Abstract: AT LEAST seven gangliosides of different molecular compositions and structures occur in normal mammalian brains. These individual gangliosides differ in the number and position of their monosaccharide moieties and the amount of NANA they contain. Because of their different molecular structures, these gangliosides also vary in polarity, solubility, and in the degree of their interactions with other chemical compounds. These seven gangliosides are clearly separated by the descending thin-layer chromatography system (KOREY and GONATAS, 1963) routinely used in our laboratory. They are numbered according to KOREY and GONATAS (1963) in the order of increasing mobility on thin-layer plate. Go has not been well characterized, although KOREY and GONATAS (1963) indicated that it is a trisialoganglioside. G,, G,, G, and Gp are the major gangliosides in normal mammalian brain. KUHN and WIEGANDT (1963a, b) proposed the structures of GI, G,, G3 and G4 as shown below. G6, a minor component in normal brain, is predominant in Tay-Sachs disease; it lacks the terminal galactose of G, (SVENNERHOLM, 1962). G6 contains no amino sugar and is probably the same compound as hematoside (KUHN and WIEGANDT, 1964).

Abstract: PREVIOUS studies have indicated that levels of activities of 5-hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO) may have a significant role in regulating tissue concentrations of 5-HT. Investigations of such enzyme activities in brain tissue have been performed on relatively gross areas (100 mg or more) which contain varying amounts of grey and white matter. For better assessment of the contribution of each of various cell types, microdissection of cortical and cerebellar areas was performed. Since the sample size of a relatively homogeneous cellular population is of the order of a few pg, new, sensitive micromethods had to be devised for the measurement of M A 0 and 5-HTPD activities. Methods currently used for the quantitative estimation of M A 0 activity by oxygen consumption, ammonia production, disappearance of amine, and fluorimetric assay of indole acetic acid formed are not sufficiently sensitive for such studies. The method frequently used for estimating the activity of the enzyme which decarboxylates 5-HTP and dihydroxyphenylalanine, is based on the fluorimetric estimation of the products 5-HT and dopamine, respectively. KUNTZMAN, SHORE, BOGDANSKI and BRODIE (1961) recently described modifications of these methods which increase their sensitivity, but, even with modifications, approximately 20-100 mg of brain tissue are required. In this paper, procedures are described for quantitative estimation of M A 0 and 5-HTPD activities in samples of nervous tissue weighing as little as 2-5 pg dry weight. Certain properties of the enzymes in various tissue homogenates, as well as distribution of activities within discrete areas of the nervous system, are presented. Preliminary descriptions of these methods have been previously reported (MCCAMAN, R. E., 1961 ; MCCAMAN, 1962). METHODS All the reagents used were analytical grade. Preparations of 14C-labelled 5-HT, 3-hydroxytyramine and tyramine with different specific activities were obtained from various commercial sources, and all tested gave equivalent answers in the described procedures. The DL-[~-'*C]~-HTP was obtained from New England Nuclear Corporation. This material is now available from ChemTrac Corporation, Boston, Massachusetts. The general techniques for handling small amounts of tissue and the appropriate volumes have been previously described (LOWRY, 1953 ; LOWRY, ROBERTS, LEINER, WU and FARR, 1954). The buffer substrate solution (0.1 M-potassium phosphate, pH 7.2; 0.8 m~-[3-'~C]serotonin) was stored at -20\" until needed. Ten pl of ice-cold buffer substrate were added to the sample, which was equivalent to 1-20 pg dry brain or 1 pl of homogenate (1 g tissue per 25 ml H,O), in a pointed microtube of 2.5 mm I.D. x 4 cm. The tube was placed in a tray of M A 0 determination.

Abstract: I N the preceding paper (HESS and THALHEIMER, 1965), a microchemical scheme was given for analysing microgram-sized samples of nervous tissue for a group of biochemical structural substances : gangliosides, RNA, DNA, cerebrosides, proteolipid proteins and residue proteins. Extraction and partition of lipids were performed by a microversion of the technique of FOLCH, LEES and SLOANE-STANLEY (1957). This article describes in detail the use of microadaptations of the orcinol-sulphuric acid reaction (S0RENSEN and HAUGAARD, 1933; BRUCKNER, 1955) for determination of cerebroside hexose (FOLCH, 1949; FOLCH et al., to be published), and of the microcolorimetric method of LOWRY, ROSEBROUGH, FARR and RANDALL (1951) for assay of proteolipid and residue proteins.

Abstract: RELATIVELY little work has been done on the metabolism and physiological disposition of norepinephrine in the intact brain because of the inability of this compound to cross the blood-brain barrier (WEIL-MALHERBE, AXELROD and WHITBY, 1961). In a preliminary communication (MILHAUD and GLOWINSKI, 1963), it was shown that after the administration of ~-~[~~C]norepinephr ine into the lateral ventricle of the rat brain, a large amount of radioactivity was present in the brain, and that deaminated and U-methylated metabolites were present soon after the injection. MANNARINO KIRSHNER and NASHOLD (1963) injected [14C]norepinephrine into the lateral ventricle of the cat and found that the principal metabolites were 0-methylated products. This report will describe the physiological disposition and metabolism of [3H]norepinephrine of high specific activity introduced into the brain ventricles. The relatively large amounts of catecholamine in previous experiments rendered the animals unconscious for long periods of time (FELDBERG and SHERWOOD, 1954; MILHAUD and GLOWINSKI, 1963). The technique of intraventricular administration and the small amount of norepinephrine (0.13 pg) used enabled the rats to recover within a short time after pentothal anaesthesia. The animals behaved in an apparently normal manner for several days; this permitted study of the fate of the neurohumour in the intact rat brain over a relatively long period of time.

Abstract: THE discovery of the highly active biogenic amines in discrete areas of the brain (AMIN, CRAWFORD and GADDUM, 1954; VOGT, 1954; CARLSSON, 1959) has led to the suggestion that they may be concerned with brain function in these areas. The finding that many psychotherapeutic agents alter the metabolism of the amines (PLETSCHER, 1963) has stimulated the interest both of pharmacologists and of psychiatrists and many attempts have been made to study their metabolism in animals and man. In vitro techniques using tissue homogenates do not take into account the spatial distribution of enzymes and substrates in vivo and there are advantages in using the intact animal. In experiments in vivo measurements of amine concentrations are of limited usefulness since they do not distinguish between different types of drug action, e.g. the fall in brain levels of 5-hydroxytryptamine (5-HT) produced by reserpine as a result of interference with storage, and the fall produced by a-methyldopa by interference with amine synthesis (PLETSCHER, 1963). Attempts have been made to extend the information obtainable from in vivo studies (a) by measuring not only the amines, but also amine precursors and metabolites, e.g. 5-HT and 5-hydroxy-indol-3-ylacetic acid (5-HIAA) (ASHCROFT and SHARMAN, 1962) and (b) by administering a ‘loading dose’ of a precursor to increase the concentration of intermediate metabolites. Previous investigations with the latter technique include studies of the effect of tryptophan administration on tryptamine levels in animal tissues (HESS, REDFIELD and UDENFRIEND, 1959), of tryptophan administration on tryptamine and 5-HT levels in rat brain (HESS and DOEPFNER, 1961) and of 5-hydroxytryptophan (5-HTP) administration on 5-HIAA levels in rabbit brain (Roos, 1962). The present paper describes an attempt to combine ‘loading’ with the measurement of precursors, amines and metabolites. Metabolites in the 5-hydroxyindole pathway were determined in the brains of rats killed at varying times after intraperitoneal injection of tryptophan. The aim was to obtain a picture of the pathway in vivo under load. Investigations of the influence of certain drugs on these parameters are reported in the accompanying paper (ECCLESTON, ASHCROFT and CRAWFORD, 1965).

Abstract: ELECTRON microscopic studies have shown that the myelin sheath is composed of a compact ordered arrangement of unit membranes (FINEAN, 1957 ; ROBERTSON, 1959, 1964). Myelin therefore provides a particularly suitable model for the investigation of the molecular organisation of biological membranes. However, until quite recently it was not possible to isolate myelin from nervous tissue and reliable information about its chemical composition was not available. Improved methods for the separation of subcellular structures have now been applied to neural tissue and a number of laboratories have described techniques for the isolation of myelin in a form suitable for direct analysis (WHITTAKER, 1959; PATTERSON and FINEAN, 1961 ; AUGUST, DAVISON and WILLIAMS, 1961 ; LAATSCH, KIES, GORDON and ALVORD, 1962; DAVISON and GREGSON, 1962; HULCHER, 1963). Some reports of the lipid composition of myelin from individual species have already appeared (PATTERSON and FINEAN, 1961 ; NUSSBAUM, BIETH and MANDEL, 1963; AUTILIO, NORTON and TERRY, 1964; EICHBERG, WHITTAKER and DAWSON, 1964; SEMINARIO, HREN and GOMEZ, 1964; CUZNER, DAVISON and GREGSON, 1965). Although it has been suggested that all membranes may have a common ‘unit membrane’ structure (ROBERTSON, 1964) both electron microscopic and biochemical evidence indicate that the various membranes found in the cell are not necessarily similar in all properties (STOECKENIUS, 1964). Earlier work from this laboratory has indicated that mammalian myelin may represent a unique class of membrane for it has a characteristically slow rate of turnover with little associated enzyme activity (ADAMS, DAVISON and GREGSON, 1963). In addition indirect evidence led to the idea that the lipid composition of myelin also differs from that of other membranes (ROSSITER, 1962; ADAMS and DAVISON, 1965; GREGSON 1965). The purpose of the present work has been to investigate this possibility more thoroughly by comparing the chemical composition of myelin with that of another cell membrane fraction from the same tissue. Myelin and microsomal fractions have been prepared therefore from the brains of a number of mammalian and more primitive vertebrates ; samples have been examined by electron microscopy and submitted to chemical analysis. The results of these and earlier observations will be discussed with particular reference to the possible molecular organisation of membranes. EXPERIMENTAL Materiais. The following animals were used in the present work: white Wistar rats, rabbits, the feral pigeon, common frog (Rana temporaria) and dogfish (Scyllium cuniculu). These animals were killed, exsanguinated and the brain removed. Ox brain, collected in ice from the slaughter house, and human frontal cerebral cortex obtained at autopsy, were brought to the laboratory and white matter removed. Human brains were obtained from patients who had died from non-neurological causes.

Abstract: THIS paper outlines a quantitative, microchemical scheme for analysing nervous tissues for six categories of biochemical structural substances : gangliosides, RNA, DNA, cerebrosides, proteolipid proteins and residue proteins. Techniques were devised for their simultaneous assay in order that their concentrations relative to one another might be studied in samples differing in histological structure. The methods were developed, primarily, for determining these components in the layers of human frontal cortex as part of a long-range study of the biochemical architecture of this tissue. An ultimate goal of this work is to establish the chemical composition of the cortex in detail sufficient to allow correlations to be made, a t increasingly precise and more meaningful levels, with cortical structure as depicted by light and electron microscopy and with cortical function as revealed by neurophysiological techniques. The intralaminar distributions of these constitutive components are expected to be of interest in relation to the cytology of the cortex and to previous, analogous studies of enzymes (POPE, CAVENESS and LIVINGSTON, 1952; POPE, 1959, 1960; HESS and POPE, 1960, 1961). Such a study of cerebrosides in human eulaminate frontal cortex is reported in an accompanying paper (LEWIN and HESS, 1965). Concomitant studies of the other components in the same specimens will be presented separately, as will results on twelve other regions of human CNS. An aim has been to explore the possibility that, like DNA, some of the other components may prove useful as quantitative indices of nervous tissue structure. Each component was selected because of evidence suggesting for it either a relatively specific histological localization or some general structural or metabolic significance. Whereas direct enumerations of histological elements and estimations of volume, surface area, and mass are tedious and fraught with methodological difficulties, certain chemical assays can be performed rapidly and accurately upon large numbers of samples. Therefore, if chemical referents can be found for myelin, neuronal cytoplasmic volume, neuronal cell surface, or other histological entities, the interpretation of regional neurochemical and neuropathological data will be greatly facilitated. Such referents are needed to supplement the methods commonly used to

Abstract: ALTHOUGH the role of 5-hydroxytryptamine (5-HT): in neurophysiology is still obscure, several lines of evidence indicate that this amine may be involved in neural mechanisms in mammals. In addition, it is well documented that 5-HT and several drugs known to alter the metabolism of 5-HT have lethal, teratogenic, and prolonged behavioural effects on developing mammals. PEPEU and GIARMAN (1962) found that the administration of 5-HT to pregnant rabbits resulted in death of the foetus at a time when the 5-HT level in the maternal blood had only doubled and at a dose which caused no change in the maternal arterial pressure. LINDSAY, POULSON and ROBSON (1963) and POULSON and ROBSON (1963) described toxic actions of 5-HT and monamine oxidase (MAO) inhibitors at all stages of pregnancy in mice. Teratogenic effects may be seen in surviving foetuses of female mice given single injections of 5-HT (25-100 mg/kg) early in pregnancy (POULSON, ROBSON and SULLIVAN, 1963). In addition, 5-HT metabolism is altered in the inherited metabolic disease phenylketonuria, and has been implicated as a contributing factor in the production of the mental defects which accompany this disorder (PARE, SANDLER and STACEY, 1957). Finally, recent evidence indicates a possible correlation between the level of brain 5-HT at birth and the extent of development of the newborn in several mammalian species. Animals such as the rat (KATO, 1960; NACHMIAS, 1960; KARKI, KUNTZMAN and BRODIE, 1962; SMITH, STACEY and YOUNG, 1962) and rabbit (KARKI et al., 1962; PEPEU and GIARMAN, 1962), which are poorly developed, possess low levels of cerebra1 5-HT at birth; whereas the guinea pig (KARKI et al., 1962; S ~ T H et al., 1962) and goat (PEPEU and GIARMAN, 1962) species which are well developed and competent at birth, possess levels of cerebral 5-HT which approach or, in the case of the goat, exceed the adult levels. M A 0 activity parallels the levels of 5-HT in these species (SHIMIZU and MORIKAWA, 1959; NACHMIAS, 1960; KARKI et al., 1962; TISSARI, 1963), while 5-hydroxytryptophan decarboxylase (5-HTPD) activity approaches adult levels in the brains of the newborn in all species studied (KARKI et al., 1962; PEPEU and GIARMAN, 1962; SMITH et al., 1962; TISSARI, 1963).

Abstract: THE investigation of 5hydroxytryptamine metabolism in the rat brain, combining the technique of tryptophan loading with the measurement of intermediate metabolites, has been described in the preceding paper (Ashcroft, Eccleston and Crawford, 1965). This paper is concerned with applications of this technique. A study was made of the effect of varying the dose of tryptophan and also of the effects of administration of drugs known to block enzymes concerned in the metabolism of tryptophan.

Abstract: ALTHOUGH differentiated for conducting excitation over long distances without attenuation, the axon in fulfilling this function appears as an enormous appendage of the cell. A comparison of the ultrastructure of the axon with that of the perikaryon reveals characteristicdifferences that reflect specialization both in terms of biochemistry and function. One of the earliest indications of regional cellular differences was the apparent lack of Nissl substance in the axon in contrast to its presence in the perikaryon and dendrites (SCHAFFER, 1893). This observation was repeatedly confirmed over the years with classical methods of staining with basic dyes. Confirmation of these observations was obtained at the ultrastructural level with the study of PALAY and PALADE (1955). These investigators, and others since, failed to discern ribonucleoprotein granules (i.e., ribosomes) in the axoplasm, in contrast to its rich distribution elsewhere in the neuron. Earlier, NURNBERGER, ENGSTROM and LINSTROM (1952) also reported that RNA was lacking in the axon on the basis of ultraviolet microspectrophotometry. The apparent absence of RNA in the axon seemed compatible with the view that axonal proteins were synthesized in the soma. An indication that all proteins were not exclusively synthesized in the cell body was shown by the studies of KOENIG and KOELLE (1961) and CLOUET and WAELscH( 1961) on the restoration of acetylcholinesterase (AChE) in the axon following irreversible inactivation by organophosphorus compounds. The results pointed to an axonal capacity for synthesizing AChE. More recently, observations were extended to axotomized, cholinergic nerves which provided additional evidence that the axon synthesizes AChE (KOENIG, 1965). I t was postulated that the axonal synthesis of AChE was an example of a local mechanism concerned with synthesis of proteins forming integral substituents of the axolemma (i.e. plasma membrane). A question at the time of the early study was how the apparent absence of RNA in the mammalian axon could be consistent with axonal synthesis of an enzyme. A qualified answer was forthcoming when .I.-E. EDSTROM, EICHNER and A. EDSTROM (1962) demonstrated that the giant axon of the Mauthner cell in young gold fish contained RNA. An extension of the work was carried on by A. EDSTROM (1964). In addition GRAMPP and EDSTROM (1963) reported that the lobster sensory stretch receptor axon contained RNA. In both species the concentration of RNA was less than that found generally in cell bodies.

Abstract: IN a previous investigation (CURATOLO, 1961a), a method of paper chromatography was developed for the purpose of studying the organic non-amino acids in protein-free extracts of animal tissue. The method led to the isolation and identification of N acetyl-aspartic acid (NAAA) (CURATOLO, 1961b) which had been identified a few years before by TALLAN, MOORE and STEIN (1956) using a different approach. In studying the distribution of NAAA throughout the rabbit neuraxis, we found a new acidic ninhydrin-negative spot in the chromatograms, so close to the NAAA spot as to cause some difficulty in its recognition. In paper chromatograms developed with butanol-acetic acid solvent, the R, values of the two spots were 0.70, and 0.75, respectively. To ascertain the identity of the spots, their breakdown products after elution and hydrolysis were subjected to chromatographic analysis. It was found that the spot with the R, of 0.70 was that of an amino-conjugated dipeptide, since two amino acids-glutamic and aspartic acids in an equimolecular ratio-were set free on hydrolysis. The identification of this substance as N-acetyl-aspartyl-glutamic acid (NAAGA) was the subject of a previous study (CURATOLO, 1964).

Abstract: : The ninhydrin positive substances present in the cerebrum, cerebellum, midbrain and pons medulla of the rat were determined with an amino acid analyser which employs a single cation exchange column. Only ten of the forty-two substances measured were uniformly distributed in the four areas of the brain. The presence of an unknown fraction, eluted from the column after arginine and concentrated in the midbrain, is reported. (Author)

Abstract: THE relationship between intracerebral levels of free amino acids and the synthesis of brain proteins has not been elucidated. Evidence has been provided from experiments in ciuo that protein synthesis in the central nervous system may be limited by restricted passage of amino acids through the blood-brain barrier (GAITONDE and RICHTER, 1956; WAELSCH and LAJTHA, 1961 ; RICHTER, 1962). In support of this view is the recent observation that the protein synthetic activities of microsomal and ribosomal preparations from rat cerebral cortex were equivalent under optimal conditions to those of similar preparations from rat liver (ZOMZELY, ROBERTS and RAPAPORT, 1964), whereas hepatic proteins were renewed much more rapidly in viva than brain proteins (LAJTHA, FURST, GERSTEIN and WAELSCH, 1957; LAJTHA, 1959). Studies with isolated cerebral microsomes and ribosomes (ZOMZELY et al., 1964) further indicated that protein synthesis in the brain may be unusually sensitive to alterations in levels of amino acids, ions, and other regulatory substances. Similar conclusions may be derived from studies of amino acid incorporation into proteins of brain cortex slices (FOLBERGROVA, 1961 ; MASE, TAKAHASHI and OGATA, 1962). Alterations in the free amino acid composition of cerebral cortex were induced in young adult rats fed diets differing in amino acid content for 4 days (ROBERTS, 1963). In some instances, the cerebral variations reflected differences in plasma levels of amino acids. Concomitant changes were not observed in the total protein content of the cerebral cortex or in the amino acidcomposition of totalcerebral protein. Although these results suggested that protein synthesis in the brain might be relatively independent of alterations in the intracellular pool of free amino acids, differences may have been produced in the rates of synthesis of individual proteins which were not detectable by the methods employed, The present investigations reveal that differences in the distribution of free amino acids in plasma and cerebral cortex, produced by dietary variations or amino acid injection, were accompanied by variations in cerebral protein turnover. METHODS Dietary dejciency. Male rats of an inbred Sprague-Dawley strain were maintained on Purina laboratory chow until they were about 6 weeks old and weighed 160-17Og. They were then fed

Abstract: ON THE basis of a number of morphological and biochemical findings (WEISS and HISCOE, 1948; SAMUELS, BYARSKY, GERARD, LIBET and BRUST, 1951 ; WAELSCH, 1958; KOENIG, 1958; LAJTHA, 1961; MIANI, 1960, 1963; DROZ and LEBLOND, 1963; and others), the concept of a soma1 origin for axoplasmic constituents, particularly proteins, has gained general acceptance in recent years. The lack of demonstrable Nissl substance (SCHAFFER, 1893) and its ultrastructural counterpart ribonucleoprotein (RNP) granules (PALAY and PALADE, 1955)-presumably requisite protein-synthesizing machinery-in the axon further seemed to strengthen the hypothesis of axonal proteins being synthesized in the cell body and convected distally in the axon. However, observations contradicting an exclusive synthesis of axonal proteins in the perikaryon emerged from studies of AChE restoration following irreversible inactivation by organophosphorus inhibitors (KOENIG and KOELLE, 1960, 1961 ; CLOUET and WAELSCH, 1961a, 1961~). As stated earlier (KOENIG and KOELLE, 1961), the rationale for using organophosphorus compounds (e.g., DFP) was based upon the importance of not producing atypical alterations in the regional disposition of AChE which appeared likely to be induced by a state of growth (i.e. regeneration). Histochemical evidence available at the time (SCHWARZACHER, 1958) showed that AChE disappears from the cell body during the period of axon outgrowth following axotomy. Hopefully, what is revealed following irreversible inactivation is a restoration of AChE a t a rate which reflects its normal turnover. If the cell body serves as a continuous source of newly synthesized AChE, then a proximo-distal gradient should be established during the early period of AChE return, and this should diminish in steepness with time. On the other hand, if no proximo-distal gradient becomes evident and the rate of enzyme restoration is uniform along the nerve, a local axonal synthesis would be indicated. The results (KOENIG and KOELLE, 1961) showed a uniform rate of AChE restoration along the nerve. In view of the importance of validating the existence of a local mechanism for protein synthesis in the axon, it was deemed essential to repeat a part of the earlier

Abstract: EARLY studies of insulin shock, particularly those by HIMWICH and his collaborators, showed that profound hypoglycaemia is accompanied by a decreased cerebral uptake of glucose and of oxygen from the blood stream. HIMWICH (1951) reviewed these studies and cited much evidence indicating that the behavioural and electrographic manifestations of hypoglycaemia are related to the reduced supply of glucose to the brain and the consequent decrease in available oxidative energy. More recently, GEIGER (1958) and his coworkers found that the perfused brain of the cat is able to function for more than 1 hr without exogenous glucose if a high perfusion rate is maintained ; this finding suggests that the brain can utilize endogenous non-carbohydrate substrates and that hypoglycaemic symptoms are due in part to the accumulation of toxic breakdown products of these substrates. ABOOD and GEIGER (1955) found decreases in the protein, lipid and nucleic acid contents of the perfused cat brain deprived of glucose and an associated accumulation in the perfusing blood of small quantities of nitrogenous substances, especially GABA, glutathione, glutamate and creatine. On the other hand, SAMSON, DAHL, DAHL and HIMWICH (1959) found that insulin shock did not significantly decrease the cerebral proteins in cats, and DAWSON (1950) made the same observation in rats. A number of other cerebral constituents are known to undergo quantitative changes during hypoglycaemia. These include glycogen, acid-soluble phosphates, and certain of the free amino acids and related compounds. Some of these substances represent possible sources of limited amounts of energy, either as oxidative substrates or as phosphorylated intermediates, while others may occur as breakdown products of proteins, lipids, or other substrates. Pertinent reports are cited in subsequent sections of this paper; in general, these chemical changes have not been studied in temporal relation to the development of electrographic abnormality. We have attempted to follow simultaneously the progression of the electrographic and a number of chemical changes in insulin shock, and their reversal on termination of the hypoglycaemia. E X P E R I M E N T A L The experiments were done on adult male dogs weighing 8-23 kg. Food was withheld for a period of 18-24 hr, after which the cranium was exposed and opened and most of the calvarium was removed, the dura mater remaining intact. Preparation was made for electrographic recording from three cortical areas and for freezing the brain in situ with liquid air. Blood pressure was measured from a cannulated femoral artery in the final stages of the experiment. The details of these procedures have been described (TEWS, CARTER, ROA and STONE, 1963).

Abstract: GLUCOCORTICOIDS in large doses are known to interfere with body growth and bone maturation (WELLS and KENDALL, 1940; HOWARD, 1962; and others). Although there have been occasional reports of cerebral atrophy in Cushing’s syndrome (TRETHOWAN and COBB, 1952; SOFFER, DORFMAN and GABRILOVE, 1961) there seems to have been no systematic study of possible effects of corticoids on brain growth. In the course of observations on the effects of steroids on bone maturation, it was noticed that corticosterone given to infant mice produced a considerable reduction in brain weight, which will be described in this paper. The effects of restriction of body growth by corticosterone were compared with the effects of a similar degree of body growth restriction produced by limitation of food intake. To evaluate the significance of the decreases in brain weight, in terms of numbers of cells (BLOCH, 1958), the DNA content of the forebrain was determined. The results to be reported show that corticosterone exerted a significant interference with the normal increase in the DNA of the infant forebrain. The Nissl substance, long known to undergo a characteristic increase in relation to neuronal maturation, has been shown to contain RNA (EDSTROM and HYDI~N, 1954). In the present work, measurements of the RNA content of the forebrain were made as an approach to an evaluation of cellular maturation. Finally, cholesterol was measured as a representative of the lipids that are known to increase in amount with the progress of myelination (FOLCH-PI, 1955). Liver was studied also, since it provides an interesting comparison to the brain.

Abstract: IT was previously reported (FLEXNER, FLEXNER and STELLAR, 1963) that the effective locus of the memory trace of simple maze learning in mice appears to spread from the hippocampi and temporal cortices to remaining areas of the neocortex in from 3-6 days after the learning experience. Memory before this period of transition has been called short-term or recent memory; after this period, longer-term memory. Recent memory can be consistently destroyed by bilateral temporal injections of puromycin which have been found to inhibit protein synthesis in the hippocampus and temporal cortex by at least 80 per cent for 8-10 hr. (FLEXNER, FLEXNER, OBERTS and DE LA HABA, 1964). Bilateral temporal injections are, however, without effect on longer-term memory, the loss of which depends upon the use of combined bilateral temporal plus ventricular plus frontal injections (FLEXNER et al., 1963). The present report is concerned with the inhibitory effects of these combined bilateral injections on cerebral protein synthesis. In addition, we have measured the rate of cerebral protein synthesis in the presence of several substances which are related to puromycin but which have no effect on memory.

Abstract: N-ACETYL-L-ASPARTIC acid (NAA) is confined solely to the central nervous system of vertebrates where, in mammals, it occurs at a concentration of approximately 1 mg/g of brain. The concentration of this amino acid derivative in various areas of the brain as well as its distribution in various species has been extensively investigated (TALLAN, MOORE and STEIN, 1954, 1956; TALLAN, 1957; JACOBSON, 1959; OKUMURA, OTSUKI and AOYAMA, 1959; OKUMURA, OTSUKI and KAMEYAMA, 1960; CURATOLO, D’ARcANGELO and LINO, 1964; TSUKADA, UEMURA, HIRANO and NAGATA, 1964). Attempts to label this compound in the brain ‘in vivo by injecting 14C-labelled precursors such as acetate, aspartate, or glucose (MARGOLIS, BARKULIS and GEIGER, 1960; BERL, LAJTHA and WAELSCH, 1961) have been unsuccessful except for one demonstration in weanling animals where both labelled glucose and acetate were incorporated into NAA (JACOBSON, 1959). These experiments suggest that in the mature animal NAA is metabolically inert. Further support for this contention arises from the observation that the concentration of NAA in the brain remains remarkably constant despite the administration to animals of a wide variety of drugs such as convulsants, hypnotics and tranquilizers, in addition to electric shock and insulin shock (JACOBSON, 1959; DE ROPP and SNEDEKER, 1961; OKUMURA et al., 1959; TEWS, CARTER, ROA and STONE, 1963). On the other hand, it was recently reported from this laboratory (MCINTOSH and COOPER 1964) that the administration of 5hydroxytryptophan (SHTP), as well as certain drugs classified as monoamine oxidase inhibitors, will cause a rise in the level of NAA in the brain, whereas drugs such as reserpine and LSD-25 will cause a fall. Since NAA is present only in the central nervous system, a connection with nervous activity has been sought. However, the compound has been shown to have no effect on the crayfish stretch receptor (JACOBSON, 1959), or on spinal neurones (CURTIS and WATKINS, 1960). OGANESYAN (1961) was unable to demonstrate a direct acetylation of choline with NAA. GEBHARD and VELDSTRA (1964) concluded that NAA did not play a role in the synthesis of carbamyl aspartic acid, arginosuccinic acid, or in purine biosynthesis as measured by formate incorporation. In this paper, some further attempts to ascribe a role to NAA in the central nervous system are presented; they lead to the conclusion that the compound serves essentially as an anion with the apparent ability to shift reversibly from a dicarboxylic to a monocarboxylic acid form. Department of Pharmacology, Yale University School of Medicine

Abstract: IN a series of studies KRECH, ROSENZWEIG, BENNETT (1960, 1962) and co-workers (ZOLMAN and MORIMOTO, 1962; ROSENZWEIG, KRECH, BENNETT and ZOLMAN, 1962; ROSENZWEIG, BENNETT and KRECH, 1964; BENNETT, KRECH and ROSENZWEIG, 1964~) demonstrated that rats maintained under conditions of ‘environmental complexity and training’ (ECT) had significantly higher btain cortex weights than isolated controls (IC). Further, they showed that ECT animals differed from IC animals in activity and distribution in the brain of acetylcholinesterase but not of total protein, hexokinase, or serotonin (BENNETT, DIAMOND, KRECH and ROSENZWEIG, 1964b). It appeared to us that these environmental conditions, being sufficiently profound to alter weights of portions of the brain, might reasonably be expected also to influence parameters of brain function. Such responses to early environment may be regarded as internalization of external stimuli (learning?, memory?) and for this reason it seemed particularly important to investigate those aromatic amines that have been associated with neurohumoral transmission and which may reflect such an internalization. In addition, it appeared reasonable to us that these environmental conditions may be stressful, and that a consequent chronic activation of the pituitary-adrenal system may occur. A series of enzymes may thereby be stimulated or inhibited leading to more widespread changes. Although BENNETT et al. (19646) reported no change in adrenal weights under IC conditions, we felt that the possibility of isolation stress should be pursued further by measuring adrenal and plasma corticoid levels and the activities of several enzymes known to be responsive to high circulatory levels of corticoid.

Abstract: THERE is evidence that under normal conditions glucose is the main substrate utilized by the brain (c j . MCILWAIN, 1959). A possible alternative substrate is glutamate, since the glutamate concentration is relatively high in the brain and it is oxidized at a high rate by brain slices in vitro (WEIL-MALHERBE, 1936). It has been inferred recently, from the specific activity of the respiratory 14C0, produced when brain slices are incubated with labelled metabolites, that glutamate is oxidized in preference to glucose (CHAIN, COHEN and POCCHIARI, 1962; SWAIMAN, MILSTEIN and COHEN, 1963). On the other hand HASLAM and KREBS (19633), who measured the net metabolic changes also, obtained evidence of an inhibition of glutamate oxidation by glucose. There are three main pathways by which glutamate can be oxidized in brain preparations. In mitochondrial systems glutamate is mainly utilized through transamination with oxaloacetate followed by oxidation of a-oxoglutarate in the tricarboxylic acid cycle (KREBS and BELLAMY, 1960; BALAZS and HASLAM, 1965; BALAZS, 1965). This pathway accounts for more than three-quarters of the glutamate utilized and with some preparations the contribution of this route has been found to be more than 90 per cent. The rest of the glutamate removed is probably oxidized mainly through the glutamate dehydrogenase, but some can also be decarboxylated and utilized through the y-aminobutyrate pathway. Since the oxidation of glutamate in viuo normally occurs in the presence of pyruvate derived from the blood glucose, the effect of pyruvate on the utilization of glutamate was investigated. The results agree with the observations of HASLAM and KREBS (19636) with brain homogenates, in that they show an inhibition by pyruvate of glutamate utilization. The mechanism of this effect of pyruvate has now been analysed. I t is suggested that the inhibitory action of pyruvate, and certain characteristic features of the operation of the tricarboxylic acid cycle reported previously, are factors affecting the steady-state concentration of glutamate, aspartate and alanine in the liver and brain in viro (BALLZS and RICHTER, 1962; HASLAM and KREBS, 1963a; BALAZS, MAGYAR and RICHTER, 1964).

Abstract: THE activities of a great many enzymes increase rapidly in the period in which the brain is growing fast and the electrical activity is attaining a mature pattern (HIMWICH, 1962; FLEXNER, 1955). Very few studies deal with the behaviour of closely related enzymes in this period of brain development. In view of the current opinion on the important function of y-aminobutyric acid (GABA) in nervous activity, we studied the levels of glutamate decarboxylase (GAD) and y-aminobutyric transaminase (GABAT) during the development of the brain of rats, rabbits and chickens. SISKEN, SANO and ROBERTS (1961) determined both enzymes in the optic lobe of the chicken from 10 days after incubation onwards. The activities of both enzymes seem to rise in a similar manner. In an earlier publication (VAN KEMPEN, VAN DEN BERG, VAN DER HELM and VELDSTRA, 1965) we reported some studies on the intracellular localization of GAD, GABAT and other enzymes in brain from adult rat, studied by centrifugation of mitochondria1 preparations on continuous sucrose gradients. This report describes the results of similar experiments with brains from immature rats. It was suspected that there might be changes in the intracellular localization of enzymes at the time when maximal changes in morphological, physiological and biochemical parameters occur.

Abstract: RECENT studies have indicated that L-phenylalanine inhibits antibody response to diphtheria toxoid in rats and rabbits (RYAN and CARVER, 1964). It was suggested, as an explanation for the inhibition, that excess phenylalanine caused a disturbance in the free amino acids of the animals with a resultant decrease in antibody (protein) synthesis. It is also conceivable that chronic alterations in the amino acids of the central nervous system induced by phenylalanine could also give rise to changes in protein synthesis. Since an excess or deficiency of an amino acid might exert an effect on the distribution of other amino acids, a quantitative study of the amino acids in rat brain and liver was conducted to assess the influence of phenylalanine on the concentration of the other amino acids.

Abstract: SEVERAL authors have published data on the lipid composition of myelin isolated from tissue of the central nervous system (AUTILIO, NORTON and TERRY, 1964; EICHBERG, WHITTAKER and DAWSON, 1964; DAVISON, 1964; O’BRIEN, 1964; SEMINARIO, AREN and GOMEZ, 1964; HULCHER, 1963; THOMPSON, KIES and ELLSWORTH, 1963; NUSSBAUM, BIETH and RANDEL, 1963; PATTERSON and FINEAN, 1961). In every case the myelin has been isolated by ultra-centrifugal fractionation of homogenised tissue but the details of techniques have been varied and the tissue has been obtained from a variety of animals. We have accumulated comparable data on myelin isolated from the brain and from peripheral nerves of a variety of animals and have also compared the myelin fractions obtained using a variety of published isolation techniques. In this paper our lipid analyses of myelin from brain and peripheral nerves are compared with some of the data already published: University of Birmingham

Abstract: EVIDENCE for altered lipid composition of malignant tissue has gradually accumulated during the last two decades. In intracranial tumours BRANTE (1949) found, in contrast with normal brain tissue, a considerable amount of esterified cholesterol. NAYYAR, MCCAMAN and HEIMBURGER (1960) found a kephalin-lecithin ratio near 1 in brain tumours, whereas normal brain tissue contained 2 or 4 times as much kephalin as lecithin. GOPAL, GROSSI, PAOLETTI and USARDI (1963) published a study on the lipid composition of brain tumours which gave further support for the conception of a change in phospholipid proportions. RAPPORT and GRAF (1955) showed that tumour lipid extracts had antigenic properties not found in normal tissue lipids. As a consequence of these findings RAPPORT, GRAF, SKIPSKI and ALONZO, (1958) described and isolated from epidermoid carcinoma a new lipid hapten, a dihexose ceramide termed cytolipin H. KOSAKI, KODA, KOTANI, NAKAGAWA and SAKA (1958) demonstrated the presence of a new choline-containing phospholipid in a wide variety of human malignant tissues which could not be demonstrated in normal tissue. In the same period, considerable evidence for alteration of the cell surface during malignant transformation has been furnished. COMAN (1 944) demonstrated that tumour tissue was less coherent than its normal homologue. Interference-microscope studies of cell contacts in tissue cultures have shown the loose and seemingly accidental nature of cell to cell contact in malignant tissue when compared with its normal counterpart (ABERCROMBIE and AMBROSE, 1958). Furthermore, there is evidence that the tumour cells, in contrast with their normal homologues, have a strongly electronegative surface charge which increases as the cells acquire more malignant properties (AMBROSE, JAMES and LOWICH, 1956, PURDOM, AMBROSE and KLEIN, 1958; AMBROSE, 1962). Finally, the normal 'contact inhibition' of cellular movement is lacking in malignant growth, where the cells are moving freely and independent of each other (AMBROSE, 1962). The aim of the present investigation has been to compare the polar lipid patterns of normal brain tissue with the patterns of gliosis, benign and malignant brain tumours and to discuss their implications to the surface properties of the cell.

Abstract: THE presence of mucopolysaccharides (MPS) in the central nervous system has been demonstrated by means of histochemical techniques (ABOOD and ABUL-HAJ, 1956; BRANTE, 1957; YOUNG and ABOOD, 1960) as well as by chemical means (BRANTE 1957; MEYER, HOFFMAN, LINKER, GRUMBACH and SAMPSON, 1959; SZABO and ROBOZEINSTEIN, 1962; CLAUSEN and ROSENKAST, 1962; CLAUSEN and HANSEN 1963). SZABO and ROBOZ-EINSTEIN (1962) isolated hyaluronic acid and chondroitin 4-sulphate from bovine brain while CLAUSEN and his co-workers (1963) demonstrated the presence of hyaluronic acid, dermatan sulphate and chondroitin 6-sulphate in human brain. On the other hand MEYER et al. (1959) reported the presence of hyaluronic acid and heparan sulphate in the normal brain of a child and dermatan sulphate and heparan sulphate in the brain of a gargoyle patient. These observations indicate the presence of different types of mucopolysaccharides in the brain although their exact na txe is not fully established. This prompted us to investigate the different types of mucopolysaccharides in the central nervous system in further detail. Earlier work from this laboratory (GUHA, NORTHOVER and BACHHAWAT, 1960) on the incorporation of [35S]sulphate into the mucopolysaccharides during the development of rat &brain suggested the importance of the sulphated mucopolysaccharides in the process of myelination and brain maturation. Recent work from this laboratory (BALASUBRAMANIAN and BACHHAWAT, 1964) has demonstrated the in vitro enzymic sulphatation of mucopolysaccharides in young rat brain. I t has been further observed that the enzyme activity was much diminished in adult brain. On the other hand SZABO and