of appropriate dimensions for incubation (less than 0.5 mm thick). One can simply cut it free at the two sides and have a very small cut surface relative to the total surface area. In contrast, if you are not careful in s!icing white matter thz tissue imbibes too much water and disintegrates. As far as the difference in results for the cortex is concerned, it occurs to me that this may be partly a species difference with in vitro preparations. Since I have not done any of these experiments with the rat, I can’t say definitely, but under our conditions there is a very distinct difference between the size of the fluid spaces accessible to chloride and the size of those accessible to sucrose (for rabbit, cat, monkey cortex). There is a further complication, which I did not originally intend to bring up here, but which, I think, may be important for our discussion as it has developed during this session. There is a difference in the size of the sucrose (inulin) spaces in incubated slices of cat cerebral cortex, depending upon when one adds the indicator solute and depending upon the time of incubation. This is illustrated in summary form in the following table.

TABLE I SLICE SUCROSE SPACES A S FUNCTIONS OF T I M E S OF SOLUTE ADDITION A N D OF SLICE INCUBATION

lncubatim Time (miti)

Time Sucrose Present ( m i n )

Sucrose Space (%)

65 125

65 125

42.3 41.9

65 125 125 I25 125

20 20 40 60 90

29.4 29.7 30.5 35.4 39.1

Slices of cat cerebral cortex were incubated aerobically at 31°C for the times indicated in bicarbonatesaline-glucose media containing 27 mM K+.Trace amounts of [14C]sucrosewere added to be present for the incubation periods specified. Slice swelling and chloride spaces were in all cases essentially similar (C’ Table VI of TOWER:this Conference). Values are means of 4 or more experiments; those above the dashed line differ significantly ( P < 0.01) from the values below the line except in the case of the final entry (incubation 125 min; sucrose present 90 min). Taken from BOURKE AND TOWER (1966a). If the sucrose or inulin is added at the beginning (when the slice is immersed in the incubation medium), there is a space of about 42%whether the slices are then incubated for one hour or for two hours. However, if the sucrose or inulin is added later on, after incubation is under way (in this example up to 20 minutes before the end of incubation), there is a significantly smaller space, and in this situation the longer the period of incubation after addition of solute the larger the mace becomes, so that eventually (90 minutes after addition) it reaches the same size as that observed wh n the solute is added initially.

462

H. M. P A P P I U S

Now I don’t know the explanation for this phenomenon, but I suspect it may be as Dr. Elliott suggested, namely that some of the channels between cells are very, very small, and these size differences may reflect some sort of diffusion problem for the solute. Perhaps Dr. Rall may have a comment on this point. But one is faced with several possible explanations. (I may point out that if we select the smaller values, then we are in even better agreement with Dr. Pappius than before.) One could also suggest that the difference between the two extremes has something to do with cut surfaces of slices, although the mechanisms involved are a little more difficult to work out. H. M. PAPPIUS:First of all, Imamrelieved that Dr. Tower thinks that the differences between his results and mine are not as striking as they seemed on paper. The point of definite disagreement is still the fact that in vitro 1find a distribution of sucrose closer to that of thiocyanate than 01 inulin, while Dr. Tower, in the cortex at least, finds that the inulin and sucrose distributions are of the same order of magnitude. I have measured the distribution of these markers in white matter also, and found the same difference between the sucrose and the inulin spaces as in cortex tissue. However, the thiocyanate space in white matter became progressively smaller with time of immersion, because I think there was relatively more tissue damage and the thiocyanate was slowly penetrating into the open ends of fibers. This can be shown quite easily with nerve preparations, where the thiocyanate will equilibrate with all of the watir if you wait long enough, or increase the extent of damage by cutting the nerve. It is also interesting to point out that if, as we suggest, thiocyanate penetrates into the glial space, this does not make glia particularly different from other cells. We found in kidney or liver slices incubated in vitro that a definite non-inulin space could be demonstrated, but that thiocyanate equilibrated with all of the tissue water. In muscle, thiocyanate and inulin had the same distribution

K. A. C. ELLIOTT: We used sucrose and thiocyanate in our early experiments, because one is a charged particle and the other is not. My picture of thiocyanate is that it is a very freely permeable ion which goes into the same places as chloride. If you calculate either the chloride or the thiocyanate space from the ordinary transmembrane potential, assuming these ions are distributed only according to the potentials, the ratio of extracellular to intracellular concentration should be 1 : 16. Supposing, contrary to my own remarks, that we think of the cell as a bag, the thiocyanate inside should be 1/16 of what it is outside. Thus, the main bulk of the thiocyanate would be extracellular. That could explain why thiocyanate occupies a little greater space than sucrose, which all stays out of the cells. If you make a slice anoxic, or maltreat it in other ways, you destroy the transmembrane potential; the thiocyanate will then enter the intracellular space in much higher concentration and you will find, as demonstrated by Dr. Pappius, that the thiocyanate space steadily increases in the anoxic slice. 1 think then that these extended thiocyanate spaces are dependent on the metabolic condition of the slice and on the extent to which the membranes are damaged. There must be cells in which the thiocyanate is distributed according to the Nernst equation. There may be cells in which there is no such distribution, whose cell membrane is freely permeable to thiocyanate, permitting a lOOu/, exchange between the ions inside and outside the cell. The two cell types together make up the apparent difference in the space. The most remarkable difference, I think, between the findings of Dr. Pappius and Dr. Tower is the observation, in vivo at any rate, that the inulin and the sucrose spaces are the same. Do I understand that you find the same also in your slices? D. B. TOWER: Yes. K. A. C. ELLIOTT: This is astonishing.

I have mentioned at the beginning of my talk that when measuring spaces in v i m H. M. PAPPIUS: it is essential to establish that equilibration has been reached between the given marker in the medium and in the water compartment in the tissue available to it. 1 have found that under aerobic conditions, provided sufficient time is allowed for the marker to diffuse into the tissue, any further increase in the duration of incubation with the marker does not affect its distribution. The relevant experimental data for the distribution of sucrose, inulin, and thiocyanate are summarized in Fig. I . When cerebral cortex slices were incubated in the presence of oxygen for 120 min, the distribution of sucrose was the same whether the sucrose was present in the medium for the last 60 min of the incubation or throughout the experimental period. If, however, the sucrose had been added only 15 or 30 min

SPACES I N B R A I N TISSUE

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463 TIME W I T H SUCROSE MIN 60

I ,

SUCROSE I

I*

SUCROSE

I20

TIME W I T H INULIN

120

INULlN

ANAEROBIC

INCUBATED

120 MIN.

I ,

'

INULIN

. I

INULIN

ANAEROBIC

!

INCUBATED 60 MIN THIOCYANATE

I

20

40 60 80 MILLIGRAMS OR MICROLITERS

I

100

-I

60 120

MIN.

I'

10

60

THIOCYANATE I

'

TIME W I T H THIOCYANATE

I

0

1

I

I

120

140

160

Fig. 1. Spaces and swelling of incubated cerebral cortex slices of rat. Total length of each bar represents the average weight of slices after incubation, per 100 mg of initial weight. The portion to the right of the vertical broken line indicates the extent of swelling. The black portion represents dry weight and the open portion represents the marker (sucrose, inulin, or thiocyanate) space. The shaded portion, obtained by difference, gives the respective non-marker space. Six o r four determinations were averaged in each case. The short marks on either side of divisions show the widest range of individual determinations. before the end of the incubation, the sucrose space would have been significantly smaller in the presence of the same amount of swelling. Under anaerobic conditions the swelling was greater and equilibration did not appear to be complete even at 60 min, although the results may also indicate slow penetration of the marker into a compartment not available to it under aerobic conditions. Similar results were obtained with inulin. Aerobically, 60 min incubation with inulin was sufficient to obtain equilibration, while after 30 min a smaller inulin space was obtained (not shown on figure). Thiocyanate can be shown to equilibrate with the compartment available to it aerobically within 10 t o 15 min, and longer incubation does not affect its distribution. Results presented in the figure show, in contrast, that under anaerobic conditions thiocyanate slowly penetrates into all of the tissue water. Thus in slices incubated for 60 min, thiocyanate space increased steadily with duration of incubation in the presence of this marker. From the foregoing it isclear that it is very important to keep tissueslices well oxygenated throughout the experimental period, as both the swelling and the distribution of markers is changed under anaerobic conditions. In our initial work on spaces in v i m , we placed the slices in the medium and gassed the incubation vessels at room temperature. We consistently obtained swelling of 40%. Subsequently, we made sure that the preparation procedure was as quick as possible and that the slices were kept cold until full oxygenation of the medium was achieved. This resulted in a decrease of 10% in the swelling and a corresponding decrease in the non-inulin space (see Fig. 2). In other words, intracel-

464

H. M. P A P P I U S

;

Preparation at room temperature

Preparation in the cold

0

20

40

60

80

100

120

140

Milligrams or microliters Dry weigh!

Tissue w a t e r equilibrated with thiocyanate only

Tissue water not equilibrated with either inulin or thiocyonate

Tissue water equilibrated with both inulin and thiocyanate

Fig. 2. Effect of initial cooling on fluid compartments in cerebral cortex slices of rat incubated aerobically for 60 min.

M a r swelling occurs in tissue which is not well oxygenated and appears as increased non-inulin space. In connection with species differences reported for spaces in vivo by Dr. Tower, it may be of interest that, under optimal experimental conditions, we found that 20% of the original tissue volume equilibrated with inulin in v i m in the case of rat cerebral cortex slices, while in cat cerebral cortex slices this figure was 29%. These were two different groups of experiments, but they may indicate that species differences also exist in spaces in v i m .

H. MCILWAIN: The question of membrane potential has been raised in discussing the distribution of electrolytes and sucrose. With our measurement, both in vivo and in vi/ro, it can be shown that two clear compartments exist in cerebral tissue: extracellular and intracellular, with a difference in potential of about 60 mV between the inside and outside. From the analysis for chloride, which again is simple to determine in vivo and in vitro, one can come to some fairly direct conclusions about the size of the two compartments. I n vivo, it does give the information that there is a 26-27% extracellular space in grey matter while in the incubated tissue the value for the space is about I5 ”/, greater. One can also weigh one’s slices before and after, and we do in fact find that the weight will increase about 15 %, so that this seems a quite simple situation.

465

Delineation of Fluid Compartmentation in Cerebral Tissues D O N A L D B. T O W E R Laboratory of Neuroclietnistry, National Institute of Neurological Diseases and Blindness, Bethesda Maryland ( U.S.A.)

INTRODUCTION

Much of the preceding discussion at this Conference has dealt with barrier and transport mechanisms. Such mechanisms usually cannot be described quantitatively or meaningfully without knowledge of the relevant compartments into or out of which transport takes place and between which “barriers” to some or all solutes appear to exist. Hence I wish to consider briefly some of the important problems encountered i n attempting to delineate these compartments in terms of size and locus. I shall deal with two general situations, studies in vivo and studies in vitro, and with factors of species differences, ontogeny, areal differences and the like. If some points have been presented in earlier contributions, the repetition will hopefully have the virtue of emphasizing the importance of the problems. I would be remiss if I failed to acknowledge here our debt to the late Heinrich Waelsch, who started us thinking again about compartmentation in the nervous system in the course of a discussion at a meeting in Amsterdam almost exactly 7 years ago (Waelsch, 1960). Studies in vivo

Turning first to studies in vivo, there are several problems peculiar to the central nervous system. Solutes when introduced directly into the cerebrospinal fluid (CSF) exhibit a distinctly non-homogeneous distribution. This behavior is illustrated for trace amounts of I “C]sucrose after intracisternal injection into monkey CSF (Fig. I). Note that levels of the solute in lumbar CSF are rapidly established at 4 to 6 times comparable levels in cisternal CSF and that within several hours after injection levels of the solute in subarachnoid CSF (over surfaces of the cerebral hemispheres) are twice those in cisternal CSF (Bourke et a/., 1965). Thus, there would be considerable error introduced by referring the solute content of a cerebral cortex tissue sample to lumbar or cisternal CSF concentrations for purposes of calculating tissue spaces accessible to the solute (Table I). Even greater complexity is imposed by delivery of the solute indicator via the cerebral circulation, particularly if tissue spaces are calculated with reference to plasma rather than CSF solute concentrations. Solute distribution is importantly influenced by factors of solute diffusion and/or Ri,/l.renres p . 478-480

D. B. TOWER

466

10:'

.h -

E

.

10':

10'

Cistarnal Lul

05

I

naurs

5

.

I

.

50

10

Fig. 1. Distribution and levels (in cpm/ml.) of UL-[13C]sucrose(S.A. 3 ,/rc/,/cM) in monkey CSF (ordinate, log scale) as a function of time after intracisternal injection of 1 pc (abscissa, log scale). Individual observations are plotted except at 6 h for which means (iS.D.) of 3 or more samples are given. The slopes have been fitted by the method of least squares. (Reproduced from TOWER1965 Fig. I.)

TABLE I F L U I D S P A C E S I N C E R E B R A L C O R T E X it1 V i V O : F A C T O R O F R E F E R E N C E P O I N T FOR C A L C U L A -

TIONS

Tiitie after i.c. injectioii

Sucrose spaces ( O4) calctrlatedfrotn siicrose coticetiti atiotir in :

of [ ' T ] s i ~ r o s e

Si~barachrioirl (adjacent) CSF

Cistertial C SF

Luiiihar C SF

Ih 6h 22 h

32.5 34.1 33.9

3.25 134.5 67.5

1.35 32.4 13.3

Derived from studies on monkeys (Fig. I ) repotted by BOURKEet a/. (1965).

transport. We need not dwell on this point but merely recall, for example, the distribution data for [Wlthiocyanate under conditions identical to those for sucrose in Fig. I . Cisternal levels of thiocyanate decrease much more rapidly after injection, primarily as a consequence of the transport system mediating its efflux from the central nervous system (Pollay, 1966; Pollay and Davson, 1963; Streicher et a]., 1964; Welch, 1962). Thus thiocyanate levels in cisternal CSF are less than half those found for sucrose at 5 to 6 h after injections of identical amounts, and very different ratios of solute levels in lumbar CSF (0.3-0.4) or subarachnoid CSF (0.6-0.9) to those in cisternal CSF are found (Bourke et a/., 1965). The two factors just cited emphasize the importance of choosing the proper point of reference, i.e., which value of extracellular fluid solute concentration is to be used for

DELINEATION O F

CSF

467

COMPARTMENTS

TABLE I1 F L U I D S P A C E S I N C E R E B R A L C O R T E X it2 ViVO: F A C T O R O F S P E C I E S D I F F E R E N C E S ~

Brain Weigh1 (g)

Species Rabbit Chimpanzee

10

380

Relative Neurotr Detrsity

Chloride Spaces

44 13

30 45

I 70)

Sucrose Spaces

f %) 23 40

-

Data taken from TOWER(1954) and BOURKEet al. (1965). All differences shown are statistically significant ( P . 0.01 or better). '

calculation of the tissue spaces accessible to the solute. The principles here involve not only the question of CSF VS. plasma but the proximity of the CSF sample to the tissue sample taken for analysis and a knowledge of the behavior of the solute after administration. I t has only recently become apparent that for cerebral cortex, at least, the sizes of tissue fluid compartments delineated by chloride, thiocyanate, inulin or sucrose vary as a direct function of species brain size. Details of these correlations have been reported by us elsewhere (Bourke et a/., 1965), so that I will simply show an illustrative example, comparing data for rabbit with those for chimpanzee(TableI1). The point to be emphasized here is that one cannot generalize about cerebral compartments but should specify and consider species individually. The factor of species differences may have a bearing on conclusions derived from electron microscopy as discussed below. Tissue heterogeneity is obviously a much more serious problem in neural tissues than i n most other body tissues, such as liver or muscle, upon which studies of fluid compartmentation have beenconducted. There are not only gross areal differences (cortical gray, subcortical white, cerebellar cortex, etc.), as discussed below, but probably also more subtle differences between various anatomically or physiologically defined areas within the same gross subdivision. In this context the differences in size of chloride space in the various layers of allocortex in the region of Ammon's horn (Lowry et al., 1954) provide an illustrative example (Table 111). T A B L E 111 C H L O R I D E S P A C E S IN C O R T I C A L L A Y E R S OF A M M O N ' S HORN ( R A B B I T ) - ~-

Layer

Morphology

CI Space ( oh)

Thicktress ( p )

Dry Wt. ( %)

200

20.6

30.6

I50 400

27.1 23.3

38.5 39.0

50 250 200

17.0 20.4 30.3

31.3 29.9 45.8

-~

Molecularis Lacunosum Radiata Pyramidalis Oriens Alveus

Terminal dendrite and axon arborization; pial vessels Dendrites; rnyelinated fibers Dendrites - closely picked Cell bodies (closely picked), giving origin to fibers of other layers Non-rnyelinated axons; dendrites Myelinated fibers

Taken from data reported by LOWRYer al. (1954). Chloride spaces have been calculated assuming an extracellular CI- (CSF) of 12'3 pequiv./ml (BOURKEel a / . , 1965). Xcqrrcmes p. 478-480

D. B. TOWER

468

Swelling

CI Space

(70)

(701

K

Na

(peq./g.)

Fig. 2. Fluids and electrolytes of cat cerebral cortex in samples biopsied immediately (open bars) or 30 min (solid bars) following circulatory arrest. All values (ordinare) are referred to fresh weight of tissue and areexpressedinthe units specified beneath each set of bar graphs. Means of 2 or more determinations are given and except for those for CI space, differences shown are statistically significant (P < 0.01 or better). (Derived from data reported by TOWER,1967.)

Finally, we should not overlook pathological factors and the artifacts which they may superimpose on such underlying complexities. Consider, for example, the effects of circulatory arrest for 30 min before sampling brain tissue and adjacent CSF (Fig. 2). Such a period of ischemia is not unusual for a tissue sample being excised incident to a neurosurgical procedure and is certainly brief compared to most intervals before postmortem sampling. The critical consequences in the tissue are intracellular (nonchloride space) edema, loss of K+ and gain of Na+, changes which are reflected by a pronounced rise of CSF K+ (from the normal 3.8 to 29.5 pequiv./ml) and a persistence of tissue swelling and abnormal electrolyte distribution during subsequent studies in vitro (Tower, 1967). The elevation of CSF K+ in the cat under these conditions is of the same order of magnitude as has been reported for human CSF postmortem (Mason et ul., 1951 ; Naumann, 1958).The edema observed here is only one of several types of edema, each with rather characteristic sites of predilection, which may be encountered (Table IV). The foregoing examples of problems associated with delineation of fluid compartments in neural tissues in vivo do not represent a complete listing, but they should suffice to stimulate thinking and discussion about these and other problems at the in vivo level. Studies in vitro

Turning now to in vitro approaches to the delineation of compartments in neural tissues, one would expect to have the opportunity to study compartments and factors affecting them under more precisely defined and more readily controlled conditions. Basically such is the case, but these advantages are countered to some extent by new problems peculiar to the in vitro situation. Such studies generally involve the use of

DELINEATION O F

CSF

469

COMPARTMENTS

T A B L E IV SOME TYPES OF C E R E B R A L E D E M A ENCOUNTERED

in ViVO

Dry Weighr (04) Cerebral Cortex Subcortical White

Species aiid Pathology

Man: Tumorn

C E Rabbit: Alkyl tin” C E Cat: Cold lesionC C E Car: Circulatory arrest“ C E

17.4 16.9 20.2 20.0 17.9 18.5 16.0 13.5

Degree of Swelling ( 74)

30.7 17.4 30.8 22.9 31.2 22. I 29.6

-

C: controls; E : experimental (italicized values differ significantly from respective controls); w: white matter swelling; c: cortical swelling. a STEWART-WALLACE (1939); ALEUer at. (1963); PAPPIUSand GULATI (1963); TOWER (1967) I ” “ ” ’ “ “ “ ‘

40

i

E.. 0,

b

4

,’

a

.-

,

I

.

Ouabain

20Control

,

0

,

I

I

0.5

,

,

I

t

I

incubation time

L

U

2

i

Ihrs.)

Fig. 3. Swellingor imbibition ofextra fluid in slices of cat cerebral cortex (ordinate) as a function of time after immersion (abscissa) in bicarbonate-saline-glucose incubation media containing 5 m M K+. Mean values ( & S.D.) for 4 or more determinations are plotted and are all referred t o initial fresh weights of tissue. Swelling attributable to “adherent” medium or of the “preparatory” type (see text) is denoted by x . Data on slices incubated at 37”are denoted by 0 for control slices incubated aerobically; by 0 for slices incubated anaerobically; and by A for slices incubated aerobically in the presence of M ouabain. (Derived from data reported by BOURKEand TOWER, 1966a.)

tissue slices (in which cellular integrity and tissue architecture are largely preserved) incubated under optimal basal or carefully specified experimental conditions. Most of the examples to follow are taken from our own studies on incubated slices of cat cerebral cortex (Bourke and Tower, 1966a; b), unless otherwise specified. When carrying out such incubations in vitro, one immediately appreciates the potentialities of neural tissues for swelling or imbibition of additional fluid. Fig. 3 summarizes the unavoidable in vitro artifacts so well investigated by McIlwain and colleagues (Varon and McIlwain, 1961 ; Keesey et al., 1965). A portion of the total swelling of inReferences p. 478480

470

D. B. T O W E R

Lc

0

I

1

20

40

I

60

Medium K’

I

I

80

100

,

120

I

140

I

(mM/L)

Fig. 4. Swelling in incubated slices of cat cerebral cortex (ordinate)as a function of K +concentration in the incubation medium (abscissa). Mean values (k S.D.)for 4 or more determinations are plotted and are all referred to initial fresh weights of tissue. All incubations were carried out aerobically at 37” for 1 hr in bicarbonate-s3line-glucose media containing as principal anion either 125 mM chloride (0) or 125 mM isethionate (0).(Reproduced from BOURKEand TOWER 1966a. Fig. 4.)

cubated slices is attributable to incubation medium “adherent” to cut surfaces of the slices-a portion which is demonstrable within seconds after immersion of fresh slices in incubation medium, which is accessible to all indicator solutes including inulin, and which amounts to about 8-10 per cent of the initial fresh-tissue weight even with greatest care of slicing. A second portion of the total slice swelling is associated with “preparatory” procedures prior to institution of optimal incubation conditions - a type of swelling which is progressive with time, which is inaccessible to inulin but is accessible to chloride,andwhich can be minimized (as in Fig. 3) to about 5-10 per cent of the initial fresh-tissue weight by rapid, careful preparation of slices. Thus, with care the total swelling or excess fluid uptake (“adherent” medium plus “preparatory” swel1ing)can be held to about 12-16 per cent of the initial freshweight of cortical slices (Keesey ef al., 1965; Bourke and Tower, 1966a). During subsequent incubation under the usual optimal conditions, little if any further swelling occurs (control, Fig. 3), but swelling of or analogous to the “preparatory” type can increase to major proportions if suboptimal conditions persist, as illustrated here by anoxic conditions (N2, Fig. 3) or conditions which interfere directly with cellular cation transport systems (ouabain, Fig. 3). Superimposable upon the foregoing is additional swelling of slices dependent upon the K+ concentration of the incubation medium and the type o f principal anion therein (Fig. 4). Note the degree o f slice swelling associated with K + concentrations of 100 or more ,uequiv./ml of incubation medium. Such high K + media have been used by some investigators to study various aspects of neural tissue metabolism, and one wonders about the significance of such data in view of this degree of fluid imbibition. The substitution of a relatively non-diffusible anion (isethionate = 2-hydroxyethane sulfonate) for the usual chloride of incubation media clearly prevents the development of such K+-dependent swelling of cortical slices (Bourke and Tower, 1966a).

DELINEATION OF

CSF

i

27mM K

Chloride; SCN

0

Sucrose; lnulin In vivo- spaces-

47 1

COMPARTMENTS

Chloride; SCN In Vitro

Sucrose; lnulin

0 Swsliinp

Fig. 5 . Comparison of fluid spaces in vivo with spaces and swelling irr vitro in slices of cat cerebral cortex. Mean values (ordinate) of 4 or more observations are expressed in per cent of initial fresh weight of tissue for biopsied slices (open bars) and for slices incubated aerobically at 37" for 1 h in bicarbonate-saline-glucose media containing either 5 mM K+ (data on left) or 27 mM K+ (data on right). Fluid spaces in incubated slices are denoted by the hatched bars and slice swelling is denoted by the solid bars, which are positioned to indicate the extra fluid added to that originally measured by chloride or sucrose irr vivo for comparsion with chloride or sucrose spaces measured in vitro. The horizontal lines through the bars denote the levels of values obtained for slices incubated under the same conditions in media containing 125 rnM isethionate instead of chloride. All differences shown are statistically significant ( P . 0.01 or better). (Derived from data reported by BOURKE ef a/., 1965 and BOURKE and TOWER, 1966a.)

I20

I

-I

125 mM K

40

20

Chloride

Sucrose

Fig. 6. Same depiction as Fig. 5 for slices of cat cerebral cortex incubated in bicarbonate-salineglucose media containing 125 mM K + .See legend for Fig. 5 for details.

Relationships between the fluid of swelling and spaces of incubated slices accessible to various indicator solutes are summarized in Figs. 5 and 6 . In the case of slices incubated in a 125 m M Ki- medium (Fig. 6), note the discrepancy in size between tissue spaces delineated by sucrose and those delineated by chloride and how these spaces in vitro relate to those measured irt vivo and to the swelling associated with incubation R&rcnrrs

p. 4711-480

D. B. TOWER

472

c 0

ICP

10''

10.'

10-8

10.'

Ovaboin (MI

Fig. 7. Contents of K+ (a),Na+ (0) and CI- (A)in pequiv./g and the swelling ( x ) in per cent of initial fresh weights of tissue (ordinate) for slices of cat cerebral cortex as functions of the molar concentrations of ouabain in the incubation media (abscissa).Mean values ( f S.D.) for 4 or more observations are plotted. All incubations were carried out aerobically at 37" for 1 h in bicarbonate-salineglucose media containing 5 mM K+. The arrows near the righthand ordinate scale denote correspondand ing mean concentrations of Na+, K+ and CI - in the incubation media. (Reproduced from BOURKE TOWER 1966b, Fig. 2A.)

TABLE V in vitro:

FLUID SPACES A N D ELECTROLYTES OF CEREBRAL CORTEX SLICES INCUBATED

EFFECTS OF A D D I T I O N S TO THE I N C U B A T I O N MEDIUM

Conditions

Control ( 5 mM K+) 10 mM glutamate* 10-5 M ouabain Anaerobic 27 mM K+

+ +

Swelling ( %)

16.8 27.8 30. I 44.4 32.7

Spaces ( %) CISucrose

62.2 59.4 104.3 107.2 64.1

47.5 47.5 46.0 46.6 42.2

(pequiv.ld

K+

Nu+

74.9 74.0 28.4 27.1

95.4 113.4 132.8 166.3 75.3

99.9

Incubation 1 h at 37" aerobically (except as noted) in bicarbonate-saline-glucose media. Italicized mean values differ significantly (P < 0.01 or better) from control mean values (from BOURKEand TOWER, 1966a, b). * Final tissue glutamate level was 29.2 pM/g compared to a mean control value of 9.55 and to a final concentration in the incubation medium of these experiments of 4.7 pM/ml (TOWER, 1962).

in vitro. Similar but less extreme examples are provided by the observations on slices incubated in media containing 5 or 27 mM Kf (Fig. 5). If during incubation the K + concentration of the incubation medium is changed from 5 to 27 mM or vice versa, no change in size of slice chloride spaces and only minimal changes in size of slice sucrose spaces occur, yet there is a significant change of slice swelling, implying shifts of fluid into (5 + 27 K) or out of (27 + 5 K) intracellular spaces of the slices (Bourkeand Tower, 1966a, b). The fact that chloride (and thiocyanate) spaces in virro are considerably

DELINEATION O F

CSF

473

COMPARTMENTS

T A B L E VI F L U I D S P A C E S A N D E L E C T R O L Y T E S O F C E R E B R A L C O R T E X SLICES I N C U B A T E D EFFECTS OF V A R I O U S A L T E R A T I O N S O F I N C U B A T I O N M E D I A

Conditions

Swelling ( %)

Spaces ( %) CISucrose

K+

NU+

Control (27 mM K) 98 mM Li/Na

32.7 36.5

64.1

42.3

99.9

75.3

84.8

58.8

59.3

19.3

98 mM Choline/Na

24. I

70.5

36.6

83.3

20.I

27 mM Rb/K

33.3

56.5

43.3

17.2

79.4

125 mM Isethionate/CI

14.4

56.2*

36.6

105.5

61.2

M Ouabain

45.1

115.2

45.9

46.1

127.2

in vitro:

f ,wuiv./d Other

117.4 (Li) 75 (Choline) 84.6 (Rb) 35 (Iseth.)

-

N

-

Incubation I h at 37” aerobically in bicarbonate-saline-glucose media. Jtalicized mean values differ significantly ( P < 0.01 or better) from control mean values. * Determined with SCN- (from BOURKEand TOWER,1966a, b).

larger than those in vivo raises the possibility that there may be a mechanism operating in vivo to exclude (“pump” out) chloride from some compartment (? glial) and that under in vitro conditions this mechanism fails. Effects related to specific transport systems are illustrated in Fig. 7 and Tables V and VI. Ouabain in concentrations which completely inhibit Na+ extrusion and K+ accumulation by incubated slices exerts comparatively much less effect on slice swelling or on slice spaces accessible to sucrose (Fig. 7; Bourke and Tower, 1966b). Addition of glutamate to the incubation medium is associated with a significant degree of intracellular (non-chloride space) swelling (Table V), which presumably reflects transport into and concentration within cells of glutamate so that it can function there as additional intracellular anion (Pappius and Elliott, 1956; Tower, 1962; Bourke and Tower, 1966a, b). When other ions are substituted for normal components of the incubation media, striking effects on swelling, spaces and electrolytes of incubated slices may ensue (Table VI). The outstanding feature of this last set of observations is the disparity or lack of parallelism between changes in slice swelling and changes in accessibility of tissue spaces to chloride or to sucrose. Thus, increased (or decreased) swelling is not necessarily accompanied by increase (or decrease) of chloride spaces, and the latter may expand markedly without any change in swelling and/or sucrose spaces. The socalled Na+ substitutes, Li+ or choline+, not only interfere with monovalent cation transport but also affect fluid and solute distribution in such slices (Bourke and Tower, 1966a, b). It would be of considerable interest to be able to examine the role of water itself in the various shifts of fluid which are encountered. In studies on plants, the use of D2O in place of H2O has been informative in this context. For example, segments of apical stem from etiolated pea seedlings normally elongate and gain weight during incubation under optimal in vitro conditions. This “growth” is almost entirely attributable to inReferences p . 478-480

474

D. B. T O W E R I

I

CI

Iseth

Swelling (%)

CI

Sucrose

Spaces 1%)

K Na lpeq./g )

Fig. 8. Comparison of fluid spaces and electrolytes in slices of cat cerebral cortex incubated in media employing HzO (open b m ) or DaO (hatched bars) as solvent. All values (ordinate) are means of 4 or more determinations referred to initial fresh weights of tissue and are expressed in the units denoted beneath each set of bar graphs. Only the differences in swelling and in K t content are statistically significant ( P :. 0.01 or better). All incubations were carried out aerobically at 37" for 1 h in bicarbonate-saline-glucose m-dia containing 27 mM K+.(From unpublished studies by TOWER, D. B. and D. A.) TOWER,

creased water content and fails to occur when the incubations are conducted in DzO media (Tower, D. B. and Tower, D. A. - unpublished). Analogous experiments with incubated slices of cat cerebral cortex yield quite different results (Fig. 8). In D20 media additional slice swelling occurs- swelling which is independent of K+concentration or anion species in the medium which is not paralleled by changes in slice spaces accessible to chloride or sucrose, but which is associated with a significant increase of tissue K + content. The import of such findings remains to be elucidated. The relevance of the foregoing examples to the problems of delineating compartments in slices of neural tissue incubated in vitro should be immediately evident. It is seldom possible or permissible to suggest direct correlations between such experiments in vitro and apparently analogous situations in vivo. Yet certain of these observations may be relevant to studies in vivo under experimentally altered conditions. The use of perfusion fluids of abnormal composition and the addition of amino acid (iontophoretically or in bulk) or extra Ki or ouabain represent a few examples ofrecorded studies where such correlations deserve consideration. Comparative and Ontogenetic Aspects

Some obvious differences of fluid compartmentation in corpus callosum of cat brain compared to cat cerebral cortex are illustrated in Fig. 9. Data on cell densities of the two tissues (Heller and Elliott, 1954) indicate that the total number of non-neuronal cells (mostly glia) per unit volume is about equal in cerebral cortex and corpus callosum. If, as has been suggested by a number of investigators, some of the swelling and some of the spaces accessible to chloride in cerebral cortex are identified with glia (? astrocytes), then glial cells of the subcortical white matter (corpus callosum) must be-

DELINEATION O F

CSF

COMPARTMENTS

47 5

I 0 White Gray

80

““i 40

20

Dry Wt.

(%I

Swelling

(%I

CI

lnulin Spaces (%I

K No (peq./g.)

Fig. 9. Comparison of fluid spaces and electrolytes in incubated slices of cat cerebral cortex (solid bars) with those of cat corpus callosum (open bars). All values (ordinate) are means of 2 or more determinations referred to initial fresh weights of tissue and are expressed in the units denoted beneath each set of bar graphs. All differences shown between cortex and corpus callosum (except for N a content) are htatistically significant ( P .: 0.01 or better). The horizontal lines across the bar graphs denote the levels of values observed for biopsy samples in vivo, which with the exception of values for dry weight ’ or better). All incubations were and for K ‘ content differ significantly from in vi/ro values (P -0.01 carried out aerobically at 37“ for I h in bicarbonate-saline-glucose media containing 27 mM K+. (Derived from data by TOWER and BOURKE,1966.)

have quite differently under comparable conditions in vitro. This deduction should not come as a surprise since morphologists have long ago recognized a number of differences for glia in these two locations, but again the differences emphasize the principle of not generalizing about brain but referring more specifically to the area or subdivision in question. I t is interesting that in vitro it is cerebral cortex samples that swell so readily (Fig. 9), whereas ill vivo it is the subcortical white matter which seems most prone to edema (Table IV). Also, in contrast to cerebral cortex where both in vivo and it1 vitro sucrose spaces = inulin spaces and both are chloride spaces, in corpus callosum in vitro chloride spaces = sucrose spaces and both are > inulin spaces (Tower and Bourke, 1966). What the morphological counterpart is for the space in corpus callosum accessible to chloride and sucrose but inaccessible to inulin remains to be determined. Ontogenetic studies, particularly in a species like the cat, may be very helpful in localizing more precisely some of the phenomena referred to above. Kittens are especially useful because brain maturation involves 3 postnatal months and henceevents are sufficiently spread out in time that some tentative correlations become possible, as discussed in details elsewhere (Tower and Bourke, 1966). Some of the data obtainable in this way for kitten cerebral cortex are illustrated in Fig. 10. By one month postnatal age, maturation of cortical neurons is complete (Noback and Purpura, 1961 ; Voeller et a / . , 1963; Purpura et a/., 1964). Coincident with this stage one can demonstrate for the first time K i -dependent swelling of incubated cortical slices and the establishment of adult levels of tissue K+ and Na+. Association of these biochemical parameters with mature cortical neurons is thus strongly suggested. Similarly the onset of additional s’

R ~ ~ f i i pw. 478-480 i ~ ~

476

D. B. TOWER

Fig. 10. Changes in the extent of swelling (Sw), sucrose spaces (Sucr) and chloride spaces (CI) in incubated slices of kitten cerebral cortex as functions of postnatal age (abscissa). Values for fluid compartments (ordinate) are means ( & S.D.) or 4 of more observations expressed as per cent of initial fresh weights of tissue. Values which differ significantly (P < 0.01 or better) from the value immediately preceding are indicated by solid symbols. Values for mature cat cerebral cortex are plotted for age 12 (+) months. All slices were incubated aerobically at 37" for 1 h in bicarbonate-saline-glucose media containing 27 m M K'. See text for discussion of morphological correlations. (Derived from data reported by TOWERand BOURKE,1966.)

slice swelling and larger chloride spaces is first demonstrable at about 3 months postnatal age well after completion of cortical myelination (Noback and Purpura, 1961 ; Tower and Bourke, 1966) but coincident with the period of glial proliferation in kitten cortex(Brizee and Jacobs, 1959a,b). Thus it is possible that the biochemical characteristics which become demonstrable at this stage may be associated with glial (? astrocytic) cells. The examples considered briefly here illustrate how comparative and ontogenetic approaches may be helpful in elucidating the significance or nature of various observations obtained on adult tissues. CONCLUDING R E M A R K S

In all that has been said here and by others at this Conference there has been little appraisal of the many intracellular compartments which are lumped together by our relatively crude space measurements into the non-inulin or non-chloride spaces of neural tissues. This complex category includes at least six different cell types (neurons, astrocytes, oligodendroglia, microglia, ependymal cells and endothelial cells), and within each cell there are a number of additional compartments (nuclei, mitochondria, endoplasmic reticulum, etc.). All these compartments have obvious relevance to problems of solute transport and the barrier systems imposed thereby. For example, various studies with radiosodium and radiopotassium indicate that only about 80-90 per cent of the cortical Na+ and Kf is rapidly exchangeable, so that the residual portion may be contained within an intracellular compartment with rather distinct permeability characteristics (Keesey and Wallgren, 1965; Bourke and Tower, 1966b).

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Recent investigations of ionic transport into and out of mitochondria clearly indicate the importance of this intracellular compartment (Ernster, 1965; Rasmussen et al., 1965; Chance and Mela, 1966; Tager et al., 1966). Although most such studies have been carried out on mitochondria from non-neural tissues, the observations by Rossi and Lehninger (1963) indicate that brain mitochondria can beexpected to conform to the general pattern for other tissues in this respect. The apparent discrepancy between sizes of fluid spaces discussed here and the much smaller sizes proposed by most electron microscopists requires a brief comment. Since this problem has been considered at length elsewhere (Bourke et al., 1965), I shall confine my remarks to three points. Firstly, the factor of species differences (v.s., Table 11) has an obvious bearing here, since most observations by electron microscopy have been carried out on rodent species with small brains, which from our investigations would be expected to exhibit relatively small interstitial spaces, whereas many of the estimates of such spaces based upon physiological (impedance) and biochemical (solute indicators) methods have involved species with larger brains for which larger interstitial spaces would be expected. Secondly, there is the problem of artifact in electron micrographs, a factor which has largely been ignored or overlooked by electron microscopers. The changes which specimens of neural tissue undergo during fixation and dehydration for embedding and examination by electron microscopy are summarized in Table VII (Bourke, R. S . , Wanko, T. and Tower, D. B. - unpublished). As originally pointed out by Bahr e t a / . (1957), these changes imply gross distortions of the tissue samples and presumably of the cells therein during the process of fixation and dehydration, so that any interpretation of the original geometry of the sections becomes very difficult. Since much of the swelling maybe intracellular, either on an asphyxial(Van Harreveld e?a/., 1965, 1966a) or fixation (Bahr et a/., 1958; Tooze, 1964) basis, extracellular spaces of neural tissue samples could be partially or completely obliterated during processing of the tissue sections. Thirdly, the dimensions involved should be considered. Recent studies demonstrate that molecules the size of ferritin can penetrate through interstitial spaces quite readily (Brightman, 1965) and that such relatively narrow clefts of interstitial fluid are entirely adequate to support neuronal activity (Nichols and Kuffler, 1964). It would not take much artifactual swelling of adjacent cells to narrow the interveningextracellular space to one-half or one-third its normal width or volume. The differences between interstitial spaces 150 A wide and those 300 A wide may not be very striking in an electron micrograph but for a cuboidal space the volume would be doubled (for example, from 10 to 20 per cent) and for a cylindrical space the volume would be tripled (for example, from 10 to over 30 per cent). That such possibilities are real has been nicely demonstrated by Rall et a / . , (1962) and by Van Harreveld et a/., (1965; 1966a), who showed the prompt and striking decrease of extracellular space postmortem or with asphyxia. Undoubtedly part of the confusion over interpretation of electron micrographs purporting to show very small (< 5 per cent) interstitial spaces arose because of apparent agreement of such estimates with space measurements in vivo based on such solutes as thiocyanate and sulfate before it was realized that the latter when referred to Rcfiwnces p . 478480

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D. B. TOWER

T A B L E V11 E F F E C T S O F F I X A T I O N A N D D E H Y D R A T I O N O N S L I C E S OF C A T C E R E B R A L CORTEX

Change in total Slice solids weight of slice ( “/o of unfixed wt) ( 0; of fresh wt)

Conditions

Unfixed Fixed 30 min in I Osoc Fixed 5 min in I <: OsO? then Dehydrated in l0-50% ethanol; then Dehydrated in 70-100% ethanol

-

( 1 18.8

16.0 22.8 19.9

1.003 1.015 1.015)

I 84.6 -20.4

10.4

0*928! 0.767

<:

-i- 29.8

1 !

S.G. l f Solutions

‘

-

Slices (150 mg; 0.5 mm thick) were fixed in bicarbonate-saline-glucose (pH 7.4) containing OSOJI 7 ; (wlv). Dehydration was carried out successively for 10 min each in 10,25,50,70,80,90,95 and I O O ~ , ethanol (vlv.). Total weight changes are corrected for specific gravity (S.G.) of fixation and dehydration solutions. All values refer t o final stages indicated and are means of 5 or more determinations. Each mean value differs significantly (P < 0.01 o r better) from any other value in the same series. (From unpublished studies by BOURKE, R. S., WANKO,T. and TOWER,D. B.)

plasma for calculation of tissue spaces accessible to these solutes yielded erroneous values because of the outward transport of these solutes from CSF (and brain) to plasma (Pollay and Davson, 1963; Streicher, et al., 1964; Pollay, 1966; Richmond and Hastings, 1960; Van Harreveld et al., 1966b). Taking the three foregoing factors into account, it seems likely that most previous estimates of sizes of interstitial spaces in electron micrographs of cerebral tissues are too low and that the discrepancy between these estimates and those obtained with impedance studies or with solute indicators like inulin is only an apparent discrepancy attributable to fixation artifacts and the like. My purpose here has been not so much to present a formal paper in the usual sense but to pick out a number of points with which, hopefully, to stimulate the discussion to follow and to recall for us the fluid environment and compartmental framework within which barrier and transport phenomena are taking place. ACKNOWLEDGEMENT

Travel provisions by the U.S. Navy, Office of Naval Research, are gratefully acknowledged. REFERENCES

R. AND TERRY,R. D. (1963) Structure and electrolyte analyses of cerebral ALEU,F. P., KATZMAN, edema induced by alkyl tin intoxication. J . Neuropathol. Exptl. Neurol., 22, 403413. BAHR,G. F., BLOOM,G. A N D FRIBERG, U. (1957) Volume changes of tissues in physiological fluids during fixation in osmium tetroxide or formaldehyde and during subsequent treatment. Exptl. Cell Res., 12, 342-355. BAHR,G.F., BLOOM, G. AND JOHANNISSON, E. (1958) Further studies on fixation with osmium tetroxide. Hisrochein., 1, I 1 3-1 18. BOURKE, R. S., GREENBERG, E. S. AND TOWER, D. B. (1965) Variation of cerebral cortex fluid spaces in vivo as a function of species brain size. Anrer. J. Physiol., 208, 682-692.

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BOURKE, R. S. AND TOWER, D. B. (1966a)Fluid compartmentation and electrolytes of cat cerebral cortex in v i m - I . Swelling and solute distribution in mature cerebral cortex. J. Neurochem., 13, 1071-1 097. -, (l966b) Fluid compartmentation and electrolytes of cat cerebral cortex in vitro-11. Sodium, potassium and chloride of mature cerebral cortex. J. Neurochem., 13, 1099-1 117. BRIGHTMAN, M. W. (1965)The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. I I . Parenchymal distribution. Atiter. J. Anat., 117, 193-220. BRizEE, K.R. A N D JACOBS, L. A. (1959a) Postnatal changes in volumetric and density relationships of neurons in cerebral cortex of cat. Acta Anat., 38, 291-303. -, (1959b) The glia/neuron index in the submolecular layers of motor cortex in the cat. Anat. Rec. 134, 97-105.

CHANCE, B. A N D MELA,L. (1966)A hydrogen ion concentration gradient in a mitochondrial membrane. Nature, 212, 369-372. ERNSTER, L. (1965)Control of cell metabolism at the mitochondrial level. Fed. Proc., 24, 1222-1235. HELLER, I. H. A N D ELLIOT,K. A. C. (1954)Desoxyribonucleic acid content and cell density in brain and human brain tumours. Canad. J. Biochent. Physiol., 32, 584-592. KEESEY, J. C. A N D WALLGREN, H. (1965)Movements of radioactive sodium in cerebral cortex slices in response to electrical stimulation. Biochent. J., 95,301-310. KEESEY, J. C.,WALLGREN, H. AND MCILWAIN.H. (1965)The sodium, potassium and chlorideof cerebral tissues: maintenance, change on stimulation and subsequent recovery. Biocheni. J., 95, 289-300. LOWRY,0. H., ROWRTS, N. R.,LEINER, K. Y.,WL', M. L., FARR,A. L. ANDALBERS, R.W.(1954) The quantitative histochemistry of brain. 111. Ammon's horn. J. biol. Chern., 207, 3949. MASON, J. K., KLYNE.W. A N D LENNOX, B. (1951) Potassium levels in the cerebrospinal fluid after death. J. clin. Pathol., 4,231-233. NAUMANN, H. N. (1958) Cerebrospinal fluid electrolytes after death. Proc. SOC.Exptl. Biol. Me(/., 98, 16-18. NICHOLS, J. G. AND KUFFLER, S. W. (1964)Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neurophysiol., 21, 645-671. NOHACK, C. R. A N D PURPURA, D. P.( 1961)Postnatalontogenesisof neurons in cat neocortex. J. Conip. Nertrol., 117, 291-307. PAPPIUS,H. M. A N D ELLIOTT,K. A. C. (1956)Factors affecting the potassium content of incubated brain slices. Canarl. J. Biocheni. Physiol., 34, 1053-1067. PAPPIUS, H. M. AND GULATI, D. R. (1963)Water and electrolyte content of cerebral tissues in experimentally induced edema. Acta Neuropathol., 2, 451-460. POLLAY,M. (1966)Cerebrospinal fluid transport and the thiocyanate space of the brain. Amer. J . Pliy.siol., 210, 275-279. POLLAY, M. A N D DAVSON, H. (1963)The passage of certain substances out of the cerebrospinal fluid. Brai~/,86, 137-1 50. PURPURA, D. P., SHOFER, R. HOUSEPIAN, E. M. A N D NOBACK, C. R. (1964)Comparative ontogenesis of structure-function relations in cerebral and cerebellar cortex. Growth and Maruration of the Brain. Vol. 4.of Progress in Bruit/ Research. D. P. Purpura and J. P. Schadk (Eds.). Amsterdam, Elsevier, pp. 187-221. RALL,D. P., OPPELT,W. W. A N D PATLAK, C. S. (1962)Extracellular space of brain as determined by diffusion of inulin from the ventricular system. Life Sci., 1, 4343. RASMUSSEN, H., CHANCE, B. AND OGATA,E. (1965)A mechanism for the reactions of calcium with mitochondria. Proc. Natl. Acad. Sci. ( U S A ) , 53, 1069-1076. RICHMOND, J. E. A N I ) HASTINGS, A. B. (1960)Distribution of sulfate in blood and between cerebrospinal fluid and plasma irr vivo. Anter. J . Physiol., 199, 814-820. Rossi, C. S. A N D LwNINcitx, A. L. (1963)Stoichiometric relationships between accumulation of ions by mitochondria and the energy-coupling sites in the respiratory chain. Biocheni. Z., 338, 698-71 3. STEWART-WALLACE, A. M . ( I 939) Biochemical study ofcerebral tissue, and changes in cerebral edema. Braifr, 62, 426-438. SmEicHER, E., RALL,D. P. A N D GASKIN, J. R. (1964)Distribution of thiocyanate between plasma and cerebrospinal fluid. Atner. J. Physiol., 206, 251-254. TAGER, J. M., VELDSEMA-CURRIE, R. D. AND SLATER, E. C. (1966)Chemiosmotic theory of oxidative phosphorylation. Nature, 212, 376-379.

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TOOZE,I. (1964) Measurement of some cellular changes during fixation of amphibian erythrocytes with osmium tetroxide. J. Cell. Biol.,22, 551-563. TOWER,D. B. (1954) Structural and functional organization of mammalian cerebral cortex: the correlation of neurone density with brain size. Cortical neurone density in the fin whale (Balaenoptern physalus L.) with a note on the cortical neurone density in the Indian elephant. J . Comp. Neurol., 101, 19-52. -, (1962) Molecular transport across neural and non-neural membranes. Properties of Membranes andDiseasesofthe NervousSystem, D.B.Tower, S. A. Luse, and H. Grundfest. New York, Springer, pp. 1-42. -, (1965) Problems associated with studies of electrolyte metabolism in normal and epileptogenic cerebral cortex. Epilepsia, 6, 183-197. -, (1967) Distribution of cerebral fluids and electrolytes in vivo and in vitro. Brain Edema. F. Seitelberger and I. Klatzo, (Eds.). Vienna, Springer (pp. 303-332). TOWER,D. B., AND BDURKE,R. S. (1966) Fluid compartmentation and electrolytes of cat cerebral cortex in vitro-Ill. Ontogenetic and comparative aspects. J. Neurochem., 13, 11 19-1 137. VANHARREVELD, A., AHMED,N. AND TANNER, D. J. (1966a) Sulfate concentrations in cerebrospinal fluid and serum of rabbits and cats. Amer. J. Physiol., 210, 777-780. VAN HARREVELD, A., CROWELL,J. AND MALHOTRA, S. K. (1965) A study of extracellular space in central nervous tissue by freeze-substitution. J. Cell Biol., 25, 117-137. VANHARREVELD, A. AND MALHOTRA, s. K. (1966b) Demonstration of extracellular space by freezedrying in the cerebellar molecular layer. J. Cell Sci., 1, 223-228. VARON,S. AND MCILWAIN, H. (1961) Fluid content and compartments in isolated cerebral tissues. J . Neurochem., 8, 262-275. VOELLER, K., PAPPAS,G . D. AND PURPURA, D. P. (1963) Electron microscope study of development of cat superficial neocortex. Exptl. Neurol., 7 , 107-130. WAELSCH, H. (1960) An attempt at integration of structure and metabolism in the nervoussystem. Structure and Function of the Cerebral Cortex. D. B. Tower and J. P. Schadk (Eds.). Amsterdam, Elsevier, pp. 3 13-327. WELCH,K. (1962) Concentration of thiocyanate by the choroid plexus of the rabbit in v i m . Proc. Soc. Expfl. Biol. Men., 109, 953-954.

DISCUSSION L. BAKAY:In connection with what Dr. Elliott mentioned at the beginning, I think we have quite a bit of evidence showing that morphologically visible extracellular spaces can enlarge. That certainly has been seen in the white matter why it is not seen in grey matter is a n enigma: But in the white matter it can visibly enlarge. Although there is perhaps no evidence, there is a hint that potentially existing, but closed, extracellular spaces (perhaps similar to the muscle capillaries that are just to be closed) do exist in the nervous system and can be expanded under abnormal conditions. In edema, fluid accumulates within the myelin sheath. This happens by splitting of the myelin sheath, and this splitting occurs at the intraperiodic line. This might be a latent extracellular space that happened to be invaginated by the rolling around of the myelin. The splitting of the myelin at that particular point might represent an expansion of the existing but closely approximated extracellular space. There was one thing I wanted to say about Dr. Tower's findings: It is true that if you ligate the carotid artery you get swelling, and the sodium and the chloride spaces increase in the swollen tissue. Now this happens t o be limited to the cortex, which he showed. However, I personally feel very uneasy about ischemic anoxia, rather than hypoxic or anoxic anoxia, because in ischemic anoxia, in addition to anoxic damage, you get other kinds of things. If you render the cortex ischemic by carotid occlusion, in addition t o the swelling you get a back-flow of blood from surrounding relatively normal area which maintains its arteriolar pressure, so that you get a dilation, particularly in the grey matter. Normally, in the cat cortex the blood content is about 34 per cent, or it can be as high as 4 per cent. You can get a lot of sodium and chloride that is intravascular, which actually might

-

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augment the sodiuni or the chloride space, not necessarily on the basis of the enlargement of the space in the tissue, but by the addition of this sodium- and chloride-containing vascular compartment. D. B. TOWER: If I may continue with that point; I think you probably arecorrect under the conditions which you specify. In the experiments which we did, we clamped all the vessels, not just the carotid - a complete clamp around the neck so that everything was trapped in the skull. Thus, at least in our experiments, the sodium and chloride concentrations in the cerebrospinal fluid were the same as in the control animals. The only change in the ions that were measured was for the potassium, which increased. I f you recall the slide, the chloride space was exactly the same size, whether the biopsy was taken immediately after clamping the vessels or 30 minutes later, so there was no increase in the space accessible to chloride. This does not say where the chloride was distributed, however. Furthermore, when one takes samples from such a preparation and incubates them, instead of finding the normal degree of slice swelling in response to extra potassium in the incubation medium (cf. the potassiumthis : conference), the swelling has already taken place, and no more depending curve of Fig. 4 in T O W ~ R swelling occurs iir virro. I think this is definitely an intracellular swelling in the potassium-dependent area and confirms the fact that the cells are swollen in the biopsy specimen.

D. P. RALL:Turning to the question of the difference between the inulin and sucrose space: with our diffusion methods wc have examined the two indicators in the caudate nucleus and mid-brain and found a very significant difference. Sucrose, is perhaps, as you suggested, moving into the smaller channels more freely than inulin. I regret that we have not done this in cortex. W. W. TOURTELOTTE: I would just like to ask Dr. Tower, when he tied the ligature around the neck, how he felt about the circulation of the vertebral arteries. In regard to Dr. Bakay's thought that there is 3 i to 4"/, of the blood in the cerebral cortex, I would say this is very different from the literature data. I t means that you have marked vaso-dilatation in the experimental set-up you have.

I,. BAKAY: The 3 "A to 4';" value of the cat cortex I took from literature data in which the blood volume was measured by haemoglobin-determination. Incidentally, in soriir grey matter, in some basal ganglia, and for example in the geniculate bodies, it is higher. As far as the white matter is concerned, true enough, it is less than f but some layers of cortex are verj, very vascular.

x,

D. B. TOWER:May I answer the one question about the ligature very simply. We did not use a ligature around the neck. Dr. Elliott many years ago invented a very fancy apparatus which instantly crushes t h e whole area, vertebral, spinal cord, spinal column, and evervthing. W. W. TOURTtLOTTE: Then I suggest the possibility that you have stimulation of nervous tissue from below, which may be driving a lot of impulses in anteriorly, and that you also now have a cerebral stimulation as well. G . Lcvi: I would like to ask about the existence of regional differences in such phenomena as swelling and inulin space, against the background of experiments that 1 am doing now in a glucose-free medium. Slices from the cerebral hemisphere do not show any change in inulin space, but there is an increase in sodium space, so that one can assume that it is intracellular sodium. But cerebellum and medulla oblongata d o show a slight increase in inulin space of the order of 7 to 8 %. Correspondingly the swelling, instead of increasing about 10:; in a glucose-free medium. increases about 15 to 1776, so that there may be intracellular undextracellular swelling. There is another question I would like to ask Dr. Pappius: I think that she did some studies on inulin in an inulin-free medium, and found that some 5 to 6 or 7% of the labeled inulin remains in the slices. I wonder if you haveany explanation for this?

H. M. PAPPIUS:I an1 well aware that if one inhibits metabolism there is a substantial increase in total swelling of the tissue. Hobever, as you point out, in cerebral cortex slices there is no increase in the inulin space. Thus, it appears that inhibition of metabolism leads to intracellular swelling. I have no experience with the cerebellum or medulla oblongata. In the experiments in which slices containing inulin were transferred to an inulin-free medium, the amounts of inulin were not trace amounts, because a t the time we were still analyzing inulin chemically. The inulin remaining in the slice was presumably in equilibrium with that which had leaked out into the medium.

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D. B. TOWER

M. BRIGHTMAN: I realize that in order to make many basic assumptions and average values, cells must become idealized spheres and clefts. I would feel a little happier if I could leave at least one little impression, for my departmental colleagues as well as myself, that the spaces, and the cell borders which make up there spaces, vary exquisitely from point to point in the same region, let alone between different species. We have come to realize very recently that we can at will open and close these spaces, depending on the way we fix them. But if the fixation is reasonably good, defined in terms of the cellular cleft, and if the organelles are in the right place, for example, then the spaces between the cellular membranes and the material between them approximate something like the normal state, perhaps even the living state. I make no claim as to the size of this gap. What I can say, based on the tracers we used, is that it is probably not under 100 A units, the size of one of the larger markers. The variation, however, is something else again. Where three processes meet, you have angles and corners, and the space there naturally is greater. Whatever it was in life, of course, we still don’t know. The modest conclusion then is that this gap is very likely a hydrated one, permitting hydrophylic molecules to wander between it. And that is a very modest conclusion.

D. B. TOWER: We have discussed informally, Dr. Wyke and the rest of us, the possibility that these spaces might pulsate or open up under certain circumstances, and 1 think this is a point which bears on this discussion. However, I don’t know how we might demonstrate such a phenomenon. It has been shown, e.g., that when fluid is passing across the gall bladder wall, the spaces between the cells can expand tremendously, and when no fluid is passing, the cells are closely apposed. This is a possibility which deserves to be considered too.

K.A. C. ELLIOTT:When we were dealing with the measurement of the40% swelling that occurs in the slice preparation, we wondered whether the electronmicroscopist could be expected to see any change in spaces. We showed quite clearly that if slices underwent the ordinary formalin fixation, afterwards one could not tell the difference between the fresh slice and a swollen slice. But if we went through the process which we believed was used by the electronmicroscopist (fixation with osmium), the tissue went through all sorts of changes in weight and volume, and nevertheless at the end one could still tell the difference between the slices that had swelled and those which had not. A. LAJTHA:I would like to add evidence from our own experiments which confirms what Dr. Tower mentioned, that the inulin space itself may be in two compartments. When b e incubated brain slices and measured the rate of uptake of inulin, we had a break in the curve. We interpreted this to be caused by an initial rapid entry and then a markedly slower entry, indicating entry is occurring in two compartments, which then could be distinguished kinetically. What probably also occurs on incubation, and this in turn may depend on the medium in which the slices are incubated, is that there is an increase in swelling and thus an increase in these inulin spaces. If slices previously incubated with inulin were transferred into inulin-free medium, the exit of inulin also showed some evidence for a biphasic rate of exit. These two compartments, the rapid and the slower one, could also be distinguished by measuring inulin flux at 37 C and 0 C since some compartment does not seem to be fully accessible at O‘C. The experimental evidence from such experiments, measuring either flux rates or differences in flux from slices preloaded at 37°C versus at 0°C is not quite clear, but it does indicate more than one inulin compartment. What these may be structurally, I don’t know. They may be the smaller clefts that were mentioned, partly damaged cells, or other elements, which would, then, add a structural dimension also to the compartmentation of our inulin space. If some of this space is in damaged cells, or at least surrounded by some kind of a damaged membrane, the picture may be further complicated, since such a space could be permeable to inulin and not permeable to some of our other markers. D. M. WOODBURY: May I ask about the problem of penetration of chloride into the brain and into the two types of cells, glial cells and neuronal cells. There is evidence that the concentration of chloride in glial cells is considerably higher than that in neuronal cells. This would suggest that if there is passive distribution, there aredifferent potentials across the neuronal membranes as compared with across glial membranes. Thereis a little bit of evidence supporting this, which is not quite clear. ilf there is a difference in potential - say neurons are 16 mV or so higher - and if glial potentials were accordingly lower, then there would be only about a 40 : 1 ratio. This would make a tremendous difference in the concentration in the different compartments, so that the over-all chloride space

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itself does not really measure a particular space at all, it measures only that part which is in the interstitial fluid. When we talk about the chloride space in general, it really does not mean very much in these terms, only that it is in the interstitial space. I did want to bring this up because it is an important point in saving a place for thiocyanate. The other point is just a matter of the sucrose versus the inulin space. As we showed, the inulin and sucrose spaces in the cerebral cortex of the rat are different in young animals. I would like to call the attention of the participants in the symposium to the W. W. TOURTELOTTE: study that Sjostrand reported in some detail about the mechanisms of demyelination and about manipulating the intracellular space. He used conditions such as hypotonicity, hypertonicity, freezing, etc. He found that the space of 150 to 200 A between the cellular membranes was very resistant. I think that it is of some importance here to note that when very definite efforts were made to make tissue artifacts, the space between the cells was very constant. B. D. WYKE:I wanted to ask whether the surviving electronmicroscopist in the group would take this nice suggestion of pylsating spaces a little more seriously. It is intended seriously, because brains do pulsate, both on a micro- and a macro-scalz. When Dr. Pappas discussed these microparticles earlier at this symposium, somebody asked the question: what is the force which propulses these particles into the spaces? I wonder if it would be thought reasonable to propose a pulsating mechanism for these spaces. R. V. COXON: I have a brief question which I think will admit a simple answer, and that is: How homogeneous is the inulin that was used in the experiments? Quite a lot of work from therenal physiologists has shown that the ordinary inulin anyone buys is rather variable as far as particle size. etc.. is concerned. H. M . PAPPIUS:I have never really tested it. I measured the inulin in the slice, and I compared it to the total inulin in the medium.

D. B. TOWER:Dr. Coxon has raised an important point. With 14CC-labelledinulin, such as Dr. Woodbury and I used, there is the problem that this is not inulin, but carboxyl-inulin which carries a charge. I don't think this makes a great deal of difference. However, it has been shown by some Japanese investigators, who used tritium-labelled inulin and [14C]carboxyl-inulin,that there is a difference between carboxyl-labeled and ordinary inulin under certain circumstances. I have to make a comment relative to this; that is that we have used both tritiated D. M . WOODBURY: and carboxyl-labeled inulin, and the spaces were the same. D. P. RALL:I would like to comment on the two compartments into which inulin mighi enter. We think that we can say from the kinetic analysis that there is only one compartment inulin is entering into in vivo in the areas we have examined. Secondly, we also can tell if this compartment is mixed (as if by a little egg beater), and we think it is not mixed. We have pretty good evidence for this, which will probably rule out the idea of a pulsating system.

Y. M . PAPPIUS:I think that the impression that there are two compartments for inulin in vitro is probably related to the fact that there is grossly damaged tissue on the edge of the slice, which would be expected to equilibrate quickly, and smaller spaces in the internal part of the slice, which would take a little longer to equilibrate. C. CRONE:In reply to Dr. Woodbury's question about the potential difference, 1 just want to remind you of the work of Kuffler and his collaborators, in which they actually measured the membrane potential and found that themembrane potential in glia wasequivalent to that of neuronal elements. While talking about glia. I would like to ask if anyone in this audience would sum up the evidence that any intracellular shift of fluid is preferentially a shift into glial cells. Also, I would like to ask if anybody would comment on the idea which was popular some time ago, that the glial cells actually make up a part of the extracellular compartment. I myself have always had great difficulties in understanding such a theory, and the more so after Kuffler showed that the membrane potential in these cells corresponds to that of other cells.

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D. B. T O W E R

K. A. C. ELL1o-r-r: May I answer that? You have to make some hypothesis. There is so much sodium and so much chloride in brain tissue, that you either have to postulate an extracellular space to contain these ions or you have to assume an intracellular space, into which these ions penetrate freely.

R. KATZMAN: The evidence that glial cells take up water and fluid under pathological conditions of swelling consists entirely of electronmicrographic evidence on a wide variety of edemas, carried out by literally dozens of electronmicroscopists in various laboratories, using different methods of fixation. I might just say that the changes that you get in terms of the initial swelling at the time of fixation with various fixatives is different. You get a swelling with osmium, you get a contraction with glutaraldehyde, and then through dehydration these things are partially corrected. Yet with all of these different kinds of fixatives and fixing-agents and different treatments, on eends up with the cortex always showing the same changes, namely a swelling of the clear astrocytic glial cytoplasm. M. BRiGtrrMAN: In connection with the point you have just raised: there is an addition. It may not be just glial swelling. When one uses a marker before fixation one gets a peculiar, what I call “colorized artifact”, in that the markers sort of enter the dendrites, and the postsynaptic process. So it seems that, in addition to astroglia, the dendrites as well swell markedly and take up the marker in an artifactual way.

485

Thiocyanate in the Brain and the Size of the Extracellular Space C. M. F R E N C H The London Hospiral Medical College, London E. I . (England)

A knowledge of the size of the extracellular space of the brain would frequently be of value in the interpretation of observations relating to brain function and structure. Unfortunately there is little agreement on this topic, and values ranging from less than 5 % to over 25 % have been suggested. It is generally believed that thiocyanate ions are mainly extracellular and the thiocyanate space of tissues has therefore been equated with the extracellular space. It is however, curious that the thiocyanate space of the brain is related to the plasma thiocyanate concentration (Fig. 1). These values are in the steady state obtained after

18

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..

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-

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-

86-

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4 t

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0

I

5

I

10

I

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Plasma Conc meq / I

Fig. 1. The thiocyanate space of rat brain determined in the steady state at various plasma thiocyanate concentrations.

the plasma thiocyanate concentration had been kept constant for at least Similar results have been reported by Streicher (1961) in the rat; Streicher, Gaskins (1964) in the dog; and Pollay (1966) in the rabbit. In the present experiments the extent of the plasma thiocyanate concentration variation Rrfercncrs p. 489

6 hours. Rall and series of is much

486

C.

M. F R E N C H

larger, ranging from the minimal detectable quantity of 0.02 mequiv/l, to the maximal tolerable amount of 20 mequiv/l which rapidly killed the animals. The relationship between the space and the plasma thiocyanate concentration holds true over the to 17 whole of this range, the brain thiocyanate space rising from about 3 During these experiments it was noticed that the brains from animals with high plasma thiocyanate concentrations were lighter than those from control animals. This phenomenon was investigated more fully, using rats selected so that they weighed exactly 200 g and maintaining the plasma thiocyanate concentration constant for 6 h (Fig. 2).

x.

Brain Wt.Grarns I .84 - , I .80I .?bI .72-

1.68-

I

I

I

I

I

0

5

10

15

20

Plasma Conc meq / I Fig. 2. The dry and wet weights of rat brain measured after the plasma thiocyanate concentration had been kept constant for 6 h (each point represents the mean and standard error of the mean for 16 observations).

The dry weight varies very little and is unaffected by the thiocyanate, so that it provides a convenient reference point to which other measurements can be related and gives an indication of the original size of the brain. The wet weight, on the other hand, shows a small but very significant fall as the plasma thiocyanate concentration rises. Hence there appears to have been a loss of fluid from the brain, which is clearly seen in Fig. 3 where the water content of the tissue is expressed as a multiple of the dry weight. This method of presenting the results also has the effect of cancelling out a number of experimental variables as indicated by the smaller standard errors of the means (compare Figs. 2 and 3). From this it appears that the increase of brain thiocyanate space coincides with a shrinkage of the brain due to a loss of fluid. In Fig. 3 the water content of a skeletal muscle (left rectus femoris) from the same rats is also shown. This, like that of the brain, decreases as the plasma thiocyanate concentration increases, although the magnitude of the change is slightly smaller. A possible inference from this similarity is that other changes produced by thiocyanate in the brain, may also occur in skeletal muscle.

THIOCYANATE IN THE BRAIN

487

Tissue W a t e r I D r y Wt

t

3.7

Fig. 3. The water content of rat brain and skeletal muscle, expressed in terms of tissue dry weight, after the plasma thiocyanate concentration had been keptconstant for 6 h (each point represents the mean and standard error of the mean for 16 observations. The same animals as for Fig. 2).

Such is in fact the case, the thiocyanate space of skeletal muscle is related to the plasma thiocyanate concentration (Fig. 4). The relationship is similar to that found in brain, but the size of the change is smaller, the thiocyanate space increasing from about 10 to 16 %. It is, therefore, less noticeable, but over the large change of plasma thiocyanate concentration used here, it is quite appreciable. Thus it seems that skeletal muscle resembles brain in its response to thiocyanate. In both there is an increase of thiocyanate space and a loss of tissue fluid as the plasma Muscle Space 'lo

.

*

I '

-0

5

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20

Plasma Conc meq 11

Fig. 4. The thiocyanate space of rat skeletal muscle determined in the steady state at various plasma thiocyanate concentrations (the same animals as for Fig. 1). Ref~~r.rc~nri~.r p. 489

48 8

C. M. F R E N C H

thiocyanate concentration increases. A simple explanation, applicable to both tissues, could be that the thiocyanate in the extracellular fluid is exerting an osmotic effect, drawing fluid out of the cells, with consequent reduction of cell size. Even though this would cause some shrinkage of the whole tissue, a much larger proportion of the change would probably occur within the tissue, the extracellular fluid expanding at the expense of the intracellular fluid. Support for this hypothesis comes from the observations of Streicher, Rall and Gaskins (1964), who reported a reversible increase of brain thiocyanate uptake following intracisternal injection of hypertonic solutions. In addition an increase of both brain and skeletal muscle thiocyanate spaces has now been found after intraperitoneal injection of 100 % urea solution. In skeletal muscle the entire change of thiocyanate space could be due to these osmotic effects. However, the larger change in brain thiocyanate space suggests that in the brain there may also be other factors. In particular there may be active transport of thiocyanate from the cerebrospinal fluid to the blood in the choroid plexus, as described in an earlier presentation (Woodbury, 1966). The present observations give no indication of the location of the thiocyanate in the brain. The osmotic distension of the thiocyanate permeable compartment could occur equally well if all or part of this compartment were intracellular. One might conclude however, that only some of the cells could be involved since the effect would not be sustained if all the cells allowed unrestricted entry of the thiocyanate. The uptake of thiocyanate by rat red blood cells has a relationship with the plasma thiocyanate concentration resembling that of the brain thiocyanate space (French, 1965). An entry into one of the types of cell present in the brain might also be related to the plasma concentration, and in part responsible for the final effect, which would then be the result of choroid plexus transport, cell entry and osmotic redistribution of the tissue fluid. Whatever the additional factors, however, an osmotic effect, like that in skeletal muscle, is likely to account for an appreciable proportion of the change, In conclusion it must be remembered that the extracellular space of brain and other tissues is not fixed and unalterable. The volume of the extracellular fluid may vary in response to a number of factors such as the extracellular fluid osmotic pressure; even the thiocyanate concentrations used in physiological experiments have a considerable osmotic effect. Hence, when extracellular space size is involved in the interpretation of results, the effect of anything introduced into the extracellular fluid on the size of the extracellular space should be assessed and due allowance made for this. SUMMARY

(1) Thiocyanate space of rat brain is related to the plasma thiocyanate concentration over the range 0.02 mequiv/l to 20 mequiv/l. (2) The increase of brain thiocyanate space is accompanied by a loss of fluid from the brain. (3) Both an increase of thiocyanate space and a shrinkage due to a loss of fluid, are also found in skeletal muscle. It is suggested that this can be explained by an osmotic

T H I O C Y A N A T E IN T H E B R A I N

489

redistribution of tissue fluid, the extracellular space increasing and the intracellular space decreasing. (4) A similar redistribution of tissue fluid may occur in brain and account for a proportion of the change in thiocyanate space observed when the plasma thiocyanate concentration is varied. (5) The extracellular space is not a rigid structure and its size can vary. Therefore, when interpreting observations, it is necessary to consider alterations of extracellular space size induced by substances entering the extracellular fluid. ACKNOWLEDGEMENT

I wish to thank Miss Deborah Goodman for her valuable technical assistance. REFERENCES

C. M. (1965) The relation of thiocyanate space in brain, muscle and erythrocyte to plasma FRENCH, concentration. J. Physiol., 178, 55P-56P. POLLAY,M. (1966) Cerebrospinal fluid transport and the thiocyanate space of the brain. Anier. J. Pliysiol., 210, 275-279. STREICHER, E. (1962) Thiocyanate space of rat brain. Anier. J. Pliysiol., 201, 334-336. E., RALL, D. P. AND GASKINS, J. R. (1964) Distribution of thiocyanate between plasma STREICHER, and cerebrospinal fluid. Amter. J. Physiol., 206, 251-254. D. M. (1966) Distribution of various substances in the brain as affected by alterations WOODBURY, in active transport of cations and anions across the choroid plexus. This volume pp. 297-313.

DISCUSSION D. B. TOWER:I think this is very interesting, but I would like to pose a reservation here in regard t o the space aspect of this problem, because Dr. French is using high concentrations of thiocyanate. However, if you use tracpr quantities of thiocyanate, which I am quite sure cannot be implicated in any osmotic effects of this sort, the behavior of thiocyanate is identical with that of chloride as far as we can tell. Its distribution in the central nervous system follows the pump mechanism out of the CSF which Dr. Woodbury has shown us. I think we are dealing with:two different phenomena here. You may use thiocyanate in tracer amounts to measure distribution, or you may use it in what I might call “pharmacological amounts” to measure some other aspects such as one might do with doses of urea or glycerol. It is important to keep this distinction in mind. K. A. C. ELLIOTT:What I want to say is pertinent to what Dr. Tower said. I would not deny with the larger concentrations of thiocyanate that you could have an osmotic effect. But I am pretty sure that thiocyanate penetrates into the cells and is distributed according to the resting potential. This would make the intracellular concentration about 1/16 of the extracellular concentration under normal conditions. If you have a very high concentration of thiocyanate surely there would be a toxic effect which would decrease the transmembrane potential. If this is true, a concentration of thiocyanate within the cell would become greater. Would you agree that more is going into the cell? A perfectly reasonable explanation would be that the resting potential has changed and that thiocyanate just occupies more intracellular space. C. M. FRENCH: In support of your view, I would like to mention that Hodgkin and Huxley have shown that if you replace the chloride in the bathing fluids, surrounding a node of Ranvier, the resting potential does fall and becomes more unstable, and the spike potential is highly reduced. This is certainly a quantitative aspect and this is in agreement with what you would expect from the normal equations of ion distributions across membranes.

490

C. M. F R E N C H

G. PAPPAS: I would like to point out that the morphological observations of the effect of urea on the brain, show that the relationship between membranes consists of a 200 A space which remains the same and also remains the same when the brain is swollen. When the brain is swollen, the glial cells are, as you know, quite expended so that I am sure that the thiocyanate is probably in the glia cells. C. M. FRENCH: But this would not affect the osmotic hypothesis in the sense that it would shrink neurons and expand glial cells. It does not matter where the thiocyanate space compartment is, whether it is intracellular or extracellular. The thiocyanate would increase this osmotic pressure compared to alternative systems. May I come back to Dr. Tower’s point about the chloride? One would agree that in low concentrations the thiocyanate distribution in the brain has the same ratio as chloride between brain and CSF, but the chloride space in the brain is quite considerable: about 35%, whereas the thiocyanate space in the brain, expressed in plasma ratio, is down to about 3 %.

D. B. TOWER:If you express it on a basis of cerebrospinal fluid, it is exactly the same as the chloride for about 7 or 8 different species. I think Dr. Woodbury has data on rats, I have data on guinea pigs, cats, monkeys, chimpanzees, and I think there is no question that, when measured this way, the thiocyanate space and the chloride space are the same. It goes along with what Dr. Elliott says, that it distributes exactly the same way as the chloride distributes.

1.BAKAY: May I add one point? The idea that thiocyanate may be involved or included in some of the cells in the central nervous system occurred to us. We were, or we still are, in the process of trying to localize labeled thiocyanate electronmicroscopically, which could give us a direct answer as to whether it is incorporated in glia cells or not.

T. Z. CSAKY:You said that at 20 mg/y{ of blood levels the rats died. Why did they die? C. M. FRENCH: This isa difficultquestiontoanswer.Theratsshowanumberofchangesat thisleve1:one in particular is that the arterial venous oxygen difference is very much reduced, and there appears to be a decreased oxygen uptake from the blood. The rats show convulsions which may be related to the thiocyanate entering neurons, which may affect their metabolism or be related to the effect of thiocyanate on the neuron potential. During these convulsions the rats stop breathing, which could bring on an anoxic effect. Another possibility is that the thiocyanate is converted by rats’ red cells in vitro to cyanide, which is suggested by studies in the dog, wherr when you increase the thiocyanate concentration in the plasma, you obtain an increased cyanide concentration.

D. P.RALL:This cyanide would probably stimulate the respiration level, since this level would be an excellent respiratory stimulus. This is probably a good indication that the cyanide gets into some cells and causes convulsions. D. M. WOODBURV: I just wanted to show a slide which summarizes the CSF to plasma relationships for this cyanate. In this figure, taken from the work of Pollay (1966) in rabbits, the ordinate is the [35S]thiocyanate space in per cent and the abscissa is the plasma unbound SCN- concentration in mM/liter. A progressive increase in the plasma concentration of SCN- resulted in a concomitant increase in the SCN space of CSF and of brain. This demonstrates that SCN- transport via thechoroid plexus anion pump, like that of I and CIO -,is self blocked by large loads of the anion. If the brain/CSF ratio is plotted, the values decrease with an increase in plasma concentratioli and level off at a space of about 27?{, equal to the CI-, I - and C104- when transport is blocked. The figures in parentheses on the brain/CSF space curve are the concentrations of SCN- in brain relative to the CSF when the concentrations in brain are corrected to the volume of brain in which SCN- is distributed, namely, 20% according to Pollay. A value of 1.0 means that the level in brain water is the same as in CSF. In the non-loaded animals with a plasma concentration of 1.06 mM/I, the concentrations in brain (0.07 mM/I) = 0.35 mM/l is 5 times that in the CSF (0.07 mM/I), but lower than that in plasma by a factor of about 3. Thus, entrance of SCN- into the CSF follows the same pathways as I-, cb-,inulin and many other substances, namely, from the brain capillaries to the CSF via the interstitial space of the brain. Other substances also inhibit SCN- transport. Thus, as shown in this figure, sodium iodide and

T H I O C Y A N A T E I N THE B R A I N

70 6050 -

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30-

g 204

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PLASMA BRA I N

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, 2 2 , 2 7 5 , 1966

-

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I ! 0

1

1

I

I

I

I

1

3.0 4.0 50 6.0 7.0 PLASMA UNBOUND SCN- CONCENTRATION I N m M l L l T E R 1.0

2.0

Fig. I . Effects of thiocyanate and iodide loading and 2,4-dinitrophenol on the distribution of SS5CNin brain and CSF of rabbits. Thiocyanate ion containing SS5CN- was infused intravenously in various doses for 5 to 60 minutes and plasma, brain and CSF concentrations were determined at 6 hours post-infusion. The S 3 T N brain and CSF space values for the sodium iodide- and 2,4-dinitrophenoltreated groups were determined at 6 hours post-infusion. The sodium iodide (3 mM/kg) was given as an intravenous infusion which also contained the SS5CN -thiocyanate solution. The 2.4-dinitrophenol was given intraventricularly in a dose of 0.1 ml of a 0.05 mM solution at 0.5, I .O and 3.0 hours post-infusion of the S 3 T N -thiocyanate solution. The ordinate is the S3j-thiocyanate space (brain/plasma, CSF/plasma, brain/CSF) in per cent and the abscissa is the plasma unbound SCN- concentration in mM/liter. The figure is drawn from the data of Pollay ( I 966). See text for discussion. D N P at a constant SCN level in plasma increase the SCN- spaces of brain and CSF. Streicher (1961) has previously shown that increasing the plasma level of thiocyanate increases the SCN- space of the brain in rats, and Streicher er a/. (1961) have noted that increasing the plasma SCN-concentration also increases the SCN - space of CSF in dogs. These data of Pollay and of Streicher and colleagues provide further evidence for the active nature of anion transport out of the CSF across the choroid plexus. POLLAY, M. (1966) Cerebrospinal fluid transport and the thiocyanate space of the brain. Aniericati Jortrtml of'Physiology 210, 215-219.

SrREichER, E. ( 1961) Thiocyanate space of rat brain. Atnericair Jortrriul of Phvsiology, 201, 334-336. S T R L I C HE., ~ RRALL, , D. P. A N D GASKINS, J. R. (1964) Distribution of thiocyanate between plasma and cerebrospinal fluid. Atiierirari Journal of Physiology, 206, 251-254. D. P. RALL:I would just like to ask one question: If the thiocyanate does go into the cells, how can it simultaneously cause the osmotic changes that we are talking about? C. M . FRENCH: A suggestion may well be that it goes into soine of the cells, and therefore, it might expand in the glial cells.

D. M . WOODHURRY: I t is a time-dependent phenomenon. Hypertonic urea, as Dr. Reed and I have shown, for example, causes a loss of water from the brain early after administration when its concen-

492

C.

M. F R E N C H

tration in plasma is high and it is distributed only in a small fraction of brain, whereas later when the urea has penztrated into the brain cells, which takes a much longer period, the water goes back into the brain with the urea. Thus, the osmotic effects on the brain of many substances depend upon the rate of their penetration into the various compartments of the brain and into which compartment they enter. The same conclusions probably apply to hypertonic thiocyanate which, like chloride, is unevenly distributed in the three brain compartments, nemaly, interstitial space, glia, and neurons.

D. P. RALL:You have two simultaneous irreconcilable explanations. D. M. WOODBURY: Not necessarily, it appears to me that it is a time-dependent phenomenon, in which transients have to be considered. H. KOENIG:I would like to make two points. First: that it may not be the anion but the cation and sodium which are responsible for the osmotic effect. Some years ago I found that a hypertonic sodium lactate injected intraperitoneally into the guinea pigs, could shrink the brain in minutes, even to the point where the bridging veins ruptured and a subdural haemorrhage would result. Second: that the shrinkage may be a blood-brain barrier effect, rather than a direct effect of the anion in the intracellular fluid of the cells. The hypertonicity of the blood with respect to the brain a t the blood-brain barrier may well be responsible for the gross shrinkage that you get with high concentrations of these substances. C. M. FRENCH: While these substances are rejected by the blood-brain barrier, it is a fact that you get the same effect in muscle.

493

Some Spaces and Barriers in Postmortem Multiple Sclerosis WALLACE W . TOURTELLOTTE

AND

J U L I U S A. P A R K E R

Departmetit of Neurolog.v, University of Michigan Medical School,

Atiri

Arbor, Michigan ( U S A )

This report will deal with the chemical anatomy of human postmortem “normal” (control) white matter, as well as postmortem normal-appearing white matter and areas of demyelination (plaque tissue) in a primary demyelinating disease (multiple sclerosis). A n attempt will be made to correlate the concentration of selected chemical constituents within an anatomical space in the normal-appearing white matter and in the plaque tissue. Furthermore, some tests to evaluate the blood-brain barrier in postmortem multiple sclerosis plaque tissue will be presented. METHODS

Patient material The left cerebral hemisphere was obtained at autopsy (12 to 36 h postmortem) from 12 patients who died of multiple sclerosis; and 11 patients whose deaths were from other causes (that is, they did not have multiple sclerosis or any other gross or microscopic structural brain damage). The unfixed brain tissue was cut into 3-mm coronal sections at room temperature by an electric slicer. Each section was placed in a plastic bag, labeled, sealed, and frozen; the storage temperature was -90°C. On a thawed section, the white matter from the control brains was dissected (50 to 500 mg samples) ; in addition, similar-sized samples of normal-appearing white matter from multiple sclerosis brains were grossly dissected free of demyelinated plaque tissue, and vice versa. Each dissection was accompanied by a punch biopsy, which was used for histological analysis to confirm the presence or absence of myelin or for estimation of the axon density. Then, the dissected bits, from each multiple sclerosis patient, of normal-appearing white matter were pooled, if insufficient material was available, and the plaque tissue was pooled, if it was necessary. The dissections were weighed and homogenized in enough 0.15 M sodium chloride or water to make 20 percent homogenates or in 20 volumes of 2 : I chloroform-methanol. A portion was centrifuged (25,000 rev./min), and samples of the appropriate supernatant fiuid were taken for the various assays. Hefrrences p. 520-522

494

W. W. T O U R T E L L O T T E

Histological methods

A 16-gauge needle was used to obtain tissue for histological analyis from the centers of the dissected samples. The specimens were fixed in 10% formaldehyde solution (formalin) and labeled by a code system unknown to the neuropathologist. The samples were then embedded in paraffin, sectioned at 8 p , and stained with the pyridinesilver nitrate method of Cajal (Addison, 1950) for axons and with the KliiverBarrera method (1953) for myelin. All the biopsies taken from the dissected plaques showed severe to complete demyelination, whereas those taken from the control white matter or normal-appearing multiple sclerosis white matter samples showed normal myelination. The number of axons per unit volume of tissue was assessed in four areas of a single microscopic section from each sample with the aid of an ocular grid. The grid consisted of squares formed by eight horizontal and eight vertical lines (1 mm apart) and was used at a magnificationof 970. All axons through the entire depth ofsection falling on one or more horizontal lines were counted. A single long axon recrossing the same line or another line was counted as many times as it crossed these lines. This technique eliminates the problem of identifying the individual axons in a field and yields a number (“axonal density index”) related to the number of axons per unit volume. Water and total lipid determination

An aliquot of the homogenate was dried in a 110°C oven. The weight of the tissue in the suspension minus the weight of the dried sample yielded the data for the water content. Two methods were used for obtaining the total lipid content. The dried samples were extracted two times each with chloroform and hexane. The lipid-free sample was redried. The dry weight minus the fat-free dry weight was used to calculate the total lipids. The alternate total lipid procedure was done on a weighed tissue sample homogenized with 20 volumes of chloroform-methanol 2 : 1 . An aliquot was filtered, and the filtrate was washed three times with aqueous potassium chloride and potassium chloride-containing “Folch upper layer” as described previously (Kishimot0 and Radin, 1950). The lower layer, which contains the total lipids, was evaporated to a small volume with a stream of nitrogen, then lyophilized with benzene and weighed. This weight is a little low because the gangliosides, and a small amount of other lipids, enter the upper layers. However, there was good agreement between the two total lipid methods used - the average per cent difference was 0.9 with a range of 0.7 to 1.1 on five replications. Hemoglobin The carboxy-hemoglobin method of Gordon and Nurnberger (1956) was used on the supernatant fluids of homogenates made in water or 0.15 M sodium chloride. The two types of homogenates gave the same results. The extent of contribution of residual blood immunoglobulin-G or albumin in the brain was calculated as follows: The con-

495

POSTMORTEM M U L T I P L E S C L E R O S I S W H I T E M A T T E R

centration of hemoglobin (Hb) per kilogram (kg) of fresh tissue was divided by the concentration per ml of the patient’s blood, and then multiplied by (1-hematocrit/lOO). This value is the ml of serum per kg of brain. The g of immunoglobulin-G or albumin per kg of brain contributed by the blood in the brain was determined by multiplying the nil of serum per kg of brain by the serum concentration of albumin or immunoglobulin-G. This value when subtracted from the immunoglobulin-G or albumin for total brain tissue value yielded the concentration in the brain itself (brain immunoglobulin-G, or albumin corrected for residual blood in the brain). It is possible that a method based on the analysis of hemoglobin such as thecarboxyhemoglobin method of Gordon and Nurnberger is not an appropriate method for determination of total hemoglobin in all postmortem brain tissue. Post-mortem brain tissue, which is acidic (pH 6-6.5), enhances the formation of methemoglobin, which is not detected by the carboxyhemoglobin method. Hence, we have tried the cyanmethemoglobin procedure of Evelyn and Malloy (1938). This method detects both methemoglobin and hemoglobin. We have applied both methods to the supernatant fluid of both control and multiple sclerosis brain tissue. In approximately ten percent of the cases there existed a significant difference. An example of a statistically significant difference(p 5 0.05)of ml of blood per kg of brain (mean standard l error of the mean) was: control white matter (Stant.), carboxyhemoglobin method 3.0 0.32 and cyanmethemoglobin 8.0 f 0.58; however, normal-appearing multiple sclerosis white matter (Wacht) contained 6.0 & 0.31 and 12.0 0.61, and the plaque (Wacht) contained 6.0 f 0.31 and 14.0 0.72, for the carboxyhemoglobin and cyanmethemoglobin method, respectively. When a significant difference was found utilizing the two methods, the bloodcorrected brain values were based on the cyanmethemoglobin method.

*

*

*

Gangliosides Weighed tissue samples were homogenized with 20 volumes of chloroform-methanol 2 : I . An aliquot of the suspension was filtered, and the filtrate was washed three times with aqueous potassium chloride and potassium chloride-containing “Folch upper layer”. The aqueous layers were combined, and removal of contaminating lipids was accomplished by a back-wash with “Folch lower phase”. This was followed by methanolysis of the dried residue of the washed upper layer, and gas chromatographic determination of the ganglioside fatty acids. A mixture of methyl esters of 19 : 0 and 21 : 0 was used as an internal standard (Kishimoto and Radin, 1966). It should be noted that the final data represent moles of ganglioside, rather than of sialic acid. Hydroxyproline The white matter from two control brains (LaJ and Campb) and the normal appearing white matter and plaques from five multiple sclerosis brains (Hi, Bo, Re, Wi and Wa) were used for the analysis of hydroxyproline. Twelve homogenates (enough 0.15 M sodium chloride to make 20 percent homogenates) were prepared. Singlicate analyses were carried out on 250 ,ul of the homogenate by the method of Prockop and Rtfcrviice.> p.

520-.’22

496

W. W. TOURTELLOTTE

Udenfriend (1960) in the laboratory of Dr. A. Sjoerdsma, Chief, Experimental Therapeutics Branch, National Heart Institute, Bethesda, Maryland. Since blood vessel walls contain 9 mg of hydroxyproline per kg (Sjoerdsma, personal communication) we have attempted to correct the brain tissue value for the presence of blood vessel walls. If one-tenth of the volume of blood in the brain per kg is blood vessel walls, then control white matter on the average should contain 45 mg hydroxyproline per kg because of blood vessels (4.0 ml of blood per kg (see Table 11) multiplied by 0.1 and divided by 9 mg per kg), and normal-appearing multiple sclerosis white matter and plaques, 54. The correction for hydroxyproline in plasma was not significant, since plasma contains 10 y/ml. Albumin and immunoglobulin-G The immunochemical procedure of Kabat et al. (1948) was used, except that a biuret reaction was carried out to determine the protein concentration of the immunoprecipate instead of a micro-Kjeldahl procedure. Furthermore, we have scaled down the procedure so that the final color reaction is read in a final volume of 60 pl. Ten pl of antibody reagent (5 mg antibody protein/ml) was added to a 50 pl aliquot of the supernatant fluid obtained after centrifugation of a 20 per cent 0. I5 M sodium chloride homogenate of brain tissue. The immunoprecipitate that results after 18 h of incubation (I h at 36°C and 16 h at 4°C) was washed two times with 100 pl of cold 0.15 M sodium chloride. The precipitate was dissolved in 60 pl of a biuret reagent and read at 540 m p in a DU Beckman spectrophotometer adapted to read 35 pl. The antibody reagents were prepared in rabbits immunized with purified human albumin or immunoglobulin-(; suspended in Freund’s adjuvant. Immunoelectrophoresisof the human serum against the rabbit immune serum was used tocheck the purity of the antibody reagent. Since the procedure of Kabat et al. (I 948) has been standardized for body fluids and not for extracts from solid tissues, it was necessary to test its applicability to 0.15 M sodium chloride brain extracts. Standard solutions of immunoglobulin-G were recovered quantitatively (90 to 110 percent) when added to homogenates of control white matter, as well as to those from normal-appearing white matter and plaque tissue from brains of patients that had died of multiple sclerosis. Bromide Seven multiple sclerosis patients were given 0.64 g of sodium bromide (orally) three times per week for one to three years prior to death to maintain a serum bromide ion concentration that was non-toxic and did not produce pharmacologic effects (2.0 to 5.4 mequiv./l). The postmortem brain tissue was cut into 3-mm coronal sections and stored frozen, as described above. The dissected specimens of normal-appearing white matter and plaque tissue were homogenized in enough water to make a 20 percent suspension. An aliquot of the homogenate was used for dry and fat-free dry weight. Another aliquot (20 to 150 mg) was taken for bromide ion analysis. The organic matter was destroyed by fusion with sodium hydroxide and potassium nitrate as recommended by Brodie and Friedman (1938). The remaining ash was dissolved in

POSTMORTEM M U L T I P L E S C L E R O S I S W H I T E MATTER

497

water, and the bromide was oxidized to bromate, followed by iodometric titration. A GilmontR microbiuret calibrated to deliver 0.2 p1 was used to titrate 2 1.0 pg of bromide; the coefficient of variation was 11.7 percent. When 15 pg of bromide were titrated the coefficient of variation fell to 4.4 percent (Haerer et al., 1964). It was also shown that brain tissue from patients who died with multiple sclerosis, but who had not received sodium bromide before death, did not contain interferring substances. Sodium, potassium, and chloride Two g of postmortem white matter were obtained from a control brain (Campb) and also from a multiple sclerosis brain (Wacht), which was separated into normal appearing white matter and plaque tissue. After obtaining the exact weight of the tissue, a 20 percent homogenate in water was made and 0.25 ml was taken for dry and fat free dry weight. Duplicate aliquots of 1.0 ml were ashed at 400°C overnight, and the ash was dissolved in 0.5 ml of 1 N nitric acid. The concentration of sodium and potassium was determined by internal flame photometry. For chloride ion determination it was necessary to neutralize the phosphoric acid generated during ashing, so 0.1 ml of 1.7 M sodium carbonate was added to 2.0 ml of homogenate before the 1.O ml duplicate aliquots were taken for ashing. The ash was dissolved in 0.5 ml of 1 N nitric acid, and the chloride ion was determined in the Cotlove (1958) titrator. (The above analyses were carried out by Dr. J. P. Chandler, Chief of the Core Laboratory, Clinical Research Unit, University of Michigan.) TABLE I SOME H Y P O T H E T I C A L W H I T E M A T T E R S P A C E S ~

__ Aiiatotriical spaces

Normal Vascular True extracellular

Glia (oligodendroglia and astrocytes) Myelin Axoplasm Multiple sclerosis (add following): Expanded true extracellular space?

Reactive glia (microglia, lipomacrophages, fibrous astrycotes, myelin forming cells) Perivascular inflammatory cuffs (mostly lymphocytes)

Some possible chemical correlates

Hemoglobin (specific) Electrolytes (chloride ion space) Bromide ion chloride ion space Albumin and immunoglobulin-G do. Total lipids (2&25 ”” not myelin lipids) (cerebrosides may be better indicator) electrolytes Gangliosides (specific) Electrolytes (potassium ion space)

-

-

Electrolytes (chloride ion space) chloride ion space Bromide ion Albumin and immunoglobulin-G do. + Hydroxyproline (“specific” for fibrous astrocytes?) + Immunoglobulin-G synthesis Immunoglobulin-G synthesis

498

W. W. TOURTELLOTTE RESULTS A N D DISCUSSION

Some possible white matter spaces

In tabular form the normal anatomical white matter spaces, and also other spaces possibly introduced by multiple sclerosis, are listed in Table I. The normal anatomical spaces in white matter have been well delineated by light and electron microscopy. The non-porous endothelium of the blood vessels is surrounded by a basement membrane to which glial feet are attached. The glial feet probably encase no more than 80% of the capillary wall (Brightman, 1965). Between plasmalemmas of adjacent glial cells there exists an electron microscopic lucent pericellular space (true extracellular space about 100-200 A wide; Katzman, 1966). However, this space is irregular, for there exist intermembrane fusions between glial feet around blood vessels and many enlarged confluences measuring as much as 400 to 800 A across (Brightman, 1965). This true extracellular space has been estimated to consist of approximately 12 percent of the volume of the white matter (Oldendorf and Davson, 1967). Inside the glial cell exists the myelin sheath. There are also periodic nodes of Ranvier permitting an exposure of the axon to the true extracellular space. On the other hand, the situation is more complex when we examine the light and electron micrographs of the normal-appearing white matter and plaque tissue of multiple slcerosis. Normal-appearing white matter near a plaque shows normal myelin sheaths, an occasional microglial cell with phagocytic activity, and a mildly expanded extracellular space (PCrier and GrCgoire, 1965). Blood vessels in the area of plaques of demyelination have perivascular inflammatory cuffs (mostly lymphocytes and lipophages). Furthermore, at the plaque edge new cells appear, such as microglia, lipophages, reactive astrocytes, and perhaps myelin-forming cells. In the plaque, fibrous astrocytes become prominent and axons without a myelin sheath are present (Adams and Kubik, 1952; PCrier and GrCgoire, 1965). The latter have demonstrated with electron micrographs, in cases studied one to three hour post mortem, an expanded true extracellular space and a thin layer of glial cytoplasm applied to the basement membrane of some of the blood vessels. The possibility exists that the true extracellular space in PCrier and GrCgoire’s material is an artefact introduced by the rupture of glial cells secondary to swelling that occurred during the postmortem period prior to fixation. Also in Table I we have indicated some chemical constituents that are probably exclusively compartmentalized to a particular anatomical white matter space. It is well accepted that the determination of the hemoglobin in brain tissue can yield an estimate of the volume of the blood vascular space if the hematocrit of the circulating blood is known. It is possible that the capillaries produce a streaming of the blood in the brain. Hence, the hematocrit of the blood circulating in the brain may be different from that in a larger vessel from which blood is sampled to determine the hematocrit. If this is the case, as some have suggested (Dobbing, personal communication), the true estimation of the blood vascular space must await the development of an ultramicrohematocrit method for capillary blood.

POSTMORTEM M U L T I P L E S C L E R O S I S W H I T E M A T T E R

499

The true extracellular space and the glial cell space in normal white matter probably have a similar electrolyte profile (sodium, potassium, and chloride). Whether bromide ion is an indicator of extracellular and glial cell chloride ion space in postmortem white matter has yet to be resolved (see section on bromide ion). Perhaps macromolecules such as albumin and immunoglobulin-(; (corrected for contribution by the blood) may reside primarily in the true extracellular space, as has been shown for other macromolecules, such as ferritin (Brightman, 1965). On the other hand, the myelin sheath is rich in lipids, and the total lipid determination can be used as a type of indicator of the myelin sheath space. Our data indicate that in completely demyelinated white matter approximately 20-25 percent (40 g of lipids per kg) of the total lipid of normal-appearing white matter is non-myelin lipid. Perhaps the use of cerebroside as a myelin sheath space indicator would be more appropriate. The cerebroside of white matter is probably located mainly in the myelin (Kishimoto ef al., 1967) and is, therefore, a more specific measure of myelin concentration than total lipids, which include axonal lipids and those naturally in glial and endothelial cells, as well as the myelin lipids. Data we have recently accumulated and that of other investigators strongly suggest that gangliosides of white matter are located primarily i n the axons (Kishimoto et a/., 1967). Within multiple sclerosis lesions the situation is vastly more complicated. If the expanded true extracellular space in plaque tissue, described by Ptrier and GrCgoire, (1965) is not an artifact, the glial cell space is essentially non-existent. However, their electron micrographs show at least in some instances that the basement membranes of the capillaries tranversing the demyelinated areas are still surrounded by glial cytoplasm. Perhaps this can function as a type of regulator of the blood-brain barrier. Hence, we may have electrolytes (sodium, potassium, and chloride ion), as well as bromide ion, albumin, and immunoglobulin-G, as an indicator of this space. In the case of plaques of demyelination an appreciable amount of material (onesixth to one-third of the total dry weight) “forms” sheets or fibers during homogenization (Kishimoto et a/., 1967). Incidentally, these sheets from the plaque tissue do not seem to have been noted by other workers. Possibly it is a conglomeration of fibers derived from proliferated fibrous astrocytes, seen in abundance with the electron microscope (Perier and GrCgoire, 1965). Even though collagen fibers have not been seen in plaques (Harken, personal communication), we have carried out hydroxyproline determination of homogenates, hesitantly suggesting that hydroxyproline may be a chemical correlate for these fibers. Other complications appear to be introduced by multiple sclerosis. Our data suggest that immunoglobulin-(; is synthesized in multifocal areas of the multiple sclerosis brain (Tourtellotte and Parker, 1965; Tourtellotte and Parker, 1966a; Tourtellotte and Parker, 1966b; Tourtellotte and Parker, 1967). We have suggested that the sites for formation are located at the edge of the plaque and in theperivascular inflammatory cuffs (mostly lymphocytes) (Simpson et al; Tourtellotte et al., 1966). Furthermore, it would appear that immunoglobulin-(; can move through the brain from areas of synthesisinto thecerebrospinal fluid(Tourte1lotte and Parker, 1966b). Hence, immunoglobulin-(; concentration compared to that in control white matter, or a ratio of References p . 520-522

500

W. W. T O U R T E L L O T T E

TABLE I1 BLOOD I N POSTMORTEM WHITE M A T T E R

Brain diagnosis

Whole blood (serum) mllkg

Number of brains

Control

10

4.0 =t1.3* (2.6 f 0.7)

Multiple sclerosis Normal appearing white matter

11

4.8 f 2.0 (3.2 i 1.2) 4.9 2.0 (3.1 & 1.3)

Plaque

*

*

Same 11 as above

Mean i s.e.m.

immunoglobulin-G to albumin, is offered as a chemical correlate to the synthetic space of immunoglobulin-G in the multiple sclerosis brain tissue. Blood in postmortem white matter The data in Table 11 show that there is on the average about 4.0 ml of blood per kg of brain in the control white matter and approximately 20 percent more in multiple sclerosis normal-appearing white matter and plaque tissue. This difference was not statistically significant (p < 0.2 and > 0.1). Relationship between lipid, water content, and fat-free dry weight in postmortem white matter The data are shown in Table 111. The control white matter contained 187 g/kg and approximately 16 percent less in the normal-appearing multiple sclerosis white matter (p > 0.05). The lipid content in the completely demyelinated plaques was markedly reduced to a mean value of 44 g/kg. Hence, on the average 143 g/kg represented the TABLE 111 R E L A T I O N S H I P B E T W E E N L I P I D , W A T E R C O N T E N T , A N D F A T F R E E D R Y W E I G H T IN P O S T MORTEM HUMAN BRAINS

Brain diagnosis

Control white matter Multiple sclerosis Normal-appearing white matter Plaque

*

Mean f s.e.m.

Fat-free dry weight glkg

Number of brains

Total lipids glkg

10

187 f 4.0*

706

12.0

107 i 1.5

11

158 f 1.6

136 f 30.6

106 f 5.1

same 11 brains as above

44 f 6.0

Water glk

850

* 56.6

106

* 2.2

POSTMORTEM M U L T I P L E SCLEROSIS W H I T E MATTER

50 1

lipid content of the myelin sheath, and on the average 44 g/kg represented the lipids in other tissue elements, such as glial cells, lipophages, blood vessels and the associated perivascular inflammatory cuffs, and the normal-appearing but demyelinated axons which course through the plaque tissue. The significant increase of the water content (p < 0.05 and > 0.02) in plaque tissue appeared to replace the loss of the myelin. Hence, the tissue volume of the multiple sclerosis white matter may stay normal because a water space moves in when the myelin sheath moves out. These observations may explain in part the clinical observation that brains of multiple sclerosis patients have only a modest reduction of the total brain weight even though there exists a severe degree of demyelination. In our series the 1 I control brains weighed on the average (* SEM) 1300 g (* 36) and the 12 multiple sclerosis brains, I150 (*45). The 11.6 percent reduction in brain weight is statistically significant (p < 0.02 and > 0.01). Hence, we must be cautious about concluding that water replaces myelin loss precisely. On the other hand, the fat-free dry weight (mostly protein) was constant for the three tissues studied, 106-107 g/kg. Ganglioside content of postmortem white matter (Kishimoto et al., 1967) Only the data for stearic acid-containing gangliosides are shown in Table IV, TABLE IV G A N G L l O S l D E C O N T E N T O F POSTMORTEM W H I T E MATTER

Brain diagnosis

Control white matter Multiple sclerosis Normal-appearing white matter Plaque

*

Mean

Number of obser varions 1 1 (3 patients)

3 (2 patients) 8 (Same 2 patients as above)

p M / k g lipid 189

16.0*

223 (126, 238, 306) 126 232

1 1 50 f 96.5

910(720,840, 1170) 4870 f 543.7

s.e.m.

calculated on the basis of brain and lipid weight. Stearate represents about 85 % of the fatty acids in gangliosides, and no notable difference was seen in the relative amounts of the other acids(l6 : 0, 18 : I , 20 : 0). The most striking finding is that the concentration of gangliosides in fresh tissue is rather similar in plaque tissue, normal-appearing multiple sclerosis white matter, and control samples. The P value for the difference between control and plaque concentrations using the student t-test for the mean obtained from each patient was > 0.05. This retention of the ganglioside is emphasized by the comparison with the weight of total lipid. Where control and normal-appearing white matter values range between Rrfrrences p . 520-522

502

W. W. T O U R T E L L O T T E

TABLE V RELATION BETWEEN A X O N A L DENSITY I N D E X A N D G A N G L I O S I D E C O N C E N T R A T I O N

Brain diagnosis

Number of ohservatioris

Axonal density index

Ratio ganglioside concenrratiori per axon density index ~~

Control white matter Multiple sclerosis Normal appearing white matter, Plaque

*

5 (3 patients) 2 (2 patients) 7 (same 2 patients as above)

116

+. 17

13 (58 and 87) 46 4 12

1.4 4 0.15

2.7 (2.2 and 3.2) 2.5 :I: 0.46

Number of axons touching a standard grid per unit volume of tissue.

610 and 1,670 ,umoles/kg lipids, the plaque tissue ranged between 2,540 and 6,890 The relationship between axon densities and gangliosides concentration is shown in Table V. The five control samples counted yielded an average axonal density index of I16 (SEM = f 17), while the two normal-appearing white matter samples examined yielded a lower value, 73 (58 and 87). The density index for seven plaque tissue samples was 26 & 12, which is significantly lower than the control values ( p < 0.01). It would appear from these data that there is roughly a 60% decrease in axon density. Since gangliosides are apparently primarily neuronal substances, it is interesting to compare the ratio, ganglioside concentration in whole tissue divided by the axonal density index. Taking the data from the actual samples counted, the average ratio is 1.4 rt 0.5 in the control samples, 2.7 (2.2 and 3.2) in the normal-appearing white matter and 2.5 & 0.46 in the demyelinated samples. The difference in ratios between control and demyelinated matter is not significant. Even closer agreement is seen in the ratios from the normal-appearing white matter and plaque tissue of the same patients. Our finding that the ratio, ganglioside concentration: axon density, remains relatively unchanged during demyelination suggests that the gangliosides are located in the axon rather than in glial cells or myelin. As yet there is no method for isolating axons in quantity or for histological staining for gangliosides, so the question cannot be settled. Preparations of myelin isolated by centrifugal methods vary in their ganglioside content, and it may be that the gangliosides appear in the myelin as the result of axonal contamination or of ionic binding of liberated gangliosides (Norton and Autilio, 1966). Since multiple sclerotic plaques contain other formed elements besides axons, particularly astrocytes, there is the possibility that some of theplaquegangliosides are derived from these cells. However, Lowden and Wolfe (1964), in a study of pathological human cortex exhibiting markedlossof neurons and heavy astrocytic proliferation,

503

POSTMORTEM M U T L I P L E SCLEROSIS W H I T E MATTER

found a great decrease in ganglioside concentration. From this it would appear that astrocytes contain little or no gangliosides. If the gangliosides of white matter are primarily contained in the axoplasm, determination of the ganglioside concentration in white matter may give a value related to the axon density or the state of the axons. A particularly sensitive index of the state of a section might be obtained if both cerebroside and ganglioside determinations were made. The cerebrosides of white matter are probably located mainly in the myelin and are, therefore, a more specific measure of myelin concentration than total lipids. Such a pair of determinations might aid in distinguishing primary from secondary demyelination. TABLE V I T OT AL H Y D R O X Y P R O L I N E C O N C E N T R A T I O N O F POSTMORTEM W H I T E MATTER

Hyrlroxyproline mglkg Control

Multiple sclerosis

~~

Brain diaprosis

Control LaJ. * Csrnpb. Multiple sclerosis Hinch.

White matter

Total

Corrected for blood vessels

62 60

17 15

Bog.

Redd. Wacht. Wi-diffuse dernyelination Mean

61

16

Normal-appearing white matter Total

Corrected for blood vessels

54 82 90 84

0 28 36 30

78

24

Plaque

Total

Corrected for blood vessels

148 204 200 118 141 162

94 150 146 64 87 108

* Patient identification

**

See text.

Total hydroxyproline concentration of postmortem white matter. The preliminary data are shown in Table VI. The control and the normal-appearing multiple sclerosis white matter had similar hydroxyproline concentrations on the average, 61 to 78 mg/kg, whereas the plaque tissue had approximately 2.5 times this value, 162. When a type of correction was made for blood vessel walls, which are rich in hydroxyproline (see Methods), there is a further relative increase to approximately 6 times the control value. Further experimentation is necessary to determine whether the increase of hydroxyproline in plaques is due to exclusive subcellular localization in the fibrous astrocytes. If the fibrous material that we have noted on homogenizing plaques of multiple Rifiwnces p . 520-522

504

W. W. T O U R T E L L O T T E

I""]

Fig. 1. Water, sodium, potassium, and chloride content of postmortem white matter. Control patient was Campb and the multiple sclerosis (MS)patient was Wacht.

sclerosis can account for the increase in hydroxyproline noted in the total homogenate, and if these isolated fibers appear like fibers of fibrous astrocytes with electron microscopy, the compartmentalization of hydroxyproline to fibrous astrocytes will be on a sounder experimental base. Sodium, potassium, and chloride content of postmortem white matter The data shown in Fig. 1 agree well with those published by Lowenthal(1961). For control cerebral white matter they found a sodium concentration of 65 to 78 mequiv./ kg and potassium of 56 to 72. Our sodium values were 77, 68 and 76 mequiv./kg for control white matter and normal-appearing multiple sclerosis white matter and plaque tissue, respe:tively. On the other hand, our potassium values were 60, 72, and 57 respectively. To our knowledge human postmortem white matter chloride values have not been reported; our values were 47, 42, and 50 mequiv./kg. It is known that death destroys electrolyte transport mechanisms of the brain (Katzman, 1966). Furthermore, to our knowlegde the concentration of sodium, potassium, and chloride ion concentration in living human white matter obtained by biopsy at the time of surgery is not available in the literature. So to determine the effect of postmortem movement of ions we have used the figures reported by McIlwain (1955) for whole human brain. The sodium, potassium, and chloride ion concentrations were 57,96 and 37 mequiv./kg, respectively. It would appear in general that our postmortem values for sodium and chloride are modestly high and our potassium values very low. Because of the artifact introduced by the postmortem state (Katzman, 1966) no attempt will be made to calculate the sodium, potassium, or chloride ion spaces. Since plaques and normal-appearing white matter contain more water than control white matter the data are expressed on the bases of mequiv./l of tissue water. It was found that control white matter contained 106 mequiv. of sodium ion, the normal-

POSTMORTEM M U L T I P L E S C L E R O S I S W H I T E MATTER

505

appearing multiple sclerosis white matter 92 and the plaque tissue, 91. The same trend was found in chloride ion concentration. The control white matter contained 64 mequiv./l, the normal-appearing multiple sclerosis white matter 58 and the plaque tissue, 60. On the other hand, the potassium ion concentration was 82 mequiv. in control white matter, 97 in normal-appearing multiple sclerosis white matter and moderately reduced in the plaque tissue to 68. No conclusions will be drawn from our meager postmortem data. However, it would appear fromacomparisonof the plaque concentration of sodium ion (mequiv./l of tissue water) to surrounding normal-appearing white matter from the same patient that the concentrations do not differ, even though both are moderately lower than the concentration in the control white matter. This same trend was shown for the chloride ion. Hence, it is possible that the postmortem sodium and chloride ion spaces in multiple sclerosis normal-appearing white matter do not differ from the plaque tissue. On the other hand, itwould appearfrom a comparison of the plaque tissue concentration of potassium ion to surrounding normal-appearing white matter from the same patient that the concentration was markedly lower in the plaque tissue, whereas the control white matter concentration did not reach the value in the normal-appearing multiple sclerosis white matter but was higher than that in the plaque. A partial explanation of the low value of potassium in the plaque tissue compared to the surrounding normal-appearing multiple sclerosis white matter might be that there was a significant reduction of axons which are known to be rich in potassium. Unfortunately, we did not estimate the axon density in this specimen, but data shown in Table V on plaque tissue from other patients indicate that a reduction of axons in a plaque can exist. SOME TESTS O F T H E B A R R I E R

Two types of experiments to test the blood-white matter or plaque tissue barrier in postmortem brain tissue will be described. A foreign anion, bromide, which is known to penetrate the brain slowly and come to a steady-state concentration below the blood level of approximately 0.3 (Haerer et al., 1964), was administered orally ante mortem, and the distribution of the ion was determined post mortem in different areas of brain coronal sections. The second test used was the distribution in brain coronal sections of endogenous serum proteins, namely, albumin and immunoglobulin-G. Both of these proteins penetrate the blood-brain barrier exceedingly slowly, if at all (Barlow, 1964). Bromide ion concentration in postmortem multiple sclerosis white matter and plaque tissue Some of the data are presented in Table VII. It can be seen that all the areas determined to be plaques of demyelination or diffuse demyelination by gross microscopic inspection had a low lipid content; conversely, the normal-appearingwhite matter had a much higher concentration. The water concentration of the dissected areas are also given. In general the plaque tissue and areas of diffuse demyelination had a higher water concentration than the normal-appearing white matter. References p . 520-522

506

W. W. TOURTELLOTTE

TABLE V I I BROMIDE I O N C O N C E N T R A T I O N I N POSTMORTEM MU LTIP LE SCLEROSIS W H I T E MATTER ~~~

Bromide ion concentration Multiple sclerosis patient identification

Lipids glkg

Water glkg

BO Normal-appearing white matter 1

2 3 Plaque 1 2 3 BU Normal-appearing white matter 1

2 3 Plaque 1 2 WR Normal-appearing white matter 1

2 3 4 5

6 7 8 9 Plaquc 1 2 3 4 5

Serum mequiv.11

CSF mequiv./l

2.0

0.6

Brain mequiv./l tnequiv.lkf of tissue water

207 169 I 60

689 736 742

0.32 0.42 0.17

0.46 0.57 0.23

52 41 38

856 869 882

0.39 0.39 0.72

0.45 0.45 0.82

2.5

not done

196 187 181

704 705 709

0.41 0.41 0.31

0.58 0.58 0.44

45 27

874 894

0.20 0.42

0.23 0.47

0.9 1 0.91

2.9

0.9

212 188 186 180 174 169 158 142 142

672 725 738 738 718 751 770 775 784

0.61 0.66 0.70 0.62 0.61 0.77 0.61 0.84 0.70

26 26 20 18 7

882 890 814 878 890

0.62 0.38 0.71 0.30 0.80

0.95

0.84 0.85 1.02 0.79 1.08 1.01 0.70 0.43 0.87 0.34 0.90

507

POSTMORTEM M U L T I P L E SCLEROSIS W H I T E MATTER

T A B L E V l I (ctnd) B R O M I D E I O N C O N C E N T R A T I O N IN P O S T M O RTEM M U L T I P L E S C L E R O S I S W H I T E M A T T E R

Bromide ion concentration

Multiple sclerosis potient irlentificritioti

Lip ids Rlk

Wuter g/kg

Seriini nieqiiiv./l

Brain

CSF nwquiv./l

tnequiv./kg

tnequivJ1 of tissue n’oler

.~

~~~

--

--

HE Normal-appearing white matter I 2 3 4 5 6 7 8 9 Plaque 1

2 3 4 5

WA Normal appearing white matter Plaque HO Normal appearing white matter Diffuse demyelination I 2 3 4 5 Plaque 1

-

~

3.6

1.1

217 215 213 20 I 196 I82 I47 141 123

674 685 683 685 717 724 756 767 790

0.84 0.88 0.89 0.92 0.90 0.69 0.89 0.76 0.92

1.25 1.28 1.30 0.74 1.26 0.95 0.85 0.99 1.16

37 33 31 28 24

849 877 870 894 882

1.26 1.12 1.18 0.99 1.22

1.49 1.28 1.36 1.11 1.38

0.94 0.78

1.19 0.89

4.0 147 24

1.8

778 880 5.4

3.8

131

77 5

1.83

2.36

95 90 86 78 55

818 833 82 I 828 864

2.04 1.42 1.70 1.84 I .71

2.49 1.70 2.07 2.22 1.98

28 24 16

89 1 880 906 907 916 918 910 914

1.86 2.32 2.20 2.34 1.84 1.82 1.98 1.95

2.08 2.64 2.43 2.58 2.01 1.98 2.18 2.14

10

7 7 5 2

It can also be seen that a direct relationship between the serum concentration of bromide ion and the concentration in the cerebrospinal fluid and in the brain existed. I t is reasonable to assume that the brain bromide ion concentrations are probably Rrfrnvi~r3.sp.: 520-522

508

W. W. TOURTELLOTTE

Fig. 2. Bromide ion concentration in multiple sclerosis white matter.

steady-state ion concentrations. The oral medication was given at regular intervals (three times per week) for no less than one year. Furthermore, for any given patient the serum bromide levels were maintained at a constant level. For example, patient WA had serum bromide ion concentrationsof 3.9 mequiv./l in August, 4.0 in September, and 4.2 in December, 1964 and 3.8 in April 1965 and 4.0 in June 1965 (12 h post mortem). Furthermore, the ratio of serum to cerebrospinal fluid was reasonably constant over this period of time too (2.2,2.3,2.2,2.4,2.2, and 2.0). In the majority of the cases the brain concentration of bromide ion is equal to or approaches that in the cerebrospinal fluid. In Fig. 2 the concentration of the bromide ion per liter of tissue water in a dissected area was plotted against its concentration of lipids per kg for each patient shown in Table VII. The type of tissue, plaque of demyelination, diffuse demyelination or normal-appearing white matter are also shown. Inspection of the plotted data reveal no obvious bromide ion concentration differences between the plaque tissue and the areas of diffuse demyelination when compared to the normal-appearing white matter for any given patient. Fig. 3 presents a photograph of a dissection plan of a coronal section of a multiple sclerosis patient (HO) in order to show the relationship between the bromide ion concentration in plaques and the adjacent white matter, as well as several selected gray matter areas. The data for each dissection are shown in Table VIII. Inspection of the bromide ion concentration per liter of tissue water for plaque tissue was not different from that in the surrounding normal-appearing white matter. In most dissected regions the brain bromide ion concentration approached or equaled the cerebrospinal fluid concentration. A notable exception to this is thevalues obtained for cerebral cortex,

509

POSTMORTEM M U L T I P L E S C L E R O S I S W H I T E M A T T E R

TABLE VIII BROMIDE ION C O N C E N T R A T I O N I N DISSECTED P A R T S OF T H E C O R O N A L S E C T I O N S H O W N I N FIG. 3

Gross and microscopic description

1

2 3 5

10 4 15

24 21 22 23 I1 12

Bromide ion

Lipids

Water

glkg

glkg

rnequiv.lkg

mequiv.11 of tissue water

28 59 106

872 841 794

1.96 1.88 1.61

2.25 2.23 2.03

Periventricular plaque Deep portion of periventricular plaque Periventricular plaque

82 62

818 838

1.86 2.21

2.28 2.64

26

874

1.85

2.12

Shadow plaque White matter adjacent to shadow plaque 15

87 38

813 762

1.71 2.10

2.10 2.76

Plaque White matter around plaque 21 White matter adjacent to 22

48 75

852 825

1.94 1.63

2.28 1.98

149

751

1.77

2.36

Dissection number see Fig. 3

Plaque in corpus callosum Plaque in corpus callosum Corpus callosum adjacent to plaque 1 and 2

Plaque White matter around plaque 11 White matter adjacent to 12 White matter adjacent to 12

48 90

852 810

1.96 I .94

2.30 2.39

I49 149

75 1 735

1.70 1.58

2.26 2.15

Plaque in caudate nucleus Anterior internal capsule Plaque in putamen Putamen

84 129 97 97

816 771 803 803

1.37 1.39 1.44 1.30

1.68 1.80 1.80 1.62

Cerebral cortex plaque Deep portion of cerebral cortex plaque 18 Cerebral cortex adjacent to plaques 18 and 19

58 63

799 820

2.59 2.65

3.24 3.23

55

742

2.32

3.20

20

Cerebral cortex plaque

63

810

2.60

3.1 1

16

Arachnoid membrane

48

852

2.05

2.41

I3 14 6 7 8 9 18 19 17

which in all cases exceeded the cerebrospinal fluid value as well as that for the basal ganglia. Again the data obtained on this patient show no obvious bromide ion concentration difference between the plaque tissue and the surrounding normal-appearing white matter. An examination of the bromide steady-state ratio (RCSF = concentration in the cerebrospinal fluid/concentration in serum) for each multiple sclerosis patient studied (data presented in Tables VII and VIII) revealed a direct relationship between R C ~ F R&rmci.s p. 520-522

510

W. W. T O U R T E L L O T T E

Fig. 3. Legend is o n the photograph.

values and the serum bromide level. Serum bromide levels of 2.0 to 3.6 mequiv./l, 4.0 to 4.8, and 5.4 gave RCSFvalues of 0.31,0.45 and 0.7, respectively. This result may be explained by a mechanism recently proposed by Bito, Bradbury and Davson (1966). Their data supported the hypothesis that bromide ions passively diffuse across the blood-brain barrier and that the low steady-state distribution ratio (RCYF= 0.7) found for bromide ion was due to an active process in the choroid plexus that tends to drive bromide ion out of the cerebrospinal fluid. If this hypothesis were true, it might be predicted that the higher the serum bromide ion concentration, the greater the inhibition of the active transport system, perhaps by saturating the limited number of carrier sites (Bito e? al., 1966); hence, the bromide ion is not pumped out of the cerebrospinal fluid, so the cerebrospinal fluid bromide ion concentration rises and the R ~ s p increases. Unfortunately, Bito et al. (1966) studied only one dosage level, which was very high compared to our doses on a per kg body weight base. They gave an intravenous dose of sodium bromide of approximately 7.5 mequiv./kg, whereas we used initially 1.25 mequiv./kg and a maintenance dose of 0.1 mequiv./kg three times a week. Since it would appear from our data that higher serum bromide levels inhibit the active transport of bromide ion out of the cerebrospinal fluid, it is reasonable to assume that Bito e? al. were studying the effect of inhibitors and the size of the bromide ion space on an active transport system initially inhibited by a high serum bromide level. Because we have studied postmortem tissue we have purposely avoided a discussion of the calculation of electrolyte spaces. However, it has occurred to us after a comparison of calculated postmortem bromide and chloride ion spaces that the bromide space

POSTMORTEM MULTIPLE SCLEROSIS W H I T E MATTER

-

51 1

WHITE MAnER

C

'E

* M + 2 S.D.

*

M e a n + 2 x Standard Deviation

Fig. 4. Stratification of albumin concentration of postmortem white matter corrected for blood albumin. M.S. = multiple sclerosis.

was larger. The postmortem chloride: ion space (100 x postmortem tissue chloride ion concentration mequiv./l of tissue water divided by 101 mequiv. chloride ion/kg of postmortem cerebrospinal fluid). The postmortem chloride space values were 57, 5 I and 53 percent for specimens of control white matter, normal-appearing multiple sclerosis white matter, and plaque tissue, respectively. On the other hand, the postmortem bromide spaces (100 x postmortem tissue bromide ion concentration mequiv./l of tissue water divided by bromide ion concentration of the cerebrospinal fluid) were larger. The mean and range were obtained from the data in Tables VII and VIII. The values were: 84 percent (63-103) and 83 percent (47-1 20) for specimens of normal-appearing multiple sclerosis white matter and plaque tissue, respectively. Perhaps, when bromide ion is administered for a long period of time (years) resulting in serum bromide ion concentrations ranging from 2 to 5.4mequiv. per 1 the bromide ion penetrates into more than the chloride ion space. Bito et al. in their experiments found at 24 hours that the bromide and chloride ion spaces were identical. Furthermore, inpatient HO (see table 8) we found that the bromide ion concentrationin thecortical mantleactually exceeded that in the cerebrospinal fluid.

Relationships between Albumin and Immunoglobulin-G in Postmortem Control White Matter, Normal-Appearing Multiple Sclerosis White Matter and Plaque Tissue and Cerebrospinal Fluid Fig. 4 presents the albumin concentration (corrected for serum albumin) for each patient's brain region dissected. The mean control white matter concentration was R&rcnccs

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M t 21.0. , A W N + 2SlANDARO DNlAIlON OF CONTROI “M;-NYAN of VAWtS<M+ZS.D. “ M i - M A N Cf VALUCS>M+ZS.D.

Fig. 5. Stratification of immunoglobulin-G concentration of postmortem white matter corrected for

blood immunoglobulin-G.

identical to that in the normal-appearing multiple sclerosis white matter, 152 mg/kg (& 28 = S.E. of the mean) and 145 f 22, respectively. On the other hand, the plaques of demyelination had a higher mean value (190 f 32), with two patients outside the mean plus two standard deviation level. However, the elevated value found in plaques was not statistically significantly different from the surrounding normal-appearing white matter or control white matter@ < 0.4 and > 0.3). Moreover, the concentration of albumin in control white matter, normal-appearing multiple sclerosis white matter, and plaque tissue were very similar, if calculated on the basis of mg/l of tissue water The values were: 215 &- 39.6, 197 f 30.0 and 212 f 35.6, respectively. Fig. 5 presents the immunoglobulin-(; concentrdtion (corrected for serum immunoglobulin-G) for each patient’s brain region dissected. The control white matter value was 162 f 26 mg/kg, the normal-appearing multiple sclerosis white matter, 433 135 and the plaques of demyelination, 434 f 98. There was a statistical difference between the control white matter value and the multiple sclerosis specimens (p < 0.01). On the other hand, the immunoglobulin-G concentration was the same for the normalappearing multiple sclerosis white matter and plaque tissue. Inspection of the frequency distribution of immunoglobulin-G data from the multiple sclerosis specimens revealed that there are probably two modes. The first mode ((‘MI”) was calculated on values which fell below the control white matter mean plus two standard deviations and the second one (“Mz”) was calculated on values which were above this cut-off point. There was no statistical difference between “MI” and the control white matter, whereas “Mz” was statistically highly different (p <

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Fig. 6. Immunoglobulin-G and albumin concentration in postmortem white matter. Ten controls and 1 1 multiple sclerosis patients. y G = immunoglobulin-G; C = control; MS = normal-appearing multiple sclerosis white matter; P = plaque tissue; s.e.m. = standard error ofthe mean.

0.003). “M2” was the same for normal-appearing multiple sclerosis white matter and plaque tissue. Fig. 6 presents the mean ratio of immunoglobulin-G over albumin. The serumcorrected ratio was 1.1 for control white matter, 3.0 for normal-appearing multiple sclerosis white matter and 2.3 for the plaque tissue. Therefore, on the average there was a significantly different distribution of immunoglobulin-G and albumin in the soluble protein fraction of multiple sclerosis brains for the regions studied. Furthermore, this redistribution in multiple sclerosis was due to an increase of immunoglobulin-G and not to a decrease of albumin. The possible factors to explain an increase of immunoglobulin-G in the multiple sclerosis brain tissue are as follows: ( I ) Hyperemia of the multiple sclerosis brain, hence, introducing serum immunoglobulin-G ; (2) Hypergammaglobulinemia with transudation of immunoglobulin-G into the brain; (3) Decrease of the blood-brain barrier to immunoglobulin-G; (4) Synthesis of immunoglobulin-G in the brain itself; ( 5 ) Selective secretion of immunoglobulin-G in the brain itself; (6) Combination of 1 through 5 . Some experiments have been done to determine which of these factors might be responsible for the high immunoglobulin-G in multiple sclerosis brain tissue. Hyperemia does not exist in multiple sclerosis brain tissue (see data in Table 11). Furthermore, multiple sclerosis patients do not have hypergammaglobulinemia (Tourtellotte and Parker, 1965). The albumin concentration whose MW is 40,000 was not increased in multiple sclerosis brain tissue (see data in Fig. 4); hence, it is reasonable to assume that the blood-brain barrier could be intact for larger molecules like immunoglobulin-G, whose MW is 140,000.The possibility exists that immunoglobulin-G is selectively secreted into the multiple sclerosis brains. No experimental data References p . 520-522

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Fig. 7. Coronal section ( 3 mm thick) through the posterior portion of the lenticular nucleus of a control cadaver (Campb). The pattern of the dissection is outlined on the right photograph. The superimposed numbers within the outline of the dissection are the concentration of imrnunoglobulin-G (corrected for serum imrnunoglobulin-G) in mg/kg.

exists to disprove this, nor any to support it. Hence, we have elected to mention it only as an outside possibility. Based on the above observations, we have postulated that in the majority of patients who die with multiple sclerosis there exist multifocal areas of synthesis of immunoglobulin-G. We have discussed the possible sites of production of immunoglobulin-G in multiple sclerosis brain tissue (Tourtellotte et a/., 1966). The data supported the hypothesis that proliferation of perivascular cells (mostly lymphocytes) around capillaries and venules is sufficient and probably necessary to produce an elevation of irnmunoglobulin-G in brain tissue from patients with multiple sclerosis. Furthermore, the results also indicated that cells in the advancing margin of a plaque other than perivascular mononuclear cells might produce immunoglobulin-(?. On the other hand, evidence for synthesis of immunoglobulin-G in some multiple sclerosis brain tissue was not found. It has been established from numerous histopathological studies (Adams and Kubik, 1952; Ibrahim, 1965; McAlpine et a/.. 1955) that plaques can be empirically classified into early and old types and old plaques can be further subdivided into active and inactive forms. We are postulating that an elevated immunoglobulin-G value in a multiple sclerosis brain is a chemical indicator of an active plaque of multiple sclerosis. It is possible that the normal-appearing multiple sclerosis white matter surrounding an active plaque has an elevated immunoglobulin-G value because the immunoglobulin-(; synthesized in an active plaque has diffused into it. To test this possibility

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Fig. 8. Coronal section ( 3 nini thick) through the posterior portion of the lenticular nucleus of a cadaver with multiple sclerosis (Wacht). I t is essentially the same level of the brain as shown in Fig. 7. On thc left photograph note the large plaque of demyelination which extends from the ventricles deep i n t o the white matter and into the insular cortex. Dorsally there are two cerebral cortex plaques which were ccmbined for analysis (937 on the right photograph). The pattern of the dissection is outlined on the right photograph. The superimposed numbers within the outline of the dissection are the concentration of immunoglobulin-G (corrected for serum immunoglobulin-G) in rng/kg.

we have dissected coronal sections from a control and a multiple sclerosis brain like a mosaic along anatomico-pathological land marks and analyzed the dissected specimens for immunoglobulin-G concentration. Fig. 7 shows the distribution in a control coronal section through the posterior part of the lenticular nucleus. It can be seen on the right hand photograph that concentration of immunoglobulin-(; was rather uniform across the section. On the other hand, when a comparable coronal section from a multiple sclerosis patient was studied, the abnormally elevated immunoglobulin-G appears to be most concentrated in the largest plaque itself (2,072, 2,237 and 2,352 and 1,892 mg/kg) or the periventricular plaques (2, 040 and 2,366) and less in the cerebral cortex plaques (937, 859 and 703). On the other hand, in the surrounding normal-appearing white matter of the largest plaque the value fell to about half of the value (1,064 and 918). Furthermore, the more distant normal-appearing white matter had even lower values, for example, 613 and 367. To carry the idea of diffusion of immunoglobulin-(? through multiple sclerosis brain tissue further, we have correlated the concentration of immunoglobulin-G in multiple sclerosis brain tissue and the concentration of this globulin in the cerebrospinal fluid (Tourtellotte and Parker, 1966). The data are shown in Fig. 9. The tentative conclusion drawn from these results was that in patients with multiple sclerosis, the inReliwrrc cs p. .52O-F22

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B

150-

a n . -. r 10062

.

0

4 .

0

. I

a

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Control M I 3 SD.

Fig. 9. A correlogram of the immunoglobulin-G concentration in premortal cerebrospinal fluid and the same multiple sclerosis pateint’s plaque tissue immunoglobulin-G concentration (corrected for blood immunoglobulin-G), yG = immunoglobulin-G; M = mean; S.D. = standard deviation.

crease in immunoglobulin-(; in the cerebrospinal fluid is a reflection of an excess of this globulin in the brain. Brightman (1965) has shown by electronmicrographs that ferritin (MW 500,000) can move within the brain between the tissue pericellular spaces (approximately 200 A wide). Hence, it is reasonable that immunoglobulin-(; (MW of 140,000) could diffuse through the same spaces. COMMENTS

The dissection of the plaque materialin theaboveanalyses were performed on plaques easily discernible by inspection. Also the edge of the plaque was grossly:cut inlorder to exclude the surrounding normal-appearing white matter. Hence, the most active areas of demyelination, which are at the edge of a chronic plaque, were probably not taken for analysis. Hence, it is also reasonable to presume that if a small amount of “active” demyelinating tissue were included in the dissection that its constituents would not significantly dilute the larger area of chronic demyelination taken for analysis. Furthermore, the biopsies of the plaques taken for microscopic histology were from the center of the plaque. They all showed severe demyelination. Hence, it is our impression that the above analyses were done on chronic plaques with insignificant contamination from “active” demyelinating tissue. Therefore, the data from this study done on postmortem tissue suggest the conclusion that chronic plaques of multiple sclerosis do not have an altered blood-brain barrier to albumin and to bromide ion. Broman (1947) perfused a multiple sclerosis brain after death with trypan blue and stained areas of demyelination, but not normal-appearing white matter. He concluded from this study that the blood-brain was decreased in plaque tissue of multiple sclerosis. However, in a subsequent publication (1964) the picture appears complicated.

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The trypan blue staining of the postmortem sclerotic plaque appeared uniform; there was no accumulation of the dye either in the parenchyma or in the vascular endothelium. This is a unique finding according to Broman, and it is the only known example in his experience of a blood-brain barrier damage without a coexisting lesion of the vascular wall. He thinks it may be consistent with a primary lesion of the perivascular glia. The electron micrographs presented by PCrier and GrCgoire (1965) may help in explaining the trypan blue staining of the plaques described by Broman in postmortem plaques. I t is possible that the trypan blue is contained in an expanded extracellular space. Furthermore, Perier and GrCgoire presented an electron micrograph that showed an intact layer of endothelial cytoplasm, a continuous basement membrane, and a discontinuous thin layer of glial cytoplasm applied to the outer surface of the vessel basement membrane. Perhaps the trypan blue moved through the vessel wall not covered by glial cytoplasma into the true extracellular space. This hypothesis places the normal exclusion of trypan blue from the brain at the level of the perivascular glial feet or at the level endothelial cells of the vessel wall influenced in some way by the perivascular glial feet. Our data indicate that in postmortem brain tissue the concentration of various ions (sodium, chloride, bromide) and albumin were normal in the chronic plaques when compared to surrounding normal-appearing white matter and control white matter. In view of the findings of PCrier and GrCgoire (1965) that the expanded extracellular space in multiple sclerosis plaques could be in direct contact with the basement membrane of endothelial vessel walls without a continuous layer of perivascular glial feet would suggest that sodium, chloride, bromide, and albumin concentrations are determined by the intact vessel wall. On the other hand, the low potassium concentration of the plaque tissue may indicate that the perivascular glial cells may somehow be involved in the maintenance of the extravascular tissue concentration. However, the posybility that we suggested earlier that the decrease of axoplasm in plaques which are rich in potassium ions is also a possibility. Of interest is the study of Gonsette and AndrC-Balisaux (1965). They injected radioactive phosphorus (3") intravenously into three patients prior to death (they studied the same patients as PCrier and GrCgoire). They then cut coronal sectionsof the brain as rapidly as possible (usually one to three hours post mortem) which showed plaques of demyelination and prepared radioautographs. They found that chronic intracerebra1 gliotic plaque tissue did not pick up the isotope; on the other hand, periventricular plaques did mark the autoradiographs. They suggested that 32P could move from the cerebrospinal fluid into the periventricular plaque, whereas it could not from the blood to the brain. The two most popular etiologies of multiple sclerosis are that it is due to autoimmunity (McAlpine et al., 1965) or a slow virus infection (Palsson et al., 1966). Perhaps in multiple sclerosis there is a destruction of the myelin or of the oligodendroglia cell which maintains the health of the myelin by an immunological mechanism, i.e., the multiple sclerosis patient is allergic to his own myelin, or the oligodendroglia related to myelin is diseased by a virus. The discovery that the majority of patients who die References p.lS20-522

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with multiple sclerosis have an elevated immunoglobulin-G in the brain supports both of these possible etiologies. The fact that the albumin content was normal but the immunoglobulin-G was elevated suggested that the elevated immunoglobulin-G was synthesized in the brain. Other experiments on postmortem multiple sclerosis brain tissue supported the hypothesis that multifocal areas of synthesis of immunoglobulin-G which are probably located in active plaques of demyelination and in the accompanying perivascular cuffs of lymphoid cells, were responsible for the increased values (Tourtellotte et a/., 1966). Moreover, our results imply that the increase of immunoglobulin-(; in the cerebrospinal fluid is the result of diffusion through the normalappearing white matter of an excess of this globulin in the brain. Extrapolation of the results of this postmortem study to the living brain must be done with some hesitation. Numerous artifacts could be introduced during the agonal state of death and the postmortem period. For example, death destroys potassium transport mechanisms and postmortem cerebrospinal fluid potassium rises at a constant. rate while the brain potassium falls (Mason et nl., 1951; Naumann, 1958; Schain, 1964). Furthermore, the limitations of getting sufficient material, such as biopsies of plaque tissue from living multiple sclerosis patients, leaves us in an experimental position to have to wait for biopsy specimens obtained at cryothalamotomy for treatment of intention tremor, which will include both normal-appearing white matter and plaque tissue. SUMMARY

I . The mean values of the vascular space, estimated by a carboxyhemoglobin method, in postmortem control white matter was statistically the same as normal-appearing multiple sclerosis white matter and the plaque tissue; the mean values and standard error of the means were: 4.0 5 1.3, and 4.8 2.0 and 4.9 f 2.0 ml/kg, respectively. 2. After complete myelin loss in postmortem multiple sclerosis white matter the total lipid value was 44 f 6.0 g/kg, whereas the normal-appearing multiple sclerosis 4.0. white matter was 158 4 1.6 and the control white matter, 187 3. When there was myelin loss in the postmortem white matter it was replaced by the water. The control white matter contained 706 f 12.0 g/kg, whereas the normalappearing white matter had 736 & 30.6 and the plaque tissue 850 rt 56.6 4. On the other hand, the fat-free dry weight of the postmortem white matter (mostly protein) was constant. The control white matter had a value of 107 i 1.5 g/kg and the normal-appearing multiple sclerosis white matter 106 & 5.1 and the plaque 2.2. l tissue, 106 5. Ganglioside concentration of postmortem white matter, which is probably compartmentalized to the axoplasm, was significantly lower in plaques. The value for control white matter was 189 & 16.0 pmoleslkg, whereas the normal-appearing multiple sclerosis white matter was 223 (range = 126 + 306), and plaque tissue, 126 23.2. However, the ganglioside concentration divided by the axonal density index was the same for the three types of tissue studied. 6. The total hydroxyproline concentration of postmortem white matter, which may

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reflect the presence of blood vessel walls and possibly the filamentous portion of the fibrous astrocytes, was 61 mg/kg (range = 60 -+ 62) for the control white matter, 78 (54 + 90) for the normal-appearing multiple sclerosis white matter and 162 ( I 18 -+ 204) for the plaque tissue. After a type of correction was made for blood vessel walls the values were 16, 24 and 108, respectively. 7. Postmortem white matter concentration of sodium ion did not appear to differ in the control white matter, normal-appearing multiple sclerosis white matter, or plaque tissue. The values were, respectively, 106, 92, and 91 mequiv./l of tissue water. These values are modestly high when compared to values available for fresh tissue. 8. The postmortem white matterconcentration of potassium ion appears to be lower in plaque tissue. This may reflect the decrease ofaxoplasm in this tissue. The values obtained for control white matter, normal-appearing multiple sclerosis white matter, and plaque tissue are as follows: 82, 97, and 68 mequiv./l of tissue water, respectively. These values are very lowwhencomparedtothe concentrationavailableforfresh tissue. 9. Postmortem white matter concentration of chloride ion did not appear to differ in control white matter, normal-appearing multiple sclerosis white matter, or plaque tissue. The values were, respectively, 64, 58, and 60 mequiv./l. of tissue water. These values are modestly high when compared to values available for fresh tissue. 10. After giving sodium bromide orally for one to three years before death, the bromide ion concentration did not appear to have a different distribution in the postmortem plaque t issue when compared to theconcentrations in the surrounding normalappearing white matter. The concentration of the bromide ion in the tissue and cerebrospinal fluid, which was very similar, was dependent on the serum bromide ion level, i.e., the higher the serum level, the higher the cerebrospinal fluid and tissue concentration. For example, serum bromide levels of 2.0 to 3.6,4.0to 4.8, and 5.4 mequiv./l gave cerebrospinal fluid (and tissue) values of 0.6 to 1 . 1 , 1.8 to 2.2 and 3.8, respectively. We have suggested that this result could be best explained on the basis that bromide ion gets into the brain by passive diffusion. Furthermore, an examination of the bromide steady-state ratio (RcsF-concentration in the cerebrospinal fluid divided by the concentration in the serum) for each multiple sclerosis patient studied revealed a direct relationship between R C ~ Fvalues and the bromide level. Serum bromide F of 0.31, 0.45 and 0.7, levels of 2.0 to 3.6, 4.0 to 4.8 and 5.4 mequiv./l gave R C ~values respectively. We have suggested that this result (the elevation of the steady-state ratio by rising serum bromide ion concentration) could be best explained on the basis that higher serum bromide ion concentrations are inhibiting the active secretion by the choroid plexus of the bromide ion out of the cerebrospinal fluid into the serum. 11. The albumin concentration in postmortem control white matter corrected for serum albumin is not statistically different from that in normal-appearing multiple sclerosis white matter of plaque tissue. The values were as follows: 152 & 28, 142 i 22 and 190 $ 32 mg/kg, respectively. Moreover, the concentration of albumin in control white matter, normal-appearing multiple sclerosis white matter and plaque tissue was very similar if calculated on the basis of mg/l of tissue water. The values were 215 f- 39.6, 197 f 30.0, and 212 =k 35.6 respectively. Rcfer.rpnrmp . 520-522

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12. On the other hand, immunoglobulin-(? concentration was increased in the

majority of the postmortem brains of multiple sclerosis patients (64 percent) when compared to control postmortem brain tissue. The values corrected for serum immunoglobulin-(; for control white matter, normal-appearing multiple sclerosis white matter, and plaque tissue were, respectively, 162 26, 433 f 135, and 434 & 98 mg/kg. Furthermore, inspection of the frequency distribution of the immunoglobulin-<; data suggested there were two types of multiple sclerosis patients brains - the majority had an increased immunoglobulin-G concentration and the minority a normal concentration. We have suggested that those multiple sclerosis brains with an increased concentration of immunoglobulin-<; have multifocal areas of synthesis of immunoglobuin-G (advancing margin of the plaque tissue and/or perivascular cuffs of mononuclear cells). Perhaps an elevated immunoglobulin-(; concentration in the brain is a chemical indicator of an active case of multiple sclerosis. Some evidence was presented to support the hypothesis that immunoglobulin-G can diffuse through the brain into the cerebrospinal fluid. A mosaic type of dissection of a coronal section of a multiple sclerosis brain showed that immunoglobulin-G concentration was highest in the plaque tissue and fell to lower concentrations in the surrounding normal-appearing white matter. Moreover, a correlogram was presented between the concentration of immunoglobulin-G in the cerebrospinal and the plaque tissue. It was found that in patients with multiple sclerosis the increase in immunoglobulinG in the cerebrospinal fluid was a reflection of an excess of this globulin in the brain. 13. The data from this study done on postmortem tissue suggests the conclusion that chronic plaques of multiple sclerosis do not have an altered blood-brain barrier to albumin, bromide, sodium, potassium, and chloride ions. On the other hand, there may be multifocal areas of synthesis of immunoglobulin-G in the majority of multiple sclerosis brains, and the excess globulin diffuses from the active sites of synthesis (advancing margin of the plaque and/or perivascular cuffs of mononuclear cells) into the surrounding normal-appearing white matter and cerebrospinal fluid. 14. Extrapolation of the results of this postmortem study to the living brain must be done with some hesitation. ACKNOWLEDGEMENT

This work was supported in part by the National Multiple Sclerosis Society (272-5), National Institutes of Health (NB 05388-03) and the Kenneth H. Campbell Foundation for Neurological Research, Grand Rapids, Michigan, U.S.A. REFERENCES C. S. (1952) The morbid anatomy of the demyelinative diseases. Amer. J . ADAMS,R. D . AND KUEIIK, Med., 12, 510-546. ADDISON, W. H. F. (1950) Neurological Technique. R. McC. Jones (Ed.). McClung's Handbook of Microscopical Technique. New York, Paul B. Hoeber, Inc. (pp. 346388). BARLOW, C. F. (1964) Clinical aspects of the blood-brain barrier. Ann. Rev. Med., 15, 187-202. BITO,L. Z., BRADBURY, M. W. B., AND DAVSON, H., (1966) Factors affecting the distribution of iodide and bromide in the central nervous system. J. Physiol., 185, 323-354.

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BRIGHTMAN, M. W. (1965) The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. 11. Parenchymal distribution. Amer. J. Anat.. 117, 193-219. BRODIE, B. B. AND FRIEDMAN, M. M. (1938) The determination of bromide in tissues and biological fluids. J. bid. Chem., 124, 51 1-518. BROMAN, T. ( I 946) Blood-brain barrier damage in multiple sclerosis. Supravital test-observations. Acta Neurol. Scand., 40, Suppl. 10, 21-24. -, (1947) Supravital analysis of disorders in the cerebral vascular permeability. 11. Two cases of multiple sclerosis. Acta Psychiat. Neurol., Suppl., 26, 58-71. COTLOVE, E., TRANTHAM, H. V. AND BOWMAN, R.L. (1958) An instrument and method for automatic, rapid, accurate, and sensitive titration of chloride in biologic samples. J. Lab. Clin. Med., 51, 461-468.

EVELYN, K. A. A N D MALLOY, H. T. (1938) Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood, J . biol. Chem., 126, 655-662. GONSEWE,R. AND ANDRE-BALISAUX, G. (1965) La permdabilite des vaisseaux cbrebraux. IV. Etude des lesions de la barriere hemato-encdphalique dans la scldrose en plaques. Acta Neurol. Psychiat. Belg., 65, 19-34. GORDON,M. W. AND NURNBERGER, J. I. (1956) Estimation of whole blood in tissue homogenates. J. Histocheni. Cytochem., 4, 84-85. HAERER, A. F., TOURTELLOTTE, W. W.,RICHARD, K. A., GUSTAFSON, G. M. AND BRYAN,E.R., (1964) A study of the blood-cerebrospinal fluid-brain barrier in multiple sclerosis. I. Blood-cerebrospinal fluid barrier to sodium bromide. Neurology, 14. 345-354. IBRAHIM, M. Z. M. (1965) Neuroglia and demyelination, Neurohistochemistry, C. W. M. Adams (Ed.), Amsterdam, Elsevier, pp. 454464. KABAT,E. A.. GLUSMAN, M. AND KNAUB, V. (1948) Quantitative estimation of the albumin and gamma globulin in normal and pathologic cerebrospinal fluid by immunochemical methods. Amer. J. Med., 4, 653-663. KATZMAN, R. (1966) Effect of electrolyte disturbance on the central nervous system. Ann. Rev. Med., 17, 197-212. KISHIMOTO, Y . A N D RADIN,N. S. (1966) Determination of brain gangliosides by determination of ganglioside stearic acid. J . Lipid Res., 7 , 141-145. KISHIMOTO, Y.,RADIN,N. S., TOURTELLOTTE, W. W., PARKER,J. A. AND ITABASHI, H. H. (1967) Gangliosides and glycerophospholipids in multiple sclerosis white matter. Arch. Neurol., 16,4454. KLUVER,H. AND BARRERA, E. (1953) A method of the combined staining of cells and fibers in the nervous system. J. Neuropathol. exptl. Neurol.. 12, 400403. LOWDEN, J. A., A N D WOLFEL. S. (1964) Studies on brain gangliosides. 111. Evidence for the location of gangliosides specifically in neurones. Canad. J. Eiochern., 42, 1587-1 594. LOWENTHAL, A. (1961) Ddterminations de la teneur du systeme nerveux central en materie skhe, potassium et sodium. Chemical Pathology ofthe Nervous System. Proc. 3rd Intern. Neurochemical Symposium, Strasbourg, 1958, J. Folch-Pi (Ed.). New York, Pergamon, pp. 299-306. MASON,J. K., KLYNE,W. AND LENNOX, B. (1951) Potassium levels on the cerebrospinal fluid after death. J. Clin. Pathol., 4, 231-233. MCALPINE,D., COMPSTON, N. D. AND LUMSDEN, C. E. (1955) Multiple Sclerosis. Edinburgh and London, Livingstone. C. E. A N D ACHESON,E. D. (1965) Multiple Sclerosis-A Reappraisal. MCALPINE,D., LUMSDEN, Baltimore, Williams KK Wilkins. MCILWAIN, H. (1955) Biochemistry and the Central Nervous System. Boston, Little, Brown and CO. NAUMANN, H. N. (1958) Cerebrospinal fluid electrolytes after death. Proc. SOC.exptl. Biol. Med., 98, 16-18.

NORTON,W. T. A N D AUTILIO,L. A. (1966) The lipid composition of purified bovine brain myelin. J . Neurochem., 13, 213-222. OLDENHOF, W. H. A N D DAVSON, H. (1967) Brain extracellular space and the sink action of cerebrospinal fluid. Arch. Neurol., 17, 196-205. PALSSON,P. A., PATTISON,I. H. AND FIELD,E. J. (1966) Transmission experiments with multiple sclerosis, Slow, latent, and temperate virus infections, in D. C. Gajdusek, C. J. Giggs, Jr. and M. Alpers (Eds.). Washington, D.C., United States Government Printing Office, (pp. 49-54) Public Health Service Publication No. 1378. P ~ R I E0. R , A N D G R ~ C O I RA. E ,( 1965) Electron microscopic features of multiple sclerosis lesions. Brain, 88, 937-952.

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PROCKOP,D. J. AND UDENFRIEND, S. (1960) A specific method for the analysis of hydroxyproline in tissues and urine. .4tial. Biochem., 1, 228-239. SCHAIN,R. J. (1964) Cerebrospinal fluid and serum cation levels. Arch. Neurol., 11, 330-333. SIMPSON, J. F., KOKMEN, E. A N D TOURTELLOTTE, W. W. Multiple sclerosis: Identification of immunoglobulin-(; in plaques by immunofluorescence. (to be published) TOURTELLOTTE, W. W. AND PARKER, J. A. (1965) Distribution and subfractionation of immunoglobulins in patients with multiple sclerosis. Trans. Amer. Neurol. Ass., 90, 107-1 12. -, (1966a) Immunoglobulins in multiple sclerosis white matter. J . Neuropathol. expll. Neurol., 25, 167-169. -, (1966b) Multiple sclerosis: Correlation between immunoglobulin-(? in cerebrospinal fluid and brain. Science, 151, 10441046. -, (1967) Multiple sclerosis brain gamma2 globulin and albumin. Nature, 214,683-686. TOURTELLOTTE, W. W., PARKER, I. A. AND ITABASHI,H. H. (1966) Source of elevation of gamma globulin in brain from patients with multiple sclerosis. Trans. Amer. Neurol. Ass., 91, 351-352. WENDER, M. AND HIEROWSKI, M. (1960) The concentration of electrolytes in the developing nervous system with special reference to the period of myelination. J . Neurochem., 5 , 105-108.

DISCUSSION M. BRIGHTMAN: It would seem to me that much of the extracellular space, at least so identified by electronmicroscopy, is intracellular. Would not you expect to see more gliosis in the plaque? We also have thought about the possibility that the extracellular space was W. W. TOURTELOTTE: intraglial and that other “spaces” are an artifact of preparation. We have tried v x y hard to get a plaque with multiple sclerosis for electronmicroscopy study. However, to get it one hour post rnortem is really very difficult. As a matter of fact, until I can use Lowry’s techniques and get the analyses down to the level where 50 micrograms of tissue can be examined with some confidence that it is reasonably homogeneous tissue, I won’t really be able t o answer all the questions. A. LOWENTHAL: I will add two things to what Dr. Tourtellotte said. Firstly, I think that if you have a reduction of the dry weight in the plaques or in some material near the plaqu:s, then the possibility exists that the white matter of the plaques has been replaced by cells. In fact, the dry weight of the plaque in your experiments is similar to that of grey matter. If you have a glial reaction in those plaques, which means that there is grey matter in such plaques, then you can explain a lot of your results. The second point concerns the results showing that the lesion in multiple sclerosis can be found not only at the level of the plaque but also in some other tissue in white matter, appearing as normal white matter. This has been shown repeatedly during the last years, and I think that your results are important.

W. W. TOURTELOTTE: I would like to speak about both of these points, because we are giving them a lot of thought, and to tell you about the controls we have carried out. All the material that we dissect for a plaque is a punch biopsy made with a 16 gauge needle, and there is no chance at all that grey matter and plaque material could have been analyzed together in this. We feel very safe about this. The fact that the dry weight of plaque material in grey matter is similar to that of white matter can be explained on the basis of biological variation. So you have control punch biopsies in order to make sure that you are in the spotted area. As far as normal-appearing white matter is concerned, I think we are all aware of the fact that by gross dissection a lot of things are thrown away. These plaques are very small, so I think that with Lowry’s technique and a guide slide to tell exactly what is being dissected out, we will be able to have more confidence in what normal-appearing white matter is truly illustrating. A. LOWENTHAL: You have no histological control of that normal-appearing white matter?

Yes, every bit of our material is obtained by punch biopsy. We have stained the W. W. TOURTELOTTE: material with Luxol-fast blue, and we were able to show the presence of myelin sheath.

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523

I. KLATZO:Dr. Tourtelotte’s finding that in plaques you have primarily an increase in y-globulin, whereas the albumin seems to be the same as in normal white matter, is slightlypuzzling tome. I think that Bronian was the first who actually demonstrated the vascular permeability disturbances in multiple sclerosis plaques. I would like to ask Dr. Tourtelotte: does he feel that there are no permeability disturbances in such plaques? Otherwise. would you have to assume that this y-globulin is produced by the brain tissue itself? If you have a vascular permeability disturbance, the albumin definitely spreads much more easily then the 1’-globulin. In multiple sclerosis you have two possibilities: either the serum proteins leave the blood vessels around the plaques or in the plaque, or they permeate from the cerebral spinal fluid. In both instances I think there is enough experimental evidence to indicate that albumin will go in to a much greater extent. Thus, for me it is very puzzling if you have to assume that the 7-globulin is produced in the multiple sclerosis plaques. The one thing which would be a source of such a production would be inflammatory cells, especially the plasma cells. However, it is well known that in multiple sclerosis plaques this inflammatory reaction is coincidential, and usually not terribly impressive. The brief answer to these complex questions goes something like this: We have W. W. TOURTELOTTE: inferred that because the albumin is the same in the different materials we have looked at, that this is evidence that we don’t have a marked leak from the vascular space into the brain tissue. Now I am sure that this is going to depend on the activity of the plaque, because there are many plaques that are dead, so to speak. The possibility that there could be a leak at some time in the course of an active lesion is certainly a reality. In that case the albumin may be moving into the tissue from the blood vascular space. And if it moves, the ;!-globulin could be expected to move. To bring up the question of what Broman showed when he perfused the brains with trypan blue - i t is true that some of his plaques did stain with trypan blue. That was his first publication. His second publication showed that this was a special type of diffusion from the vascular space to the brain. I t was different from the diffusion of trypan blue in mechanical lesions of the brain, where there is a true breaking of the blood vessels, after which the trypan blue moves out. He is very unspecific about the special type of moving out of trypan blue from the vascular space to the brain. Maybe somebody from his laboratory could tell me more what it means. As far as the sources of y-globulin are concerned, we are postulating two reasonable sources: 1 think that where there is active demyelination it is possible that the microglia are making y-globulin. This is a new thought because nobody has rcally been able to establish this. It is more reasonable to think in ternis of perivascular cuffs, which are very common in this disorder. In the perivascular cuffs, it is true, there are no plasma cells, but the latest thought is that you don’t need plasma cells to make jqlobulin anymore; it can be made by small lymphocytes. About the problem that albumin moves through the brain more easily than y-globulin: I have learned a lot at this conference, and I think maybe this will help us to design some experiments to make intracerebral injections of albumin and y-globulin in the guinea pig brain, using different antibodies, and to follow the guinea pig-y-globulin, as well as the human y-globulin we will inject

B. JOHANSSON:I just want to add something to what Dr. Tourtelotte said yesterday about a case of multiple sclerosis and jaundice. Recently we had a similar case. We looked at very thin sections with the fluorcscence microscope. The striking thing was the very big difference in the plaques. In severeal of the plaques you could see the staining of the vascular wall. For example, in one plaque thecentral vein stained very nicely and in others you could definitely observe that the bilirubin had left the vascular wall. I cannot define the relationship between the age of the plaque and the difference in tracers, because the histopathological sections are not yet ready.

D. B. TOWER: I have two points. One is related to the sodium values that were shown. I think Dr. Tourtelotte will agree with me and I think it should be emphasized that one cannot necessarily draw conclusions from the values, since they are postmortem. Differences which depended upon pump-niechanisms would be obliterated at this time, so that it is a little confusing to try to draw much of a conclusion from such data. And in regard to the ;,-globulin, it has been shown in the literature that there is an immunologically distinct component of the ;!-globulin complex in the cerebrospinal fluid, a component which is not found in the gcneral circulation. I think this factor should be recognized and perhaps investigated in this type of study.

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W. W. TOURTELOTTE: I am certainly more aware than anybody here that we are dealing with postmortem material. Our efforts to get good cerebral biopsies have already been discussed this morning. It is very difficult, and as far as the specific y-globulin is concerned, this appears in normal CSF, but is no worse in multiple sclerosis CSF. As a matter of fact, the y-globulin fraction still reacts with regular y-globulin reagent. It is mostly an electrophoretic difference: the antibody is immunologically still like all the others, but yet it has a little different mobility. 0. STEINWALL: I will restrict myself to a comment on the trypan blue and postmortem perfusion which Broman performed a number of years ago. He used trypan blue in saline and found out later that this reacts with the tissue and enters the tissue in quite another way than if you use a tracer bound to a protein. In fact it moves, as far as we could detect, much slower. We have just taken it up again and will try to use it even in human material in which we can compare this new technique with what you have found. G. LEVI:Just a brief answer to Dr. Lowenthal and Dr. Tourtelotte. Amaducci showed in plaques an increase in the number of glial cells, and a corresponding increase in proteins.

M. BRIGHTMAN: I would like to make an additional remark about the dynamics of spaces in the brain. We don’t know what the viscosity is of the fluid in these narrow clefts, or whether a peristaltic-like movement actually does occur in vivo, although one can show movement in at least one cell; the oligodendrocyte, in tissue culture. Such effects as diffusion, versus a passive bulk movement between the clefts, represent just a few of the unknown factors in fluid movement, so what could be said here would be pure guess. I don’t know how you could hope to get some pictorial “static evidence” of peristaltic-like membranes, even if they work in phase. H. M. PAPPIUS: I would like to mention one of my own experiments. I was asked to make an investigation of the effect of intracranial infusion of methotrexate. We studied the water content and the electrolytes in the brains of monkeys that had had long methotrexate infusions. These animals were not killed at any specific time, but they died, and the analysis was carried out at variable times after death. I could correlate very well the water content of both the white matter and the cortex and the increase in sodium with the time that elapsed between the estimated time of death of the animal and the time that the tissue was taken out of the cranium. I think the maximum time in our case was six hours. By twelve hours you may be reaching a point where all the sodium that could get in has gotten in, and it says very little about the state of affairs at time of death. A. LOWENTHAL: I want to say something about the comment of Dr. Klatzo: In soluble protein extracts of white matter of multiple sclerosis patients, there is no increase of albumin, but a decrease of y-globulin. Dr. Tower was speaking of a specific y-globulin in multiple sclerosis, bound or free. This is still a controversial matter. I don’t think we can accept the evidence about a specific y-globulin in normal CSF or in CSF of multiple sclerosis patients. This problem could only be solved by isolating such a y-globulin and studying it immunologically. As to the question of the electrolytes, I think that there are changes in electrolyte distribution in the brain that occur post mortem. Thus, when one compares biopsy material and postmortem material there are differences.

H. M. PAPPIUS: I found no difference between normal animals and methotrexate perfused animals. The increase in water and sodium occurred in both, depending on the time that had elapsed since the death of the animal. A. LOWENTHAL: Yes, but in multiple sclerosis there are differences.

I. KLATZO:Just for the record: about the pulsation and possible movements in the CSF. It is true that it would be very difficult to show pulsation by electronmicroscopy. However, we have the evidence, from electronmicroscopy, that there is a movement of cell membrances; I am referring specifically to the evidence of pinocytosis that Dr. Brightman so beautifully showed in many of his photographs. Now pinocytosis implies invagination of cell membranes; in other words, there must be a movement incorporating these invaginated vacuoles.

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525

K. A. C. ELLIOIT:Dr. Tourtelotte, I think this is the end of the discussion of your paper. Would you like to tidy anything up?

W. W. TOURTELOTTE: No, I would only like to say that I think that we are certainly the first to appreciate that this is postmortem tissue. I have the feeling that only micro-determinations could settle the problem that all plaques are not the same. Then we may be able to find a quantitative increase in perivascular cuffs, which might again support the idea that the brain tissue now has a reticuloendothelium type of cell in it, and can have multifocal areas of generation of y-globulin. Then we have the problem of whether this macro-molecule can move through the brain. The theory that albumin itself is metabolized and moves back into the blood vessel is certainly an interesting idea. I don’t know how to test it right now; it is a new idea; but we certainly should test that too.

This Page Intentionally Left Blank

527

Inhibition of Sheep Brain 5-Nucleotidase by Nucleoside Triphosphates P. L. I P A T A ltistiiiite of Biological Cliemisfry, University of Pisa, Pisa, Italy

INTRODUCTION

It has been shown by Mandel at this meeting that the mononucleotides d o not penetrate the brain barrier, but they undergo a prior degradation to the corresponding bases or nucleosides, which are readily incorporated into the RNA of the Central Nervous System. The conversion of bases and ribonucleosides to ribonucleoside triphosphates, the substrates for RNA synthesis (Grunberg-Manago, 1963), probably involves one of the so called “Salvage Pathways”, which appear to be important biosynthetic mechanisms in some tissues (Abrams and Bentley, 1951 ; Lajtha and Vane, 1958): free bases may be converted to mononucleotides by reacting with a5-phosphoribosyl pyrophosphate (Flaks et a/., 1957; Lukens and Herrington, 1957), or to nucleosides by reacting with ribose-I-phosphate (Kalckar, 1947; Friedkin, 1952; Korn and Buchanan, 1955), and nucleosides may be converted to mononucleotides by reacting with ATP (Caputto, 1951 ; Kornberg and Pricer, 1951). Nucleoside monophosphates are readily phosphorylated to nucleoside triphosphates by action of appropiate kinases (Goldwait, 1957; Lowy et al., 1958), but they arealsoacted upon by group specific nucleoside-5-monophosphatases (Heppel and Hilmoe, 1951 ; Bodansky and Schwartz, 1963), to give back the corresponding nucleosides and orthophosphate. An enzyme activity, catalyzing the dephosphorylation of AM-5-P, with a pH optimum around the neutrality, was originally found by Reis (1951) in crude brain homogenates, and partially purified from sheep brain (Ipata, 1967). The enzyme has a low Km value (0.008 mM), and it would lower the concentration of intracellular AMP, available for ADP and ATP synthesis, if its activity were not inhibited by some physiological metabolite. This paper shows that sheep brain 5-nucleotidase is strongly inhibited by ATP and other nucleoside triphosphates. MATERIALS AND METHODS

Chenticals Nucleosides and nucleotides were obtained either from Sigma Chemical Co,, or from Boehringer and Soehne. Adenosine deaminase (10 mg per ml of stabilized R e J i w n w s p. 532-533

528

P. L. I P A T A

solution) was obtained from Boehringer and Soehne, and was diluted 1000 fold with water before use. All other chemicals were standard commercial products. Preparation of 5-nucleotidase The procedure described in a previous paper (Ipata, 1967) was followed, with minor modifications. The final preparation contained 1.1 mg proteins per ml, and was free of any detectable AM-5-P or adenosine deaminase activity. Assay of activity The 5-nucleotidase activity was assayed in two ways. First, the amount of inorganic phosphate released from AM-5-P was measured after incubation of the substrate with the extract. The standard reaction mixture contained, in a final volume of 0.910 ml, 0.033 M Tris-HCI buffer pH 7.4, varying amounts of substrate, adjusted to the same pH, and 0.080 ml of the enzyme preparation. After 15 min at 37” the reaction was stopped, by adding 1.8 ml of 10% trichloroacetic acid; tubes were centrifuged, and inorganic phosphate was determined on two ml aliquots of the supernatant fluid (Method A). Secondly, the activity was measured spectrophotometrically, by coupling AM-5-P dephosphorylation to the deamination of adenosine formed, in the presence of an excess of adenosine deaminase. The assays were conducted in 1 cm I-ml cuvettes; 30 pl of the diluted commercial adenosine deaminase were added to the standard reaction mixture described before, and the decrease in optical density at 265 mp was followed with a recording Zeiss PQM I1 spectrophotometer, at room temperature. AM-5-P was omitted in the reference cuvette. The velocity of the reaction was strictly proportional to the amount of 5-nucleotidase, up to rates higher than 0.200 absorbance units per minute. All rates studies reported here were conducted at rates of less than 0.070 absorbance units per minute (Method B).

0 06

005

004

. E

E 0 03

W

a

002

001

002

OC4

006

0 08

AMP coneenfrolion ( m M )

Fig. 1 . 5-nucleotidase activity as a function of AMJ-P concentration (Method B). Insert: double reciprocal plot of the initial velocity of the reaction versus AM-5-P concentration. The calculated Km is 0.008 mM.

SHEEP B R A I N 5 - N U C L E O T I D A S E

529

Modifications of the standard conditions are described in the presentation of the experimental data. One enzyme unit is defined as that catalyzing the release of I pmole PI per minute in the conditions of method A. RESULTS

General kinetic properties 5-Nucleotidase displays uncomplicated reaction kinetics : the form of the substrate saturation curve conforms with Michaelis Menten kinetics (Fig. 1). The calculated Km is 0.008 mM. The reaction rate is proportional to the enzymeconcentration up to I 10 pg of proteins per reaction mixture, and the time course of the reaction is linear for 15 min (Fig. 2). No disproportional loss of enzyme activity occurred with the

YINUTES

ENZYME (ppr.)

Fig. 2. Left: 5-nucleotidase activity as a function of time. The reaction mixtures contained, in a final volume of 0.910 ml., 2.16 mM AM-5-P, 33 mM Tris-HCI buffer p H 7.4, and 110 pg of the enzyme preparation. Right: 5-nucleotidase activity as a function of enzyme concentration. The reaction mixturecontained, in a final volume of 0.910 ml, 2.16 mM AM-5-P, 33 m M Tris-HCI buffer p H 7.4, and the amounts of 5-nucle~tidaseindicated in the abscissa. The initial velocities were determined by stopping the reaction after 15 minutes had elapsed (Method A).

dilution of the extracts. In 0.033 M Tris-acetate buffer a sharp pH optimum at ca. 7.4 was observed. No requirement for Mg++or Mn++ions for maximal activity could be demonstrated at pH 7.4. Inhibitors 5-Nucleotidase from sheep brain is strongly inhibited by ATP, UTP and CTP. GTP is without effect. The inhibition by nucleoside triphosphates is freely reversible, as shown by the fact that treatment of the enzyme with 20 p M ATP or UTP, a concentration more than sufficient to cause maximal inhibition, followed by dilution, resulted in restoration of enzyme activity. Similar results were obtained by treatment of the enzyme with nucleoside triphosphates, followed by extensive dialysis. References p. 532-533

530

P. L. I P A T A

LL7

OC6

c ._

5

005

004

W 4

003 002 001 -d--

002

--L.

004

006

. . A

008

AMP concentration ( m M )

AMP concentration ( m M )

Fig. 3. L e f t : 5-Nucleotidase activity as a function of AM-5-P concentration measured in the absence of nucleoside triphosphates (A), and in the presence of 2 , I ~ MATP (B), or 4 p M ATP (C). Righr: 5-nucleotidase activity as a function of AM-5-P concentration measured in the absence of nucleoside triphosphates (A) and in the presence of 4 pM UTP (B), or 6 p M UTP ( C ) (Method B).

0.080

1

I

3.080

0.060

.-d

E

0.020

I

0

I

3

6

9

ATP concentration (JIM 1

12

1

0

5

10

15

20

UTP concentration ( p M )

Fig. 4. Left: effect of varying concentrations of ATP on the initial velocity of 5-nucleotidase. Right: effect of varying concentrations of UTP on the initial velocity of 5-nucleotidase. The final concentration of AM-5-P in the assays was 0.12 mM (Method B).

Even in the presence of inhibitors, there is no indication of the sigmoid kinetics, which implicates the cooperative binding of substrate molecules on the enzyme molecule (Fig. 3). Fig. 4 shows the sigmoid form of inhibition, obtained when 5-nucleotidase is assayed in the presence of increasing concentration of ATP or UTP. According to Monod et al. (1965) such sigmoid curves of inhibition may be ascribed to cooperative interaction between different inhibitor sites on the enzyme molecule. While the general shape of the inhibition curves by each nucleoside triphosphate is similar, there is a significant quantitative difference in the concentration of the inhibitors required for 50% inhibition (Fig. 4).

53 I

SHEEP BRAIN 5-NUCLEOTIDASE

Studies on desensitization of 5-nurleotidase to nucleoside triphosphates inhibition I n studies of "allosteric enzymes" it has sometimes been possible to affect a desensitization of the enzyme to its inhibitors by physical or chemical treatments (Changeux, 1961: Gerhart and Pardee, 1961). Table I shows the influence of p-chloromerTAB L E I EFFECT OF ~

PCMB

U P O N S E N S I T I V I T Y OF 5 - N U C L E O T I D A S E T O I N H I B I T I O N BY N U C L E OSlDE TRIPHOSPHATES

__

~~

~

"', itiitial

Concentration of pCMB"

activity

None 0.24 0.80 I .20 I .60

I00 98 95 89 72

-

~~

inhibition of initial activity by ATP

45 35 32 22 10

% inhihition of initial activity by UTP

% inhibition of initial activity by CTP

40 32 29 22 8

28 28 28 29 28

ATPconcentration,4 pM; UTPconcentration, 8 p M ; CTPconcentration, 30,uM. TABLE I 1 EFFECT OF I N O R G A N I C P H O S P H A T E O N 5 - N U C L E O T I D A S E A C T I V I T Y A N D O N I T S I N H I B I T I O N BY N U C L E O S I D E T R I P H O S P H A T E S ~~

~

-

~

__

~

None ATP 5 ,uM Pi 0.2 mM Pi 2.0 mM ATP 5 p M ATP 5 p M I UTP 8,uM UTP 8,uM I UTP 8 p M I UTP 8 p M t UTP 8 pM CTP 3 0 p M CTP 30pM i CTP 30pM CTP 3 0 p M CTP 3 0 p M I

+

~~

~

~~

52

mM PiO.20 m M

Pi0.002mM Pi 0.02 mM Pi 0.20 mM Pi 1.00 mM Pi 0.002 mM mM mM Pi 1.00 mM

+ PiO.02 + PiO.20

~-

~

100

+ Pi 0.02

-

__

~~~~~~~

"/, of initial activity

Additions

~~

100 100

82 98 54 55 56 55 57 61 62 63 62 61 ~

~~~

~~

curibenzoate (pCMB) upon activity of 5-nucleotidase, measured in the absence and in the presence of nucleoside triphosphates. While pCMB caused almost complete desensitization to inhibition by ATP and UTP, the inhibition by CTP was unaffected. Heating of the enzyme at 56°C for various time intervals, or treatment with mercuric acetate did not lead to any desensitization of the enzyme to nucleoside inhibition. Refcrenc.i>s p. 532-533

532

P. L. I P A T A

Eflect of inorganic phosphate on enzyme activity The data presented in Table 11 show that inorganic orthophosphate does not affect the activity of 5-nucleotidase, measured in the absence of inhibitors. If the activity is measured in the presence of inhibitors, the addition of inorganic orthophosphate at 0.2 mM completely overcomes the inhibition by ATP, but not that by UTPand CTP. SUMMARY

The data presented above indicate the presence in sheep brain of a 5-nucleotidase, characterized by inhibition by nucleoside triphosphates. The substrate saturation curve can be fitted with a rectangular hyperbola (Fig. I), rather than the sigmoid curve characteristic of cooperative interaction between substrate molecules. However, a cooperative effect between inhibitor molecules is apparent in the sigmoid form of the inhibition curves (Fig. 4) by nucleoside triphosphates. This also indicates that more than one inhibitor site is present on the enzyme molecule. In some regulatory enzymes the effector site is more fragile than the catalytic site, and conditions have been achieved, favoring the selective destruction of the former. Almost complete desensitization to inhibition by ATP and UTP was observed by treating the enzyme with pCMB; desensitization to CTP has not been observed (Table I). This is interpreted to mean that the inhibitor site for CTP might be different from those for ATP and UTP. The existence of different sites for the inhibitors on the enzyme molecule is further strenghthened by the observation that inorganic phosphate overcomes the inhibition by ATP, but not the inhibition by CTP and UTP. ACKNOWLEDGEMENT

This work was supported by a research grant from the “Impresa di Enzimologia del Consiglio Nazionale delle Ricerche, Italy”. The Author wishes to thank Prof. C. A. Rossi for stimulating discussion and encouragement. REFERENCES ABRAMS, R., AND BENTLEY, M., (1951); Biosynthesis of nucleic acid purines. 11. Role of hypoxanthine and xanthine compounds. Arch. Biochem. Eiophys., 58,109- 1 1 8. 0..AND SCHWARTZ, M. K., (1963); Comparative effects of L-histidine on the activities BODANSKY, of 5-nucleotidase and alkaline phosphatase. J . Biol. Chem., 238,3420- 3427. CAPUTTO, R., (1951); The enzymatic synthesis-of adenylic acid by adenosinekinase. J . Biol. Chem., 189,801-814. J. P., (1961); The feedback control mechanism of biosynthetic L-threonine draminase CHANGEUX, by L-isoleucine. Coldspring Harbor Symp. Quant. Biol., 26.31 3-31 8. FLAKS, J. G . , ERWIN, M . J., AND BUCHANAN, J. M., (1957); Biosynthesis of the purines. XVI. The synthesis of adenosine 5’-phosphate and 5’-amino-4-imidazole carboxamide ribotide by a nucleotide pyrophosphorylase. J . Biol. Chem., 228,201-213. M., (1952); Enzymatic synthesis of desoxyxanthosine by the action of xanthosine phosFRIEDKIN, phorylase in mammalian tissue. J. Amer. Chem. SOC.,74,112-1 15.

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533

GERHART, J. C., A N D PARDEE, A. B., (1961); Separation of feedback inhibition from activity of aspartate transcarbamylase (ATCase). Fed. Proc., 20,224. GOLDWAIT, D.. (1957); Mechanism of synthesis of purine nucleotides in heart muscle extract. J. Clin. Invest., 36, 1572-1 578. GRUNBERG-MANAGO, M., (1963); Enzymatic synthesis of nucleic acid. Progr. Biophys. M o l . Biol., 13, 175-239. HEPPEL,L. A,, AND HILMOE,R. J., (1951); Purification and properties of 5-nucleotidase. J. Biol. Chein., 188,6655616. IPATA. P. L., (1966); Resolution of 5-nucleotidase from non specific phosphatase from sheep brain, and its inhibition by nucleoside triphosphates. Nature, 214, 618. KALCKAR, H. M., (1947); The enzymatic synthesis of purine ribosides. J. Biol. Chem., 167, 477486. KORN,E. D., AND BUCHANAN, I. M., (1955); Biosynthesis of the purines. VI. Purification of liver nucleoside phosphorylase and demonstration of nucleoside synthesis from 4-amino-5-imidazolecarboxamide, adenine, and 2,6-diaminopurine. J. B i d . Chem.,217, 183- 191. KORNBERG, A., AND PRICER, W. E., Jr., (1951); Enzymatic phosphorylation of adenosine and 2,6diaminopurine riboside. J . Biol. Chem., 193,481-495. LAJTHA,L. G., AND VANE,J. R., (1958); Dependence of bone marrow cells on the liver for purine supply. Natrrre, 182, 191- 192. LOWY,B. W., RAMOT,B., A N D LONDON,1. M., (1958); Adenosine triphosphate metabolism in the rabbit erythrocyte in vivo and in vitro. Ann. New York Acad Sci., 75, 14&155. LUKENS, L. N., A N D HERRINGTON, K. A., (1957); Enzymatic synthesis of 6-mercaptopurine ribotide. Biochim. Biophys. Acta, 24,432433. MONOD,5.. WYMAN, J., A N D CHANGEUX. J. P., (1965); On the nature of allosteric transitions: a plausible model. J. Mol. Biol., 12,88-118. REIS,J., (1951); The specificity of phosphomonoesterase in human tissues. Biochem. J . , 48, 548-551. DISCUSSION P. 1.IPATA: Perhaps the control mechanism found at the level of the adenine nucleotides might be a more general one. We have found that the dephosphorylation of G M P is inhibited by GTP, and that the dephosphorylation of CMP is specifically inhibited by UTP. I won’t go any further into this subject, I only wish to add that the inhibition of 5‘-nucleotidase by nucleoside triphosphates might have a physiological role. In fact, AMP, which is synthesized from adenosine, or even from inosine by one of the so called “salvage pathways”, must be transformed into ATP in order to be incorporated into RNA, and therefore its breakdown must be inhibited. A. LAJTHA:Would not this scheme work the wrong way too? If ATP concentration is suddenly lowered, wouldn’t you retain inhibition and use up more ATP? P. I.. IPATA:I understand your point: the inhibition of 5’-nucleotidase is observed at such low concentration of ATP ( I /molar ATP gives about 504( inhibition) that one would expect that its activity is practically absent in the nervous cell. A. LAJTHA: I t is a constant control, not a variable control then? P. L. IPATA: Yes. And this fact perhaps will also explain the findings with respect to uracils, that when you kill the animal and immediately look for the adenosine or inosine, you don’t find inosine. Because if you kill the animal immfdiately then this cycle is still inhibited by this metabolite. H. KOENIG : In histological preparations, stained for 5’-nucleotidase the enzyme is found to have rather specific cellular sites. It is largely absent from nerve cell cytoplasm (except in lysosomes, which may be monospecific acid phosphatase) but is found in the neuropil and in white matter, where it occurs in glia that seem morphologically to be astrocytes (Koenig, H. and Barron, K. D. (1962) Acta Nerirol. Scaud., Srrpp. I , 72). In prcliminaryelectronmicroscopic studies, Dr. Kevin Baron found that 5’-nucleotidast seems to be associated uith the endoplasmatic reticulum of astrocytes. P. MANDEL: Where is the 5’-nucleotidase? Because to protect the soluble ATP it should be a cytoplasm. And if it is in the endoplasmic reticulum it cannot protect, unless the cell is destroyed,

534

P. L. I P A T A

P. L. IPATA: We have performed some experiments with subcellular fractions, prepared in isotonic sucrose. and have always found the enzyme activity in the soluble fraction. However, also in the particulate fraction there was some AMP-ase activity. The possibility of the existence of a particulate and a soluble AMP-ase cannot be excluded. Let me point out that in the histological detection of 5’-nucleotidase some of the activity may be ascribed to non specific phosphatases acting upon AMP, as well as upon other phosphorylated substrates. The purified enzyme, however, does not split phenyl phosphate or glycerophosphate. It is possible that it cannot be detected histologically in the cytoplasm because of its strong inhibition by ATP.

P. MANDEL: The non-specific phosphatase is in the cytoplasm.

535

Conclusions A. L A J T H A

AND

D. F O R D

The general discussion of many aspects of a problem that occurs at a conference and is later presented in a book usually raises more questions than it answers. Perhaps the success of the conference can be measured by the extent to which the problems are clarified, and also by the number of participants who return to their laboratories with new ideas and decide to investigate these problems further. The discussions presented in this book are no exception. They clearly show not only the complexity but also the multiplicity of the barrier system, i.e., that there are many different pathways and mechanisms involved in the passage of substances; and they show this multiplicity and complexity in all aspects of the system. A number of contributions describe multiplicity in membrane structure and morphology, and initial studies of the chemical composition of membranes in the nervous system also attest to their complex composition, which varies from one membrane to another and also changes during the function and the alteration of the function. Many contributions in this volume, perhaps for the first time in the history of investigations of the barriers, show the multiplicity of the mechanisms involved in passage of substances in the brain. They clearly show that these mechanisms vary with different substances and that the properties of passage show selectivity and specificity. The multiplicity of mechanisms is also shown in the reaction of the barrier system to stimuli, in its various aspects being influenced differently during development, under outside influences, and in various pathological states. Clearly, the barrier system is a dynamic entity that can be altered under many conditions, physiological and pathological. I t emerges from many of the contributions of the present volume that alterations may affect only specific aspects and specific substances and that the mechanism of various alterations can be widely different. A particular change could affect movement in only one direction, only one type of mechanism, or one substance. Other changes, on the other hand, may affect movement in a different direction, a different type of mechanism, and perhaps different substances. What we study is not a single barrier with a single property, but a complex and multiple system; the further understanding of all of the properties and mechanisms is the challenge of the future for the workers in this field. It would clearly be impossible to summarize briefly all the recent accomplishments that are described in the book. Instead, let us very briefly and in an arbitrary and random fashion choose a few of the questions that have been raised i n the book. In each area of the various questions, great advances have been made in the past years, but in each of them the final and lucid answer still evades us.

536

A. LAJTHA A N D D. F O R D

The following questions perhaps illustrate the range of the problems that were discussed and which are the subject of so much excellent continuing work: I. In what way does permeability of a membrane depend on membrane constituents? Does diffusion through membranes change through alteration of membrane composition? How do reversible changes in membrane structure reflect such changes in membrane permeability? 2. In what way do differences in membrane structure from one brain area to another, or one membrane to another, explain differences in permeability characteristics? 3. Do membranes participate in binding of substrates and can some permeability phenomena be explained by mechanisms of binding? 4. Do chemical changes in membranes parallel physical and morphological changes, and in what way could such changes affect membrane selectivity? 5. Are all or some membrane constituents in a dynamic state; what is the relationship of membrane turnover to membrane function; and what are the possible alterations of membrane turnover? 6. In what way are the levels of cerebral metabolites important for cerebral metabolism, and in what way do alterations in the concentration of cerebral metabolites affect cerebral function? 7. What are the mechanisms that determine the level of cerebral constituents? In particular, what roles do transport processes play in cerebral homeostasis and in the determination of the level of particular cerebral components? 8. What is the compound supplying energy directly for transport? Is there a single source of energy for all kinds of transport, and how much energy is used in the brain for transport? 9. How do observations made in in vitro systems relate to similar observations made in a living animal? Which properties of permeability and transport are altered in isolated systems, and in what way? 10. Do the properties of the barrier system explain the pattern of alterations under pathological conditions? For example, does the substrate specificity of transport explain changes in aminoacidurias? 1 1. What are the processes that determine the constitution of the cerebral spinal fluid? 12. How many different kinds of transport processes exist in brain, and in general, are the mechanisms of uptake identical with mechanisms of exit? Is there a separate mechanism for exchange, and is exchange always independent of available energy? 13. What is the ionic composition of cells in brain, and what are the differences in such composition of various cells? Can the differences be explained by differences in distribution of ion pumps? As an example of this problem, is there any evidence that glia have different ion and metabolite composition from neurons and could this be explained by differences in transport? 14. How can the observed differences in barrier function during development and the regional differences be explained by differences in transport mechanisms? 15. Do the various transport mechanisms and various ion pumps work through different mechanisms, or are most transport mechanisms very similar? Can any of such mechanisms be separately influenced or separately controlled?

CONCLUSIONS

537

16. What is the mechanism of alteration in the properties of the barriers? Is the mechanism of decreasing penetration through the barrier a single one or are there different ways of altering barriers, and can we clinically utilize methods of altering such barriers? 17. What is the mechanism of action of drugs on permeability? 18. How can we define the spaces in the brain, and what is the meaning of such spaces for structure, for permeability, and for transport? These questions, and many more, were raised and discussed. The ones here were arbitrarily chosen and are in the sequence in which they came up during the conference. On purpose the conference discussed only a few functions of membranes and did not touch others; there was no discussion of the roles membranes may play in growth, in differentiation, in changing structures, and degeneration, although these are all important. Even though in many of these processes the permeability of the membrane may play crucial roles, limitations of space and time obviously had to be observed. Similarly, only certain aspects of the barriers are discussed in the present book, although we have to recognize that many other processes may contribute to the regulation of the composition of the cerebral components, their level as well as their access to brain. Some of these may be enzymatic (rates of metabolism); others may deal mostly with the participation of other organs (such as rapid removal of substances by liver, preventing entry to brain). It was not the purpose of the meeting and this book to make a final definition of what membranes the brain barrier system actually include, and indeed, there are some disagreements between the various workers whether even intracellular membranes, mitochondrial, nuclear, etc., ought to be considered as part of the barrier system. Another conference and another book in the not too distant future are needed to bring us closer to the answers to these and other important questions. This book summarizes much of the important information that was gained recently and which clearly emphasized the importance of permeability processes in the nervous system. Beyond pointing out the complexity of the system and the need for further knowledge, the conference was very helpful in elucidating the many roles the brainbarrier system plays in brain function and the many roles the alteration of the system plays in the alteration of brain function under diverse conditions. The system is an integral part of the brain which serves not only as a defense mechanism inhibiting the entry of foreign substances, not only as a homeostatic mechanism inhibiting large changes in the composition of the brain, not only as a supply mechanism mediating the access of substrates and the exit of metabolic products, but as a control mechanism which, by determining the composition of the various metabolic pools, can significantly influence cerebral metabolism and can react to the varying needs of the organ. With these many roles, and perhaps many more, it is also clear that pathological or pharmacological agents affecting some part of the barrier system will affect some aspects of brain function. The central role the brain-barrier system plays in the central nervous system is clearly underscored in the present volume.

This Page Intentionally Left Blank

539

Author Index Abadom, P. N., 429, 201, 245 Abrams, R., 527 Acheson, E. D., 5 17 Adams, R. D., 498, 514 Addison, W. H. F., 494 Aigner, P., 394 Aird, R. B., 401 Akedo, H., 440 Albers, R. W., 125, 467 Aleu, F. P., 322 Allison, A. C., 87, 275 Allman, J., 410 Alousi, M. A., I1 1 Alvord, E. C., 7, 323 Anies, A., 137, 284 Andre-Balisaux, G., 517 Arnaiz, G. R. de L., 125 Arsove, S., 2 Askanazy, M., 75 Autilio, L., 5, 502 Bachelard, H. S., 430 Bacher, H. van, 404 Bakay, L., 19, 315-336, 343, 357,401, 417 Baer, S., 2 Bahr, G. F., 477 Bailey, P., 68 Baima-Bollone, P. L., 53 Bak, A. F., 385-395 Baker, J. R., 87 Baker, P. F., 27 Balfour, Y. M., 2 Ballantine, H. T., 325, 327 Ballou, C. E., 3, 8 Barham, G. B., 309 Barlow, C. F., 321, 323. 367-378, 505 Barnes, J. B., 322 Barrera, E., 494 Barrett, P. V. D., 317 Barrnett, R. J., 42, 63, 65, 264, 265,401 Baudhuin, P., 1 1 1 Baxter, C. F., 274, 429448 Becker, H., 322 Becker, K., 344 Becker, R. A,, 401 Begin, N., 179 Behnsen, G., 417 Behring, E., 349 Behrman, R. E., 318, 417 Bell, H., 327 Bellamy, D., 432 Bench, K., 63

Bendixen, H. H., 323 Bentley, M., 527 Berg, C. J. van den, 430 Bering, E. A. Jr., 19, 31, 33, 35, 135, 136 Bert, S., 267, 429 Berlin, N. I., 317 Bernfeld, P., 94 Bernsmeier, A., 344 Bessman, S. P., 437 Beyer, K. H., 185, 186 Bhattacharya, B. K., 283 Birge, W. J., 68 Bito, L. Z., 510, 511 Blane, W. A., 317 Blank, M., 279 Blasberg, R. G., 174, 185, 188, 189, 201, 245-255, 401, 429,440 Blau, M., 362 Bloom, G., 477 Bodansky, O., 527 Boell, E. L., 43 Boldizsar, H.,283 Bonting, S. L., 66 Bornichon, A., 53, 55 Borrel, A., 401 Bosczko, M., 349 Boucher-Firly, S . , 432 Bourke, R. S., 459, 465467, 469, 470, 472, 473,475477 Bowen, W. R., 317 Bowsher, D., 19 Boyd, D., 320 Brachet, J., 87 Bradbury, M. W. B., 135, 140, 283, 287, 510, 51 1 Bradley, D. F., 87, 92 Brattgard, S. O., 404 Breemen, V. L., van, 63, 125-129 Brendel, W., 385, 394 Brewer, D. B., 108, 1 1 1 Brierley, J. B., 323 Brightman, M.W., 19-36, 51, 65, 66, 75, 477, 498,499 Britton, H. G., 141 Brizee, K. R., 404, 476 Brockerhoff, H., 3, 8 Brodie, B. B., 496 Broman, T., 19, 322, 326, 358, 401, 516. 517 Brooks, C. McC., 135 Brown, A. K., 316 Brown, J. L., 186 Brown, P., 19

540

A U T H O R INDEX

Bryan, E. R., 497, 505 Brzin, M., 41-59 Buchanan, J. M., 527, 529 Buniatian, H. Ch., 433 Burch, G. E., 438 Burk, D. J., 175, 189, 250 Burton, R. M., 2 Byrne, J. M., 88, 93, 108, 111 Cajal, S. R., 275 Campbell, J. W., 279 Cantore, G., 141 Caputto, R., 527 Carr, S., 4 Case, N. M., 63, 66 Casey, T. A., 141 Cassen, B., 324 Chaikoff, I. L., 417 Chance, B., 409,477 Changeux, J. P., 530, 531 Changus, G. W., 417 Chaplin, A. E., 434 Chargaff, E., 2 Chen, H., 318 Cherayil, A., 429 Chirigos, M. A., 201, 401 Chow, S. Y.,307 Christensen, H. N., 185, 187, 188, 245, 362, 431,440 Clark, S. L., 79 Clarke, D. D., 429 Clarkson, T. W., 360 Claseu, R. A,, 320 Clayman, S., 173-181 Clemendson, C . J., 323, 377 Clemente, C. D., 63 Clydesdale, F. M., 40Y Colburn, R. W., 129 Coles, H., 409 Collewijn, H., 385, 393 Colon, E., 404 Colvin, R. B., 267 Compston, N. D.. 524 Cooke, P. M., 320 Coombs, J. C., 456 Copenhaver, J. H., 66 Cotlove, E., 497 Covell, W. P., 87 Cowan, D., I13 Coxon, R. V., 135-142 Cragie, E. H., 274, 275 Cremer, J. E., 430 Crone, C., 363 Crowell, J., 394, 477 Csaky, T. Z., 147-152, 362 Cserr, H . F.,283, 285, 287, 290 Cummings, J. N., 322 Cummins, J. T., 279 Cutler, R. W. P., 321, 367-377

Dahl, D. R., 430 Danielli, J. F., 87 Davidsson, D., 283 Davison, A. N., 142, 419 Davitian, M. A., 433 Davson, H., 87, 135, 136, 138, 140, 147. 283, 284, 287, 297, 301, 385, 436, 457, 466, 477, 498, 510, 51 1 Dawson, R. M. C., 2, 3, 13 Day, R. L., 317 Deams, W. Th., 11 1 De Duve, C., 87, 93, 108, I 1 1 Dell, M., 369, 376 De Lorenzo, A. J. D., 42 Dempsey, E. W., 19, 63, 66, 401 De Robertis, E., 401 Dettbarn, W. D., 41 Deul, D. H., 125-129 Diamond, I., 317, 417 Dickens, F., 409 Dickerson, J. W. T., 423 Diezel, P. M., 2 Dingman, W., 437 Dittrner. J., 3 Dixon, M., 250 Dobbing, J., 142, 264, 318, 401, 417-424, 498 Dossetor, J. B., 451 Doust, J. W. L., 345 Downer, J.. 283 Downing, S. J., 186 Draskoci, M., 386 Duchlteau-Bosson, Ch., 434 Duffy, P. E., 41-59 Dunn, E. R., 142 Eavenson, E., 437 Eccles, J. C., 456 Edstrom, R., 315, 326, 404 Efron, M. L., 186 Eggleston, L. V., 245 Ehrlich, P., 401 Eichberg, J., 2, 13, 125 Elliott, K. A. C., 142, 201, 245, 259, 267, 274, 430,451454,456,413,474 Endo, S., 137 Erbsloh, F., 344 Ernster, L., 317, 477 Erwin, M. J., 527 Escriva, A., 285, 287-289 Esquive de Gallardo, F. O., 318, 417 Essner, E., 107, 11 1 Evans, J. P., 321, 332, 333 Evelyn, K. A., 495 Ewald, A., I 1 Farquhar, M. G., 20, 65, 76, 106, 401 Farr, A. L., 467 Fatt, P., 456 Feindel, F., 320, 321

A U T H O R INDEX

Feldberg W., 283, 286 Fencl, V., 137, 138, 290, 300, 301 Ferguson, R. K., 297, 298, 310 Fernando, J., 111 Ferris, P. J., 386 Field, E. J., 517 Finch, L. R., 187 Finerman, G. A. M.,437 Fisher, M. A., 267 Fisher, R. G., 66 Fishman, R. A,, 19, 31 Flaks, J. G., 527 Fleischauer, K.,167, 386 Flodmark, S., 359, 360 Florkin, M., 431, 434 Folch-Pi, J., 1-13 Ford, D. H., 401411,535-537 Forgrave, P. R., 369, 376 Foroglou-Kerameos, C.. 42 Fox, M.,186, 187,437 French, C. M., 485-489 Friberg, U., 477 Friedenwald, J. S., 42 Friedkin, M.,527 Friedman, V., 135, 136 Friedmann, M. M., 496 Fries, B. A., 417 Frohlig, A., 349 Fugelli, K., 431 Fukuda, T., 57 Funahashi, S., 5 Furst, S., 437 Gad Andresen, K. L., 140 Gahen, D. Q., 267 Gaines, D., 87, 110 Gaitonde, M. K., 401, 429, 430 Galambos, J. T., 318 Giinshirt, H., 330 Garoutte, B., 401 Gaskins, J. R., 466, 478, 485, 488 Gelder, M.van, 259-268, 245 Gerebtzoff, M.A., 53, 55 Gerhart, J. C., 531 Gerschenfield, H. M., 401 Gerstein, A., 437 Giacobini, E., 41 Gianetto, R., 1I 1 Gibson, I. M., 275-277 Gilles, R., 434 Ginsburg, S . , 283-293 Glees, P., 126 Glusman, M.,496 Goldberg, M.A., 332, 368, 372 Goldmann, E. E., 19, 75, 315, 349, 401 Goldwait, D., 527 Goldworthy, P. D., 401 Golmstedt, B., 45 Goldstein, N. P.,321

54 1

Gomez, G. J., 2 Gommi, B. W., 125 Gonatas, N. K., 88, 321 Gonras, P., 394 Gonsette, R., 517 Gordon, M. S., 431,435 Gordon, M.W., 266,437,494,495 Gracia Argiz, C. A., 267, 386, 394 Gray, R., 87, 93, 110 Graziani, L., 283-293 Greenberg, E. S . , 459,477 Greenfield, P., 43 Greengard, P., 201,401 Grkgoire, A., 498, 499, 516 Grillo, M. A., 79 Grindlay, J. H., 321 Grontoft, O., 322,422424 Grossmann, A., 316 Grunberg-Manago, M.,527 Guidetti, K. L., 140 Gulati, R., 452 Gunn, C. K., 317 Guroff, G., 245,401,436 Gustavson, G. M., 497, 505 Hagen, J. H., 141 Hager, H., 103, 107, 108 Hagerstrand, I., 329 Haisma, J. A., 125-129 Hansson, E., 360, 361 Haranath, P. S. R., 386 Harden, H., 87 Harmon, E. A., 4 Harper, A. M., 409 Harreveld, A., van, 307,385,393, 394,477,478 Harris, R. C., 316 Hartelius, H., 323, 377 Hartman, J. F., 401 Hartstein, M.,401, 407 Hastings, A. B., 478 Hauser, H. M.,321 Hawkins, N. M.,66 Hayner, R., 321 Hearer, A. F., 497, 505 Heath, D., 108, I 1 1 Heinz, E., 173, 187, 246 Heiser, W. J., 335 Heisey, S.R., 136-138, 148, 151, 283, 285 Held, D., 136-138, 151, 283, 285 Heller, I. H., 274, 474 Hems, R., 245 Henderson, E. S., 284 Heppel, L. A., 527 Herlin, L., 317 Herrington, K. A., 527 Hibbard, E., 318, 417 Highashi, K., 284 Hillman, H. H., 276, 279 Hilmoe, R. J., 527

542 Hindmarsh, J. T., 187 Hirano,'S., 245, 429 Hird, F. J. R., 187 Hodges, P. C., 322 Hodgkin, A. L., 274,279 Hollenberg, P. F., 437 Holmberg, G., 323, 377 Holst, R. J., 141 Horecker, B. L., 173 Horstmann, E., 275 Hosie, R., 125, 128 Housepian, E. M., 475 Howard, R. E., 2 Hoyer, S., 344 Hren, N., 2 Hsia, D. Y.,316 Huggins, A. K., 434 Hughes, I. E., 283 Hugh-Jones, K., 316 Hurlebaus, A., 110 Hydkn, H., 404

AUTHOR I N D E X

'

Ibrahim, M. Z., 514 Inui, Y.,437 Ipata, P . L., 527-532 Jraldi, A. P. de, 125 Ishii, S., 321, 330 Israely, R. A,, 405 Itabashi, H. H., 499, 501, 514, 518 Jacobi, W., 135 Jacobs, L. A., 476 Jacobson, I., 409 Jamieson, D., 409 Janigan, D. T., 111 JanZik, H., 126 Jasper, H. H., 451 Jeanette, A., 125 Jenkins, D. A., 326 Jeuniaux, Ch., 434 Joanny, P., 267,277-279 Johannisson, E., 477 Johnson, B., 376 Johnson, L., 317 Johnston, P.V., 41 Jones, I. C., 432 Jones, M. E., 436 Jordan, E. F., 137, 151, 283 J6rgensen, C. E., 362 Junet, W., 79 Kabat, E. A., 496 Kakefuda, T., 453 Kalberer, F., 148 Kalchar, H. M., 527 Kalsbeck, J. E., 322 Kaplan, R., 285, 287, 288 Kappers, J. Ariens, 68, 75, 79

Karler, R., 311 Karnovsky, M. J., 19, 20, 30, 35 Karrer, H. E., 111 Katzman, R., 283-293, 321, 322, 498, 504 Keesey, J. C., 278,469,470,476 Kelentei, B., 349 Kelly, W. A., 321 Kety, S. S., 343, 409 Kemp, J. W., 436 Kenemy, A., 283 Kernohan, J. W., 322 Keynes, R. D., 274 Kharetschko, X.,404 Kies, M. W., 7 Kilby, D., 187 King, L., 19 Kirkland, R., 125, 127, 129 Kiser, W., 186 Kishimoto, Y.,494,495,499, 501 Kitano, M., 362 Klarner, P., 344 Klatzo, I., 35, 320, 331, 357, 385-395, 452, 455,456

Kleeman, C. R., 140,436 Klenk, E., 1, 11 Kliiver, H.,494 Klyne, W., 468, 518 Knaape, H. H., 436 Knaub, V., 496 Koa, F. F., 135 Koelle, G. B., 42, 45, 57 Koenig, E., 57, 113 Koenig, H., 87-117 Kokman, E., 499 Korn, E. D., 527 Kornberg, A., 527 Koval, G. J., 125 Kovic, M., 41, 42 Krebs, H. A., 66, 148, 245 Kreutzberg, G. W., 103, 107, 108 Kristiansen, K., 386 Krog, J., 386 Krogh, A., 401 Kubik, C. S., 498, 514 Kuffler, S. W., 35, 51, 320, 477 Kuhn, R., 2 Kuhne, W., 11 Kuttner, R. S. J. A., 266, 437 Ladman, A. J., 20, 63, 66 Lahiri, S., 245, 362, 429, 436 Lajtha, A., 174, 185, 188, 189. 201-214, 245, 246, 249, 253-255, 264, 267, 362, 363, 386, 401,429,436,437,440,527,535 Landau, B. R., 362 Lange, R., 431 Larsen, J. A., 141 Laster, L., 187 Laurence, D. J. R., 126

A U T H O R INDEX Lauter, C. J., 128 Lazansky, A., 51 Le-Baron, F. N., 1 I Leblond, C. P.,404 Ledeen, R., 1 Leduc, E. H., 19,401 Lee, J. C., 20, 35, 320, 322, 326, 331, 332, 377 Lees, M., 4, 5 Lehninger, A. L., 477 Leiderman, P. H., 283 Lein, I. N., 318 Leiner, K. Y.,467 Lemmon, W. W., 438 Lennox, B., 468, 518 Leusen, I., 137, 283 Levi, G., 174, 201, 245, 246, 249, 401, 429, 440 Levin, E., 140, 267, 386, 436 Levine, S., 321 Lewandoswky, M.. 401 Lewis. P. R., 42, 43 Li, C. L., 216, 385-395 Liang, M., 185 Lin, C. S., 318 Lin, E. C. C., 187 Lindsay, T. R., 430 Lineweaver, H., 175, 189, 249 Lipmann, F., 436 Lloyd, B. B., 135 Locksley, H. B., 19 Loeper, M. M., 75 Loeschcke, H. H., 137 Lolley, R. N., 279 Lombardo, G. van, 438 London, 1. M., 527 Long, D. M., 385 Long, R. J., 330, 333, 334 Lorenzo, A. V., 367-377 Love, W. D., 438 Lowden, J. A., 502 Lowenthal, A., 504 Lowry, 0. H., 467 Lowy, B. W., 527 Lucey, J. F., 316, 318, 417 Luck, C. P.. 138 Luft, J., 20 Lukens. L. N., 527 Lumsden, C. E., 514, 517 Lund, I., 386

MacLean, 9. S., 140, 142, 276, 277 MacLean, J. R., 316 McMurray, W. C., 422 Maeda, T., 42 Magee, P. N., 322 Magnus, G., 135 Malaniud, N., 318 Malhotra, S. K., 374, 411, 478 Malloy, H. T., 495 Mallucci, I., 87 Marchbanks, R. M., 125 Marchesi, V. T., 265 Marcus, P. I., 88, 1 I 1 Mason, J. K., 468, 518 Massion, W. H., 137 Matsumoto, R., 7 Matsutani, T., 245, 429 Matthews, C. M. E., 330. 332-334 Matthews, D. M., 187 Maxwell, D. S., 20, 25, 63 Maynard, E., 401 Meath, J. A., 2 Mela, P., 245, 430, 440, 477 Menken, M., 317 Messner, K., 385, 394 Michaelson, I. A., 125, 127, 129 Miguel, J., 19, 35, 320, 331, 386 Millen, J. W., 63, 147, 326, 417 Millichap, J. G., 31 I Mitchell, R. A., 137 Mizell, S., 433 Mizung, N., 88 Molinaro, G., 330, 332-334 Monod, J., 530, 173 Moody, R. A., 321 Morelos, B. S., 430 Morgan, W. S., 1 I 1 Mori, S., 42 Morley, T. P., 331 Mossakowski, M. J., 385-395 Moulton, M. J., 333 Muhleman, D. R., 429 Mullan, S., 332 Muller, Ch., 385 Mulnard, J., 88 Munday, K. A., 434 Mundinger, F., 333 Murakami, M., 5, 14

Maas, J. W., 129 MacAfee, J. G . . 330, 333, 334 McAlpine, D., 514, 517 McCandless, D. W., 317 McDonald, T., 87, 98, I10 McDowall, D. G., 409 McGuckin. W. F., 321 Mcllwain, H., 2, 273-280, 394, 469, 504 Maclntyre, I., 283 McKenzie, 9. F., 321

Nachmansohn, D., 41, 43 Nagata, Y.,245, 429 Nagayama, M., 429 Nakamura, R., 429 Naurnann, H. N., 468, 518 Neame, K. D., 185-194, 245, 429,437 Neff, R., 324 Neptune, E. M., Jr., 409 Nesbett, F. B., 284 Netsky, M. G., 68

543

544

AUTHOR INDEX

Newey, H., 185, 187 Nicholls, J. G., 35, 51, 320, 477 Niemer, W. T., 369, 376 Nims, L. F., 285 Noback, C. R.,475, 476 Nogueira. G. J., 267, 294, 386 Norton, W. T., 502 Novikoff, A. B., 43, 66, 107, I 1 I Nurnberger, J. I., 494 Nystrom S., 329 Obrador, S., 451 O’Brien, R. D., 125 Ochs, S., 393 Ogala, E., 477 Ogawa, K., 88 Oh, J. H., 451 Ohta, T., 386 Okamoto, M.,88 Oksche, A., 75 Oldendorf, W. H., 362, 498 Olsson, 0.. 358 Olszewski, J., 20, 35, 326, 331, 333, 377 Oman, S., 41, 42 O’Meallie, L. P., 438 Omrnaya, A. K., 284 Oppelt, W. W., 386, 394, 459, 477 Orkland, R. K.,51 Ortiz. C. L., 432, 437 Osinchak, J., 107 Otenasek, R., 19, 35, 320 Otila, E., 75 Oxender, D. L., 187, 245 Pakkenberg, H., 405 Palade, G. E., 63, 65, 76 Palay, S. L., 79 Palsson, P. A., 517 Pandolfi, S., 320 Pappas, G. D., 19, 20, 29, 33, 51, 63-83,401, 475 Pappenheimer, J., 136-138, 151, 283,285, 290, 300, 301 Pappius, R., 417 Pardee. A. B., 531 Parker. J. A., 493-520 Parker, L. O., 385-395 Parker, P., 283 Passow, H., 360 Patlak, C. F., 386, 394 Patlak, C. S., 459,477 Pattison, I. H., 517 Pavlin, R.,41 Pease, D. C., 20, 25, 63, 401 Pdrier, 0.. 498, 499 Pethes, G., 283 Pfeiffer, B., 187 Pichler, A. G., 187 Pick, J., 111

Pine, A. B., 43 Pi-Suner, J., 451 Pitts. R. F., 186 Plum, F., 323 Polani, P. E., 318 Pollay, M.,283, 310, 457, 466, 477, 485 Posner, J. B., 323 Prados, M., 320, 321 Pricer, W. E., Jr., 527 Pritchard, E. G., 8 Prockop, D. J., 495 Prokop, J. 0.. 386 Pryrot, R., 417 Purpura, D. P., 29, 51, 429. 475, 476 Quadbeck, G., 322, 343-346, 349-352 Quarles, R., 3 Quastel, J. H., 185 Quinn, J. J., 335 Quintana, N., 43, 107 Radin, R. S., 494, 495. 499, 501 Raimondi, A. J., 320. 331, 332 Rall, D. P., 159-167, 284, 301, 386, 394, 459, 466, 477, 485, 488 Rall J. E., 401, 478 Rarnot, B., 527 Rasmussen, H., 477 Rasmussen, L. E., 385-3Y5 Reed, D. J., 290, 299, 302-304 Reese, T. S., 12, 30, 33, 35 Reis, J., 527 Reulen, H. J., 385, 394 Rhines, R., 401, 404, 405, 407, 408, 410 Rhodes, A., 401,404,407,410 Richard, K. A,, 497, 505 Richardson, L. A,, 33, 76, 79 Richer, D.. 401 Richmond, J. E., 478 Riggs, T. R., 440 Rigor, B. M.,147-153 Rijssel, Th. G. van, 1 I 1 Ritchie, J. M., 274 Robbins, E., 88, 1 1 I Robbins, J., 401 Robertis, E. de, 125 Roberts, E., 429-431,453 Roberts, G. C. K., 129 Roberts, N. R., 467 Robson, E. B., 186 Rodnight, R., 277 Rodriguei, L. A., 19, 30, 401 Rogers, G. E., 63 Rogers, H. J., 94 Roots, B. I., 41 Rose, G. A,, 186 Rosenberg, A., 2 Rosenberg, D. G., 318 Rosenberg, L. E., 186, 187. 437

AUTHOR INDEX

Rosenberg, P., 41 Rosenberg, T., 185. 245 Rosenblatt, F., 125 Rosenbluth, J., 43, 51, 65 Rosengren, H., 137 Rosenhagen, H., 66 Rossi, C. S.. 477 Rostgaard, J.. 265 Roth, L. J., 323, 367, 368, 372 Rothman, A. R., 160 Rothstein, 4., 360 Rothstein, M., 429 Roux, E., 401 Rozdelsky, B., 326 Rubin, R. C., 284 Russo. H . F., 185, 186 Rutschman, J., 148 Sabatini, D. D., 63 Sadler, K., 33, 301, 302 Sakanoue, M., 137 Salganicoff, L., 125 Samiy, A. H., 187 Samojarski, T., 321 Sass-Kortsak, A,, 316 Sato, O., 135, 136 Schade, J, P., 274, 385, 394, 402 Schaepfer, W. W., 43 Schafer, I . A,, 186 Schain, R. J.. 301, 518 Schenker, S., 317 Schmid, R., 317, 417 Schmidt, C. F., 343, 409 Schmidt, W., 88, 1 1 1 Schofer, R., 475 Schoffeniels, E., 43 I,433, 434 Scholefield, P. G.. 173-181, 201, 245 Schoolar, J. C., 367 Schultz, R. L., 401 Schwab, J. H., 93 Schwartz, M. K., 527 Schwerin, P., 437 Scott, G. H., 87 Scott, J. Jr., 87, 110 Scriver, C . R., 186 %gal, S., 186, 187, 437 Sekine, H., 5 Sellers, R. D., 322 Selverstone, B. 333 Seminario, L. M., 2 Severinghaus, J. W., 137 Shafig, S. A,, 87 Shaner, G. A,, 185, 186 Sharrett, B. M., 432 Shaw, T. I., 271 Shen. S. C., 43 Sheridan, M. N., 125 Shimizu, N., 42 Sholl, D. A., 274

Shuangshoti, S., 68 Shute, C . C. D., 42, 43 Simon, K. A., 66 Simonds, W. J., 136 Simonsen, D. G., 431 Simpson, J. F., 499 Simpson, K., 316 Sims, J. A., 266 Sinapius, D., 93 Sjoqvist, F., 45 Skeggs, H. R., 185, 186 Slack, J., 316 Slater, E. C., 477 Smith, D. E., 320, 386 Smith, D. S., 43, 51 Smith, R. E., 106 Smith, S. E., 429 Smyth, D. H., 185 187 Sokoloff, L., 367, 377 Sosa, D., 325 Spaziani. E., 385 Spector, L., 436 Spence, M. W., 2 Spensley, J. C., 94 Sperl, M. P., Jr., 321 Spitznagel, J. K., 93 Sporn, M. B., 437 Stanley, P. H., 322 Starzl, T. E., 369, 376 Steinwall, O., 326, 357-363, 387 Stempfel. R., 316 Stern, J. A., 245 Stern, L., 417 Stoner, H . B., 322 Story, J., 322 Streicher, E., 357, 466. 478, 485, 487 Strong, L. H., 68 Strowger, B., 320, 321 Strumia, E., 53 Stubbs, J., 283 Sudduth, H. C., 409 Sundberg, C . , 75 Suyter, M., 141 Svennerholm, L., I Svien, H. J., 321, 326 Svien, J. J., 321 Swanson, A. G . , 137, 138 Swarm, R. L., 317 Sweet, W. H., 37, 19 Tager, J. M., 477 Takagaki, G., 429 Tallan, H. H., 245 Tamburino, G., 438 Tanaka, H., 88, 1 1 1 Tani, E., 330 Tator, C. H., 331, 333 Tauxe, W. N., 326 Taylor, J. L., 137

545

546

A U T H O R INDEX

Teller, J. D., 151 Tenenbaum, D., 10 Tennyson, V. M., 19,20,33,41-59,63-83,299 Tercafs. R. R.. 431. 434 Terry, R. D., 322 ' Thiele, 0. W., 93 Thier, S., 186, 187, 437 Thomas, J., 173 Thomas, J. J. Jr., 409 Thomas, 0. L., 87 Thompson. E. B., 7 Thompson, J. W., 142 Thudichum, J. L. W., 274 Toews, C. J., 142 Tonnis, W., 330 Tooze, J., 477 Torack, R. M.,42,43,65, 108,329,330 Toschi, G., 57 Toth, J., 201, 245, 362,401,429,436 Tourtellotte, W. W., 493-520 Tower, D. B., 274,459,465479 Trams, E. G., 128 Treherne, J. E., 43, 51 Trevor-Roper, P. D., 141 Trump, B. F., 111 Tschirgi, R. D., 19, 137. 325, 401 Tsubo, I., 65 Tsukada, Y.,245,429 Tusher, M., 79 Tyszkiewicz, E., 149 Udenfriend, S., 201, 245,401,436,496 U1 Haque, I., 320, 325 Valverde, F., 275 Vane, J. R., 527 Varon, S., 429, 453, 469 Veldsema-Currie, R. D., 477 Vidaver, G. A., 437 Villaverde, H., 43 Virkar, R. A., 431 Virno, M., 141 Vliet, P. D. van, 326 Voegtlin, C., 142 Voeller. K., 475 Vogt, J., 404 Waelsch, H., 267, 401, 429, 430, 437, 465 Wald, F., 51 Walker, M. D., 284 Wallgren, H., 278, 469, 470, 476 Walsh, P. M., 173 Wanko, T., 477 Warren, L., 126 Waters, W. J., 317 Watson, W. J., 409 Watters, G. V., 321 Wattiaux, R., 111

Watts, D. C., 436 Watts, R. L., 436 Webb. E..249 Webb&, W. A,, 186 Webster, G. R., 5, 8 Weed. L. H., 72, 75, 135 Weinstein, P., 429, 436 Welch. K., 33, 135, 136, 301, 302, 310, 466 Werdinius, B., 360 Westrum, L. E., 30 Wheeler, K. P., 437 Wherrett, J. R., 2 Whittaker, V. P., 2, 125, 127, 129, Wibo, M., 111 Wiechert, P., 429, 436 Wiegandt, H., 2 Wieland, 0..141 Wilbrandt, W., 185, 246 Wilson, C., 322 Wilson, J. E., 149 Wilson, T. H., 187, 362 Windle, W. F., 318, 417 Winkelman, J., 330, 333, 334 Wiseman, G . , 187 Wislocki, G. B., 19, 20, 63, 66, 401 Wisniewsky, H., 320 Wissig, S. L., 51 Wolf, A., 87, 113 Wolf, M. K., 92 Wolfe, D. E., 65 Wolfe, L. S., 2, 142, 502 Wolff, J., 20 Wollenberger, A., 279 Wood, J. D., 409 Woodbury, D. M.,70, 297-312,417, 436, 488 Woodman, R. J., 279 Woodward, D. L., 299 Woollam, D. H. M., 147 Woolley, D. W., 3, 125 Wright, L. O., 185, 186 Wright, R. L., 329 Wu, M. L., 467 Wyman, J., 530 Yamamoto, C., 275 Yasuzumi, G., 65 Zajicek, J., 42 Zak, E., 349 Zand, L. P., 10 Zeiger, K., 87 Zetterstrom, R., 317 Zeuthen, E., 41, 42 Zeya, H.I., 93 Zieher, L. M., 125 Zill, L. P., 4 Zimmerman, H. M., 321 Zollman, P. E., 317 Zuelzer, L. M., 125

547

Subject Index

Adenine, and neuron, regeneration, 405407 Adenosine triphosphate, activity, and choroid plexus, 65,66 Albumin, uptake, blood-brain barrier, and metrazol, 370 Amino acids, brain, accumulation, changes, 401-415 and neuron, regeneration, 404406 and thyroid function, 407409 areas, distribution, 224,225 and CSF, concentrations, 225,226 levels, 202-205.219 and administration, 236-240 and ATP, 206,207,217,218,257,258 and circulation, 235-243 comparison, 209-212,214 and fasting, 237,238 and membrane potential, 216 and membrane transport, 235 and membrane transport, 241 metabolic inhibitors, 206, 207, 213, 216, 241-255 metabolism, 267-270 pools, intrinsic and extrinsic -, 429431 slices, exit rate, 220-222 transport, and CSF, 245 kinetic analysis, 245-256 and Michaelis-Menten kinetics, 249-254 transport constant, 245-254 uptake, 240,241 graphical analysis, 252 intracellular -, 257 cerebral transport, and cerebral pool, 212-214 and energy supply, 205-207 regional differences, 21 9-233 exchange diffusion, and methionine, 175-181 possibilities, 181-184 and tryptophan, 175-177, 183 metabolism, and brain, activity, 241

and mental performance, 241 and tissue, uptake, 173-184 transport, and brain, 185-199 levels, 202-205 slices, 246, 247 and intestine, 185-199 and kidney, 185-199 mechanism, 173-1 84 and various tissues, 185-199 uptake, and blood-brain barrier, 401-41 5 and brain slices, 207-214, 219-225 and hypothalamus, 229 Anion exchange, CSF, and choroid plexus, 300-31 1 inhibition, 302-31 I , 314 Arachnoid villi, and CSF, transport, 135, 136 ATP, activity, and nucleosides, blood-brain barrier, 527-534 Barrier effect, pathology, changes, 315-341 Basic dyes, CNS, subcellular distribution, 109 and lysosomes, biochemistry, 108-1 10 metachromasia, 91, 92 Basic dyes, transport, CNS, and blood-brain barrier, 132, 133 localization, intracellular, 89, 90 and lysosomes, 87-1 1 I , 121 and membranes, activity, 132 physiological effects, 88 Bilirubin, and blood-brain barrier, 316319 and CNS, phosphorylation, 317-319 Blood-brain harrier, albumin uptake, and metrazol, 370 alterations, 343-348 and amino acids, intrinsic level, 429431, 436-441 isotope studies, 429-43 1,440,445,447449 level, 429450 uptake, 401415 and chemical injury, 357-365 and convulsions, 367-384

548

SUBJECT INDEX

Blood-brain barrier, and metrazol, 371, 372 definition, anatomy, 343 function, 343 development, 417-427 and cholesterol accumulation, 4 1 9 4 2 3 and DNA-P accumulation, 4 1 9 4 2 3 and growth spurt, 418, 419 drugs, influence, 349-355 and glucose uptake, 343-346 and hypercapnia, 372-376 and hypothermia, 385-399 and inulin uptake, 446 and multiple sclerosis, 493-525 nucleosides, and ATP, activity, 527-534 kinetic properties, 529-534 transport, 527-534 pathophysiology, 357-365 permeability, changes, 367-384 and chlorpromazine, 352, 354 and drugs, influence, 349-355 and GABA, 355 and glucose, transport, 352-354 isotope studies, 350-355 and reserpine, 351-353 and sodium exchange, 352-354 and trypan blue, 349 and vitamin BE, 354, 355 sulfate concentration, and convulsions, 367-370 sulfate uptake, and metrazol, 368 and strychnine, 368, 369 transport, experiments, 358-363 and glucose, 36C364 inhibition, and chemical injury, 357-365 and glucose, 361, 362 mechanisms, 358, 363 and organic ions, 359, 360 tracer studies, 36C364 and vegetative functions, 345-347 and schizophrenia, 345, 346 and vitamin BE, 4 4 7 4 4 9 Brain, amino acids, accumulation, changes, 4 0 1 4 1 5 mechanisms, 434-436 and neuron, regeneration, 404-406 level, and environmental effects, 431-435 uptake, and thyroid function, 407-409

Brain, cation exchange, 283-296 extracellular space, and thiocyanate, 485-492 and glucose, transport mechanism, 343-348 uptake, and mental disorder, 344, 345 and pyrithioxine, 344, 345, 348 injuries, and blood-brain barrier, 324-328 and isotope, distribution, 324-328 ion-barriers, observations, 275, 276 leucine, accumulation, and exercise, 404 mammals, ion movement, 273-28 I metabolite levels, and transport processes, 201-216 plasma space, and environment, 4 3 W 1 , 4 4 5 spaces, and cerebral edema, 4 5 1 4 5 4 experiments, 455-464 and inulin, distribution, 4 5 6 4 6 4 and multiple sclerosis, 493-525 and sucrose, distribution, 45-64 and thiocyanate, distribution, 4 5 6 4 6 4 and thiocyanate, plasma concentration, 485-489 tissue preparations, appraisal, 273-275 triiodothyronine, uptake, isotope studies, 4 0 2 4 0 4 tumor, blood content, 330 and cellular metabolism, 333-336 and extracellular space, 331, 332 and lactic acid, production, 344 pinocytosis, 330, 331 and vascular permeability, 328-332 water content, and thiocyanate, plasma concentration, 486489

white matter, and multiple sclerosis, 493-505 and albumin, 496, 499, 500, 505, 51 I 516

and bromide, 496,497, 505-510 and electrolytes, 497, 504-51 1 and gangliosides, 495, 501, 502 and hemoglobin, 494, 495 histology, 494, 498, 499 and hydroxyproline, 495, 496, 499, 503

and immunoglobulin-G, 496, 499, 500, 505, 511-516

and lipid content, 494, 500, 505-509 and water content, 494, 500, 505-509

S U B J E C T INDEX

Cation exchange, blood-brain barrier, 283-296 CSF, inhibit ion, 300-302 isotope studies, 283-296 emux coefficient, 285, 288, 289 influx coefficient, 288, 289 isotope fluxes, 286-293 transport coeficient. 285, 289 transport system. inhibition, 290 and ventricdocisternal perfusion, 283-285 Cationic dyes, transport, CNS, 87- I 1 8 and blood-hrain barrier, 132, 133 localization, intracellular -, 89, 90 and lysosomes, 87, I 1 I , I2 I and membrane, selectivity, 132 physiological effects, 88 Cerebral cortex, fluid compartments, experiments, 455-460 Cerebral edema, and brain, spaces, 45 1-454 CNS, and anoxia, 322-324 and cold, 320, 321 inflammation, 321 pathology. 319 324 toxicity, 321, 322 and trauma. 321 mechanism, theses, 453 and tissue, heterogeneity, 469, 470 Cerebral tissues, and fluid, conipartmcnts, 465484 Cerebrospinal fluid, amino acids, concentration, and brain, levels, 225, 226 anion exchange. and choroid plexus, 300-31 1, 314 and hrain, extracellular fluid, 297-314 functional relations, 159-1 67 tissue slices, inflation, 451454 cation exchange, 283-296 inhibition, 300, 301 and electrolytes, distribution, 297-3 14 exchange, 297, 298 and choroid plexus, 297-300 developmental studies, 305-314 formation, 135, 136 and choroid plexus, 63. 135 development, 72 inhibitors, 138, I39 glucose concentration, 147, 156 and inulin uptake, 298, 299, 304-31 I, 314 ionic composition,

549

and choroid plexuq, developniept, 70 and non-electrolytes, distribution, 297 -314 secretion rate, inhibition, 300, 301 and sucrose, distribution, 465468 transport, 135-146 and arachnoid villi, 135, 136 and choroid plexus, 136, 137 hydrodynamic aspects, I36 and inulin, clearance, 135 isotope studies, 138-142 and PAH, clearance, 137, 164 t hiocyanate, and choroid plexus, 488-491 and ventriculocisternal system, 136, 140 Chloride, distribution, and fluid, spaces, 467-474, 480484 comparative aspects, 474476 Chlorpromazine, and blood-brain barrier, permeability, 352, 354 Cholesterol, accumulation, and blood-brain barrier, development, 419423 Cholinesterase, activity, and catecholamines, 61 and developing nervous system, 53-58 and isolated neurons, 45-52 and permeability barrier, 43, 51 CNS analysis, 4 1-62 and ion exchange. studies, 51, 52, 61 ultracytochemistry, 41-62 and isolated neurons, studies, 41. 42 localization, 43 cytochemistry. 42-45 studies, 4 2 4 5 and sympathetic ganglia, studies, 42 synthesis, and endoplasniic reticulum, 55-58, 61, 62 Choroid plexus, and ATP-ase, activity, 65, 66 capillaries, 66-68 and CSF. electrolytes, exchange, 297-300 formation, 63, 135 thiocyanate, transport, 488491 transport, 136, 137 development, 68-83 and capillaries, 75, 76 and glycogen content, 72-75 stages, 70-75 epithelial cells, basement membrane, 6C68 characteristics. 63-68 fluid transport, 81, 82 junction, 65, 67 and peroxidase, movement, 65- 67

550

S U B J E C T INDEX

Choroid plexus, epithelial cells, and pinocytosis, 65, 67 and transport processes, 66 and ferritin, movement, 65 fine structure, 63-85 and GABA, transaminase. 261 and glucose, accumulation, inhibition, 150, 151 concentration ratio, 147, 148, 157 transport mechanism, 148-152, 154, 157 and glucose barrier, 147-158 human -, development, 7 6 8 3 and interstitial area, 79-81 and NRG, content, 97 and PAH. transport, 164-166 and proteins, movement, 19-37 rat, development , and CSF formation, 72 ionic composition, 70 interstitial area, 75, 76 stages, 70-75 structure, 147 and sugar pump, 148-150 direction, 151, 155, 157 transport, inhibition, 300, 301 Convulsions, and blood-brain barrier, 367-384 DNA, accumulation, and blood-brain barrier, development, 419423 Electrolytes, CNS, ultramicroscopic studies, 319-324 exchange, and CSF, 297, 298 transport, and CNS, 319-324 Endothelium, and proteins, movement, 20-23, 35 Ependyma, and PAH, transport, 164 passage, and proteins, movement, 27-30, 35 and transport, barrier, 161-164 and extracellular fluid, 161-164 mechanism, 159-169 and pinocytosis, 165, 166 Erythroblastosis fetalis, and bilirubin, indirect -, 317-319 Ferritin, movement, and choroid plexus, 65

entry, 26, 27, 39 and ependynia, passage, 27-30, 37 and pinocytosis, 30-40 Fluid, compartments, cerebral cortex, experiments, 455-460 and cerebral tissues, 465-484 comparative aspects, 474-476 ontogenetic aspects, 474- 476 and tissue, heterogeneity, 467, 468 spaces, and chloride, distribution, 467474, 480484 and electrolytes, 468-484 and incubation media, 472-474 and inulin, distribution, 467-474, 480-484 and sucrose distribution, 466468,470-474 and thiocyanate, distribution, 467, 468 GABA, and blood-brain barrier, permeability, 355 CNS, enzyme barrier, 259-271 transaminase, CNS, activity, 260-262 and choroid plexus, 261 and formazan, precipitation, 260-265 kidney, 259, 262-264, 267, 271 and tetrazolium salts, 262-264, 266 liver, 259, 262, 264-267, 271 localization, 259, 260, 269, 270 transamination reaction, 269 Gangliosides, chemistry, 1. 2 and membrane functions, 2, 3 and nervous membranes, 1-3, 17 properties, 2, 3 and acetylcholine, 2 and cations, 3 and serotonin, 2, 3 and serotonin, physiology, 125-129, 13I Glucose, and blood-brain barrier, transport, 36CL364 inhibition, 361, 362 choroid plexus, accumulation, inhibitors, 150, 151 concent rat ion, and CSF, 147, 156 and transport, 170-173 concentration ratio, and choroid plexus, 147, 148, 157 intestines, accumulation, inhibitors, 149, 150 transport, and blood-brain barrier, permeability, 352-354 mechanism, and brain, energy. 343-348

SUBJECT INDEX

Glucose, transport, mechanism, and choroid plexus, 148-152, 154, 157 uptake, CNS and pyrithioxine, 344, 345, 348 Glucose barrier, and choroid plexus, 147-158 Glutamate, incubation media, and fluid, spaces, 473, 474

55 1

CNS,and NRG, content, 96, 97 metachromasia, and chromotropes, 93 and electrostatic forces, 93, 94 inhibition, 91 vital -, mechanism, 91-94 morphology, 87, 89, 90 changes, 94-97, 111 ultrastructure, 98-108, 11 1 Metabolite levels,

H ypercapnia, and blood-brain barrier, 372, 376 Hypothermia, and blood-brain barrier, 385-399 Inulin, clearance, and CSF, transport, 135 distribution, and brain, spaces, 456-464 and fluid, spaces, 467474. 480484 comparative aspects, 474476 uptake, and blood-brain barrier, 445 brain/CSF ratio, 299-3 I 1 and CSF, 298, 299, 304-31 I , 314 ventriculocisternal perfusion, 299, 300 Ion concentration, and neocortex, 276-278 Ion movement, brain, and chloride concentration, 281 mammals, 273-281 and potassium concentration, 276-281 and sodium concentration, 276-281 Kernicterus, and blood-brain barrier, 3 16-319 development, and cerebral metabolism, 317-319 Leucine, brain accumulation, and exercise, 404 Lysine. and neuron regeneration, 404-406 Lysosomes, and acid phosphatase, activity, 89, 90, 96, 103, 105-107 and Golgi apparatus, 107 and membrane hypothesis, 107, 108, 132 and NRG,90, 96, 108, 109 and sedimentation, 110 and basic dyes, transport, biochemistry, 108-1 10 biochemistry, 87, 108-1 10 and cationic dyes, transport, 87-1 11, 121 biochemistry, 108-1 10

CNS,

and transport processes, 201-216 Metabolite transport, mechanisms, 173-184 Metrazol, and blood-brain barrier, albumin uptake, 370 Mitochondria, rat brain, orthochromasia, 9 1-93 and tetrazoliuni salts, transport, 87, I 11-118, 120, 121 Multiple sclerosis, and blood-brain barrier, 493-525 and brain spaces, 493-525 white matter, albumin, 496, 499, 500, 51 1,-516 bromide, 496,497, 505-510 electrolytes, 497, 504-51 1 gangliosides, 495, 501, 502 hemoglobin, 494, 495 histology, 494, 498, 499 hydroxyproline, 495,496, 499, 503 immunoglobulin-G, 496, 499, 500, 505, 51 1-516 lipid content, 494-509 spaces, 497-505 water content, 494, 5 W 5 0 9 Neocortex, and ion, concentration, 276278 Nervous membranes, composition, 1-17 gangliosides, 1-3, 17 myelin components, 3-13 neurokeratin, 11-13 neuronal components, 1-3 polyphosphoinositides, 12-15 proteolipids, 3-1 1, 15-17 Neuraminic acid, and gangliosides, chemistry, I, 2 Neurokeratin, and nervous membranes, 11-13 Neuron, regeneration, and amino acids. 404-406

552

SUBJECT INDEX

N R G , neutral red granules, and lysosomes, acid phosphatase activity, 90, 96 N ucleosides, kinetic properties, and blood-brain barrier, 529-534 transport, and blood-brain barrier, 527-534 uptake, and RNA, synthesis, 231-233 Peroxidase, movement, barriers, anatomical -, 30-35 and choroid plexus, entry. 25-27, 39 and endothelium, entry, 23-25, 39 passage, 20-23 and ependyma, passage, 27-30 intercellular - , 25, 28 and pinocytosis, 30-35, 3 7 4 0 and pinocytotic vesicles, 23, 30 Pinocytosis, and brain, tumor, 330, 331 and ependyma, transport mechanism, 165, 166 and proteins, movement, 30-40 Polyphosphoinositides, and nervous membranes, 12-1 5 Proteins, intracerebral movements, 1 9 4 0 niovement. barrier, 1 9 4 0 and choroid plexus, 19-36 and choroid plexus, entry, 25-27, 39 CSF, investigations, 19-25 and endothelium, entry, 23-25, 39 passage, 20-23, 27-30 intercellular 25, 28 intracerebral -, 19-40 and pinocytosis, 30-35, 37, 38 Proteolipids, chemistry, 4, 5 chromatography, 7, 8 composition, 8-10 distribution, 4, 5 extraction, 5-7 and nervous membranes, 3- I I , 15-1 7 properties, 8- 1 1 Pyrimidine nucleotides, metabolism, roles, 231-233 Pyrithioxine, and brain, glucose uptake, 344. 345, 348

-.

Reserpine, and blood-brain barrier, permeability, 35 1-353 Schizophrenia, and blood-brain barrier, vegetative functions, 345-347 Serotonin, ganglioside binding, 125-129, 131 physiology, and chemical energy, 125-1 29 and enzynie. activity, 125-1 29, 130, 131 and gangliosides, 125- 129, I3 I and synaptic vesicles, 125-1 29 Sucrose, distribution, and brain, spaces, 456464 and CSF, 465474 and fluid, spaces, comparative aspects, 474476 Sulfate, uptake, and blood-brain barrier, 367-370 Synaptosomes, morphology, 125, 129, 130 Tetrazolium salts, CNS, localization, 112, 1 I3 transport, and CNS, 87, 1 I I- II8 and mitochondria, 87, 111-118. 120, 121 morphology, changes, 113, I14 Thiocyanate, and brain, extracellular space, 485--492 distribution, and brain, spaces, 456-464 and fluid, spaces, 467, 168 plasma concentration, and brain, water content, 486-489 and brain space, 485489 and skeletal muscle, 486-489 transport, CSF, and choroid plexus, 488491 Tissue heterogeneity, and cerebral edema, 469, 470 and fluid, compartments, 467, 468 Triiodothyronine, uptake, brain and muscle, 402404 Trypan blue, and blood-brain barrier, permeability, 340 Ventriculocisternal perfusion, and cation exchange, 283-285 inulin, 299, 300 Vitamin BI;, and blood-brain barrier, 447-449 permeability, 354, 355