Showing papers in "Journal of Neurochemistry in 1961"

Amino acid and protein metabolism‐vi cerebral compartments of glutamic acid metabolism

Abstract: IN RECENT studies of glutamic acid metabolism in uivo, the fate of tracer amounts of rqlabelled glutamic acid administered intravenously to rats and mice was investigated in experiments of short duration (LAJTHA, BERL and WAELSCH, 1959). The major portion of the amino acid apparently entered brain, liver, kidney and muscle as the acid and conversion to the amide was not an essential prerequisite for exchange across the blood-brain barrier. The metabolic changes which then ensued were quite rapid, for within 2 min after injection of [14C]glutamic acid, the glutamic acid, glutamine, and glutathione* fractions of brain, liver and kidney and the y-aminobutyric acid fraction of brain were significantly labelled; the specific activity of the organ glutamine was closest to that of the dicarboxylic acid. In many experiments with [14c]glutamic acid the specific activity of plasma glutamine was higher than that of glutamine and glutamic acid in the brain and liver. Such a result would be obtained if the plasma glutamine were being formed in organs other than those analysed, or if the glutamine were synthesized from (14C]glutamic acid of higher specific activity than that resulting from the mixing of the administered labelled amino acid with the organ glutamic acid. In addition, the newly formed amide may have been released to the blood before being equilibrated with the organ glutamine. The latter explanation suggests cellular compartmentalization of glutamine synthesis. In this report data are presented on the metabolism of [14C]glutamic acid after intracisternal administration of p4C]glutamic and aspartic acids and p4C]glutamine to rats and monkeys. As in the study of protein turnover and the previous investigations, the animals were exposed for only short periods of time to the isotopic amino acids in order to avoid equilibration of the label and to study the initial fate of the administered glutamic acid. EXPERIMENTAL

The cerebral defect in Tay-Sachs disease and Niemann-Pick disease.

Abstract: THE nature of the functional and anatomic disturbance in the central nervous system which occurs in the constitutional syndromes referred to as ‘lipidoses’ has not been explained. There has been an understandable tendency to assume that the same hereditary defection which produces an accumulation of lipid in the solid viscera of these patients is also responsible for the changes in nervous tissue. This is the traditional view (HSIA, 1959), although THANNHAUSER (1953), in a penetrating review of the problem, raises important doubts about the certainty of this conclusion. Descriptions of Niemann-Pick disease have offered major support to the concept of the unitarian hypothesis for brain and visceral lipid derangement, since the classical patients with this diagnosis invariably had cerebral handicaps and many of the early studies showed brain sphingomyelin increases (KLENK. 1954). In Tay-Sachs disease, the failure to find ganglioside accumulations in liver and spleen was explained by the probable absence, in these regions, of a significant natural synthetic locus. In chronic Gaucher’s disease, it was stated (THANNHAUSER, 1953) that the cerebroside defect is not fully manifest until the critical period of cerebral maturation is completed (and thus the brain is spared). These generalizations are found to be unsatisfactory, however, as more patients receive careful study. Furthermore, the existing published reports on brain lipid analysis in children with lipidosis problems are difficult to summarize because of the small number of patients, and the differences among authors in clinical classification, preparation of specimens for analysis, chemical techniques employed, and method of recording results. It therefore seems to be of value to present recent material from this hospital which bears on the thorny problem of the type of nervous system handicap found in certain of the lipidoses.

Microanalytical procedures for fluoro‐metric assay of brain dopa‐5htp decarboxylase, norepinephrine and serotonin, and a detailed mapping of decarboxylase activity in brain

Abstract: THE presence of the amines, norepinephrine (NE)? and serotonin (5HT), and of the enzymes, 3 :4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5HTP) decarboxylases, in brain has aroused considerable conjecture regarding the role of the arnines in the central nervous system. A detailed mapping of the decarboxylases and the amines in brain might outline the regions where the amines act and throw some light on their normal function. Previous papers have described the fluorometric estimation of tissue NE (SHORE and OLIN, 1958) and 5HT (BOGDANSKI eta/ . , 1956) and the Auorometric (BERTLER and ROSENGREN, 19594, or manometric (HOLTZ and WESTERMANN, 1956), estimation of SHTP and DOPA decarboxylases. Although these methods are suitable for the estimation of these substances in relatively gross areas of the brain, they are not sensitive enough for their estimation in more precisely defined brain regions. This paper describes modifications of the fluorometric procedures for NE and 5HT estimation whereby they can be assayed in amounts of brain containing as little as 40 pmg of NE or 200 pmg of 5HT. In addition, methods are described for measuring the activities of SHTP and DOPA decarboxylases in 20 to 100 mg of brain tissue. With these methods the activity of DOPA decarboxylase in the cat brain has been mapped in considerable detail and has been related to the activity of 5HTP decarboxylase and to the levels of 5HT and NE.

The brain barrier system--II. Uptake and transport of amino acids by the brain.

Abstract: THE UPTAKE and release of amino acids by the brain has only recently attracted the interest of the neurochemists. It has been shown that increase in concentration of most amino acids in the brain, after their elevation in plasma, is inhibited. The penetration into the brain of glutamic acid is strongly restricted (SCHWERIN, BESSMAN and WAELSCH, 1950), that of lysine (LAJTHA, 1958) and proline (DINGMAN and SPORN, 1959) fairly strongly restricted, that of glutamine (SCHWERIN et al., 1950) and tyrosine (CHIRIGOS, GREENGARD and UDENFRIEND, 1960) not as much. The restriction is not absolute. Cerebral methionine, histidine, lysine and arginine were found to increase during continuous infusion (KAMIN and HANDLER, 1951). The restriction to uptake, measured with glutamic acid (HIMWICH, PETERSON and ALLEN, 1957) and lysine (LAJTHA, 1958), is not as strong in newborn as in adult animals. Apart from these data, very little is known about the mechanism and the rate of uptake and release under physiological conditions. As part of a study of the mechanism of passage of metabolites into and from the brain, this paper reports changes in the concentrations, of amino acids in the brain after their administration to the intact animal in various ways. It was found that part of the administered lysine and leucine leaves the adult brain against a concentration gradient of elevated plasma levels. Phenylalanine in the adult and leucine in the newborn brain did not show such behaviour under the experimental conditions employed.

The uptake of circulating [3H]norepinephrine by the pituitary gland and various areas of the brain.

Abstract: IT WAS shown previously (WEIL-MALHERBE, AXELROD and TOMCHICK, 1959) that circulating [3H]epinephrine did not cross the blood-brain barrier to any significant extent except in the hypothalamic region. Even there the uptake was slight in comparison with extracerebral tissues (AXELROD, WEIL-MALHERBE and TOMCHICK, 1959). These experiments have now been extended to [3H]norepinephrine; furthermore the effect of a series of drugs on the uptake of circulating [3H]norepinephrine by the pituitary gland and by various areas of the brain has been studied. Preliminary accounts of this work have been presented at two symposia (WEIL-MALHERBE, 1960; WEIL-MALHERBE, WHITBY and AXELROD, 1961).

Fluid content and compartments in isolated cerebral tissues.

Abstract: ION MOVEMENTS are fundamental to neural activity, and their study in cerebral tissues (CUMMINS and MCILWAIN, 1961) has prompted the following re-examination of tissue fluids. This is because, although potassium loss and assimilation can be investigated with minimal knowledge of concomitant changes in fluids, the fluid movements must be known in order to appraise changes in sodium and chloride ions. In addition, some theories of ion movement (MILLER, 1960) propose that the primary point at which metabolically derived energy is applied in performing osmotic work, is on fluid rather than on ions. The present study involved appraising: (1) Changes occurring during the initial contact of the cut tissue with fluids to be used in incubation. Changes of this category have received relatively little attention, but their study is made more feasible by the recently developed technique of slicing without contact with fluid, using a narrow blade mounted in a bow-cutter (RODNIGHT and MCILWAIN, 1954; MCILWAIN, 1961). (2) Change during subsequent incubation has been examined by measuring total fluid, and also the inulin content of the tissue after its incubation in inulin-containing media. The use of inulin to obtain a measure ofintraand extra-cellular spaces in cerebral tissue was based on the studies of KOREY and MITCHELL (1951), ALLEN (1955), PAPPIUS and ELLIOTT (1956) and MCLENNAN (1957) and is appraised further below. (3) The methods developed have been applied to determine fluid changes during electrical excitation of the tissue, and during the action of basic proteins: both these agents were found by THOMSON and MCTLWAIN (1961) to alter tissue weight, presumably by fluid changes. E X P E R I M E N T A L Media and tissue Glycylglycine saline contained 124 m-NaCI , 5 m-KCI, 1.24 mM-KH,PO,, 1.3 mM-MgSO,, 2.8 mM-CaCI,, 10 m-glucose, and 30 m-glycylgtycine taken to pH 7.4 with M-NaOH and was saturated with 0,. Bicarbonate saline, unless otherwise specified, differed only in containing 26 m-NaHCO, in place of glycylglycine and was equilibrated with 95 % 0,: 5 % CO,. In media which contained inulin (see below) its concentration was 1 % unless stated otherwise. Adult guinea pigs of either sex, weighing 25o-u)o g were stunned with a blow on the neck and exsanguinated by cutting the neck; the brain was removed as described by MCILWAIN and RODNIGHT (1962) within 1.5 min of stunning. A hemisphere was taken, subcortical structures removed, and it was placed on a cutting table (MCILWAIN, 1961) with the cortex uppermost. From this, three successive slices 0.35 mm thick were cut at room temperature (17-20\") in one of two ways. (1) With a bow cutter and glass guide (MCILWAIN, 1961), avoiding all contact with fluid. The slices were picked up with a bent wire rider of a torsion balance and weighed to the nearest mg; this is the method used in most experiments. (2) With a blade and the same glass guide. lubricated with one of the incubating media described above; after cutting, these slices were floated from the blade or guide into a shallow dish of

The inhibitory action of 3‐hydroxytyramine, gamma‐aminobutyric acid (gaba) and some other compounds towards the crayfish stretch receptor neuron*

Abstract: THE concept of inhibitory processes occurring in the nervous system of various organisms is part of classical neurophysiology, but the idea of inhibitory neurons with axons whose endings release a specific inhibitory transmitter substance is relatively new (ECCLES, 1957). Evidence for the presence of inhibitory cells in the central nervous system of mammals is now abundant but, as yet, no inhibitory transmitter has been chemically identified. CURTIS and WATKINS (19604 have, however, suggested some criteria that such a substance should fulfil. An inhibitory transmitter might be contained in Factor I, a crude preparation from mammalian brain described originally by FLOREY (1954) and FLOREY and MCLENNAN (1955u, b), and which has been shown by MCLENNAN (1960) t o meet some of the criteria set up by CURTIS and WATKINS (1960~1, b). Identification of inhibitory transmitter substances is an important objective of current neurophysiological research, and suitable bioassay procedures are essential for such work. One convenient and widely used preparation is the slowly-adapting stretch receptor neuron of the crayfish. Inhibition of the discharge of this neuron was used by BAZEMORE, LLIOTT and FLOREY (1957) to identify GABA as an active component of Factor I preparations. This and subsequent work (ELLIOTT, 1958) led to the suggestion that GABA might account for most or all of the activity of Factor I-containing brain extracts. However, it was shown later that not all of the actions of Factor I are duplicated by GABA (MCLENNAN, 1957~). Moreover, a number of other amino acids, some of which occur in mammalian brain, have also been shown to have inhibitory effects on the crayfish stretch receptor neuron (BAZEMORE, ELLIOTT and FLOREY, 1957; EDWARDS and KUFFLER, 1959). The effects of GABA have since been investigated on many biological systems (ROBERTS, 1960), and the concept is developing that this amino acid functions, a t least in the central nervous system of mammals, not as a specific inhibitory transmitter substance, but as a neuronal depressant (CURTIS, PHILLIS and WATKINS, 1959). It now appears that the crayfish stretch receptor neuron is not a specific bioassay system; such lack of specificity may be of advantage in indicating the inhibitory characteristics of compounds not previously suspected of possessing such properties. In the study now reported, a series of compounds has been tested for inhibitory

Activation and inhibition of adenosine triphosphatases of subcellular particles from the brain.

Abstract: EXPECTATION that the adenosine triphosphatases of neural tissues, like those of muscle, might participate in the tissue's utilization of metabolically derived energy (see for example GORE, 1951; MCILWAIN, 1957) were supported by the findings of SKOU (1957, 1960) and of HESS and POPE (1957), that the adenosine triphosphatases of crab nerve and rat brain required the presence of sodium salts for maximal activity. The enzymes also showed the requirements for magnesium and potassium salts which are more usual in reactions involving adenosine triphosphate (LOWENSTEIN, 1958, 1960); the sodium requirement might connect the enzymes with the performance of osmotic work in neural tissues. Recent studies from these laboratories have shown that ion movements in cerebral tissues can be modified by substances which include basic proteins, gangliosides and suramin (WOODMAN and M C I L W ~ N , 1961 ; MCILWAIN, WOODMAN and CUMMINS, 1961 ; and unpublished). The effects of these agents on the adenosine triphosphatases of cerebral tissues have therefore been examined. The microsomal fraction of ground cerebral tissues contains much of the tissues' gangliosides and is involved in the interactions of basic proteins and gangliosides with the tissue (WOLFE and McILwAIN, 1961 ; WOLFE, 1961). JARNEFELT (1960) showed that microsomal fractions of ground cerebral tissues contained an adenosine triphosphatase activated by sodium salts, but the degree of activation was not large. During the present studies attention has therefore been paid to the conditions under which the sodium activation is manifested, and also to the enzyme level and to the degree of sodium activation in different subcellular fractions of the ground tissue. E X P E R I M E N T A L

A histochemical study of DPN-diaphorase in human white matter with some notes on myelination.

Abstract: THERE is some indication of differences in the anatomical and chemical organization of individual fibre tracts in the brain, such as the outstanding activity of alkaline phosphatase in the retroflex bundle of the guinea pig (SHIMIZU, 1950) and the different rates of synthesis of acetylcholine in various fibre tracts (FELDBERG and VOGT, 1948). This paper reports on the distribution of DPN-diaphorase in human white matter. The distribution of enzyme activity in human grey matter will not be touched upon. It resembles, in principle, that of succinic dehydrogenase in the guinea pig brain which, in turn, is similar to that of several oxidative enzymes in cat brain as documented recently in an histochemical atlas (FRIEDE, 1961). The activity of oxidative enzymes is much less in white matter than in grey matter, as shown by both biochemical and histochemical data. Histochemical studies revealed detailed differences in the cytological enzyme distribution in the various tracts. Some data on myelination and on certain features of enzyme transport in axons were included in this study because they imply a difference in the chemical organization of the tracts.

Mode of regeneration of acetylcholinesterase in cholinergic neurons following irreversible inactivation.

Abstract: THE concept of somato-axonal convection of cytoplasm as a means by which the cell body supports the metabolic needs of the axon is by no means novel, and in fact, was advanced by GOLDSCHEIDER during the era of acrimony surrounding the neuron doctrine in the 1890's (BARKER, 1899). Indeed, the concept may be considered a direct consequence of the neuron doctrine, since one of its basic tenets states that the axon and all its parts, no matter how remote from the cell body, are an integral part of the nerve cell. It is only recently, however, that a number of attempts have appeared to demonstrate that axonal constituents are derived from the perikaryon. Those that have been reported fall essentially into two categories: (i) efforts to establish the general phenomenon of cellulifugal axoplasmic streaming; and (ii) studies designed to demonstrate somato-axonal migration of proteins or specific enzymes. It was the comprehensive histological studies of WEISS and HISCOE (1948) that established the likelihood that a continuous peripherally directed movement of axoplasm exists. Additional support for this hypothesis based on morphological considerations was obtained recently by VAN BREEMEN, ANDERSON and REGER (1958) and LUBINSKA (1954). Although WEIS and HISCOE (1948) postulated that axonal proteins are synthesized in the cell body and are carried peripherad by axoplasmic streaming, they offered no evidence to support their contention. More direct evidence for somato-axonal migration of protein was obtained by the use of isotopically labelled amino acids (SAMUELS et a/., 1951 ; WAELSCH, 1958; KOENIG, 1958; VERNE and DROZ, 1960; and MIANI, 1960). In all instances, proximo-distal displacement of labelled protein with respect to time was observed in peripheral nerves. Studies serving as a basis for somato-axonal migration of preformed enzymes have centred principally around acetylcholinesterase (AChE)$ (SAWYER, 1946; LEWIS and HUGHES, 1957) and choline acetylase (ChAc) (HEBB and WAITFS, 1956). In both the studies by SAWYER (1946) and HEBB and WAITES (1956), the distal accumulation of AChE and ChAc, respectively, was inferred from an observed increase of enzymic activity in the proximal, regenerating stump of previously axotomized peripheral * This investigation was supported by research grant B-282 (C6 and C7) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U.S. Public Health Service. t Predoctoral fellow of the Institute of Neurological Sciences. The work reported in this communication served as a basis for a dissertation submitted in partial fulfilment for the degree of Ph.D. by E. KOEHIC in the Department of Physiology. : Abbreviations used: acetylcholine, ACh; acetylcholinesterase. AChE; acetyl-,!l-methylcholine, MCh; butvrvlcholinesterase. BuChE: cholinesterase. ChE: choline acetvlase. ChAc; diirooroovlfluoroohomhate, 1)FP.' dir,opropylpho,phobtigmtne, DPS; endopldtmic retiLulum. ER, ribonuclsi; d&i, RNA'; ribonucIcoprotein, RNP; turnover number, T Y ; 2-diethoxphocphinvlthiocth~l~imc~hyl~mine dc d oxaldte, 217AO.

Some metabolic changes accompanying Leao's spreading cortical depression in the rat.

Abstract: IN 1944 L E ~ O described a particular electrical reaction of the cerebral cortex to stimuli acting directly on its surface. Electrical, mechanical and chemical stimuli cause a local decrease in amplitude of the spontaneous EEG. This decrease spreads slowly at a rate 3-6 mm/min in all directions over the whole cortex. Complete recovery of the original activity occurs after from 10 to 20 min. The front of the wave of spreading depression is accompanied by a striking slow potential change. The initial surface negativity, attaining 5-10 mv in amplitude, is replaced after 1-2 min by a lower wave of longer lasting positivity. A new wave can be produced from the same focus after a minimum recovery period of 4-5 min (L&o, 1944). Since the discovery of this phenomenon various aspects of it have been studied (see recent review by MARSHALL, 1959). The electrophysiological properties of spreading depression are already well known but the mechanism of initiation and spreading is still obscure. Some properties of spreading depression seem to indicate that metabolical changes are involved: ( I ) The rate of spreading is slow; (2) the slow potential change is temperature dependent with a Qlo = 1.8 ( B u d , 1956); (3) the decrease of cortical PO, during spreading depression is not caused by vasoconstriction, as was assumed by VAN HARREVELD and STAMM (1952), but rather by increased oxygen utilization due to vasodilatation (BURFSOVA, 1957); (4) the analogy between the initiation of spreading depression in vivo and the increase in respiration and aerobic glycolysis of cerebral cortex slices in vitro (BUR&, 1956). A decrease in glycogen in the rat brain during spreading depression has been observed (K~IVANEK, 1958). In the experiments reported in the present paper a more precise correlation between electrical and chemical changes has been attempted.

Proteins, lipid-protein dissociation and proteinase activity in wallerian degeneration

Abstract: THE process of demyelination in Wallerian degeneration may be pertinent to the aetiology of the human dernyelinating diseases, since the study of this process may indicate where the causes of these diseases should be sought. As far as the myelin lipids are concerned, it has been shown that there are no chemical changes during the first week after nerve section, but thereafter the lipids are progressively degraded to cholesterol esters (JOHNSON, MCNABB and ROSSITER, 1949, 1950; ROSSITER, 1955). Likewise. the histochemical characteristics of the myelin lipids remain unaltered during the first week of demyelination (NOBAK and MONTAGNA, 1952; NOBAK and REILLY, 1956) but in the second week, cholesterol esters accumulate in the degenerating sheath as is shown by the positive Marchi reaction of the myelin break-down products at this stage (ADAMS, 1958, 1960). The early events in the first week of demyelination were attributed by ROSSITER and his colleagues to ‘physical’ disintegration of the sheath. However, although they showed that the myelin lipids were chemically unaffected in this preliminary phase no account was taken of changes in the myelin proteins or the bonding between lipid and protein in the sheath. It could be postulated that a protein constituent of myelin is chemically degraded during this early phase of dernyelination or, alternatively, that there is a disruption of lipid-protein bonds, such as those of CNS proteolipid (FOLCH and LEES, 1951) or the trypsin-resistant protein residue (TRPR)? lipid complex of the PNS (TUQAN and ADAMS, 1961). In support of the former hypothesis, it was shown by TUQAN and ADAMS that proteolytic enzymes cause in uitro ‘dernyelination’ apparently as a result of the liberation of lipid bound to a trypsin-digestible protein in the myelin of both CNS and PNS. The purpose of this investigationwas to determine by biochemical and histochemical means (a) whether there is any degradation of proteolipid protein and TRPR protein in degenerating myelin of the CNS and PNS, (b) whether there is disruption of the TRPR-lipid complex during demyelination and (c) whether proteolytic enzymes are present or become active in the degenerating nerve.

Quantitative histochemical studies of the morphogenesis of the cerebellum. II. Two beta-glycosidases.

Abstract: IN THE preceding paper (ROBINS and Low, 1961), the changes in malic, lactic, and isocitric dehydrogenases, in dipeptidase, and in total lipid of the individual layers of the developing cerebellar cortex of the rat were described. In the present study changes in two B-glycosidases-8-glucuronidase and 8-galactosidase-will be described. These enzymes have been shown to increase greatly in peripheral nerves (HOLLINGER and ROSSITER, 1952; MCCAMAN and ROBINS, 1959) and in central nerves (MCCAMAN and ROBINS, 1959) undergoing Wallenan degeneration. It was of interest, therefore, to ascertain the changes in these enzymes in developing cerebellar white matter as well as in layers of the cerebellar cortex.

The action of some alpha-methyl and other amino acids on cerebral catecholamines.

Abstract: IT HAS been shown recently that AMDPt, a t first studied as an inhibitor of mammalian DOPA? decarboxylase in z’itro and of the tyrosine decarboxylase of Str. fiecalis R (SOURKES, 1954), acts in vico upon the decarboxylation of DOPA (DENGLER and REICHEL, 1958 ; MURPHY and SOURKES, 1959), 5-hydroxytryptophan (WESTERMANN, BALZER and KNELL, 1958; SMITH, 1960; OATES, GILLESPIE, UDENFRIEND a,nd SJOERDSMA, 1960), tyrosine and tryptophan (OATES et a/., 1960). This action on the decarboxylation of DOPA in riro is also displayed by a-methyl-3-hydroxyphenylalanine (a-methyl-m-tyrosine) (SOURKES, MURPHY and WOODFORD, 1960) as well as by amethyl-2-hydroxyphenylalanine (a-methyl-0-tyrosine), a-methyl-2:5-DOPA, and amethyl-5-hydroxytryptophan (MURPHY and SOURKES, 1961). By use of fluorometric methods it has been shown (MURPHY and SOURKES, 1959) that AMDP administered parenterally to the rat has two effects upon the cerebral catecholamines: one of decreasing the concentration of the endogenous amines and, secondly, of interfering with the conversion of exogenous DOPA to cerebral dopamine and noradrenaline. Although the injected AMDP is found in the brain in concentrations known to be effective for the inhibition of DOPA decarboxylase the rapid decrease in cerebral catecholamines could conceivably be due to some other mechanism than through inhibition of the decarboxylation. In this paper the amine-depleting action of AMDP has been further studied. Moreover, some other amino acids have been tested to compare their ability with that of AMDP in depleting the brain of its catecholamines.

The high-energy phosphates in developing brain.

Abstract: FOR SOME time investigators have studied the changes in energy metabolism which occur during development of the brain. A rapid rise in the rate of cerebral oxygen consumption has been demonstrated (TYLER, 1942; HIMWICH, 1941a, b) and the susceptibility of the brain to oxygen deprivation has also been shown to increase during this time. The implication is that the cerebral power (energy per unit time) requirement increases with the maturation of the brain (SAMSON, 1957, 1958). The steady state concentration of ATPt is about the same throughout the maturation period (SAMSON, 1960). The triphosphate compounds of guanosine, cytidine and uridine are now known to be present in brain (SCHMITZ, 1954; THOMAS, 1957; GERLACH, 1958). Indeed, the work of HEALD (19576) established that the GTP concentration in the brain is about 20 per cent of the ATP concentration. However, the other compounds have not been extensively studied. It seemed interesting, therefore, to determine the pattern of these high-energy-phosphate compounds, including phosphoryl-creatine, during neonatal maturation. With this in mind, the following experiments were carried out on rats in different stages of development.

Amino acid and protein metabolism of the brain--VIII. The recovery of cholinesterase in the nervous system of the frog after inhibition.

Abstract: IN THIS PAPER the results of a study on the recovery of cholinesterases in the nervous system of the frog, after inhibition with a lipid soluble ‘irreversible’ inhibitor, are summarized. While there is no conclusive evidence that the reappearance of esteratic activity after inhibition should be considered protein synthesis, a study of the sequence of inhibition and recovery of the cholinesterases offers information which may form the basis for an understanding of the origin of these enzymic activities in the central nervous system and peripheral nerve. In the preceding communication the time sequence of inhibition of true and pseudocholinesterases in the central nervous system, sciatic plexus, and nerve, after administration of lipid soluble and lipid insoluble inhibitors, was described (CLOLJET and WAELSCH, 1961). Within 75 min after the intraventricular administration of 21 7A0 (obtained through the courtesy of Dr. R. A. LEHMAN of the Campbell Pharmaceutical Company of New York), acetylcholinesterase activity in the plexus and nerve was inhibited in rapid progression in a proximodistal direction. It was suggested tentatively from an analysis of the inhibition of the pseudo and true esterases that the inhibitor may travel within the epior endo-neural spaces along the plexus and the nerve and enter the axon at the nodes of Ranvier. This paper is concerned with the reappearance of enzymic activity after inhibition of the esterases by 217AO. In addition, data on the localization of the esterases in fractionated brain tissue from various animals are presented.

Quantitative histochemical studies of the morphogenesis of the cerebellum i—total lipid and four enzymes*

Abstract: THE HISTOLOGICAL appearance of the layers of the developing cerebellar cortex in the albino rat has been described in detail by ADDISON (191 1). In the foetal rat, as late as the nineteenth day, only the presumptive cerebellar layers--ependymal, mantle, and marginal-can be readily distinguished. At birth, or just before, relatively defined layers appear. These are the external granular layer, the molecular layer (including the Purkinje cells at its inner margin), the internal granular layer, and the subjacent white matter. Once these 4 layers have appeared (the white matter immediately subjacent to the internal granular layer will be referred to as a layer in this report), it is possible to follow discretely each into adult life or until disappearance (external granular layer). The early segregation of the layers of the cerebellar cortex offered, therefore, an unusual opportunity to study the development in the central nervous system of relatively homogeneous histological regions. To understand the chemistry of the developing nervous system in detail, it will be necessary ultimately to study it in terms of its individual histological elements. While this observation may be true for any tissue, it is particularly applicable to the nervous system because of its extreme architectural complexity. Because of the minute size of these individual cerebellar layers, their quantitative chemical study required the use of sensitive microchemical procedures and their isolation, under direct histological control, in a chemically intact state (LOWRY, 1953; ROBINS, 1957). The use of the rat offered an advantage, since almost the entire development of the cerebellum occurs postnatally. In this report, we will describe changes i n total lipid, lactic dehydrogenase (LDH)S, malic dehydrogenase (MDH), isocitric dehydrogenase (ICDH), and dipeptidase (PEPT) in the individual cerebellar layers of the @day (newborn), 9-day, 14-day, and adult rat. These quantitative histochemical data will be supplemented with data from

A biochemical and morphologic study of myelination and demyelination. III. Effect of an organo-phosphorus compound (Mipafox) on the biosynthesis of lipid by nervous tissue of rats and hens.

Abstract: THE HISTOPATHOLOGY of ‘ginger paralysis’, that is, of the central and peripheral lesions caused by organo-phosphorus compounds, has been described in considerable detail (SMITH and LILLIE, 1931; BARNES and DENZ, 1953; CAVANAGH, 1954). Biochemical investigations of this condition have focused largely on the concomitant inhibition of cholinesterases (ORD and THOMPSON, 1952; EARL and THOMPSON, 1952; EARL, THOMPSON and WEBSTER, 1953; DAVISON, 1953; ALDRIDGE, 1953 a,b,c; AUSTIN and DAVIES, 1954); less attention has been given to disturbances of lipid metabolism in the nervous tissue (WEBSTER, 1954; M m o and KARNOVSKY, 1955; AUSTIN, 1957; MAJNO and KARNOVSKY, 1958 a). This aspect seems worthy of further investigation, since the organophosphorus compounds have been referred to as ‘myelin poisons’ (SMITH and LILLIE, 1931). Their primary site of action, however, has not yet been conclusively established. Previous studies of this series have dealt with lipid biosynthesis in tlitro by various preparations of central and peripheral, normal, and pathologic nervous tissue. The incorporation of labelled acetate and phosphate into the total lipids of these preparations was found to be a sensitive indicator of tissue damage even before the appearance of histological lesions in Wallerian degeneration, in the proximal stump of transected nerves (MAJNO and KARNOVSKY, 1958 b,c), in experimental diphtheric polyneuropathy (MAINO, WAKSMAN and KARNOVSKY, 1960), and in central tracts after partial chordotomy (KARNOVSKY and MAJNO, 1959). In the present study the same techniques were applied to nervous tissue of rats and hens treated with Mipafox (bismonoisopropylaminofluorophosphine oxide). This substance, a powerful cholinesterase inhibitor, is used commercially as an insecticide; it is known to cause the typical paralytic syndrome in man and experimental animals (BARNES and DENZ, 1953).

Correlation of brain monoamine oxidase activity with behavioural changes after iproniazid and 5‐hydroxytryptophan administration†

Abstract: WHEN brain levels of 5-hydroxytryptamine (5-HThS are experimentally elevated in animals by injecting its precursor, 5-hydroxytryptophan (5-HTP), the animals exhibit behavioural disturbances (UDE\"D et al., 1957; BOGDANSKI et al., 1958). The resulting atypical behaviour has been described in subjective terms. In other experiments of this type, numerical ratings of the visual observations have been made in dogs (COSTA et al., 1959; HIMWICH, 1960). Recent studies from our laboratories have established the quantitative relationship between the dose of intramuscularly administered 5-HTP and its behavioural effects by employing operant (learned behaviour) techniques (APRISON and FERSTER, 1960,1961). The same authors have also quantitatively measured the enhanced behavioural effects of 5-HTP following iproniazid administration (FERSTER and APRISON, 1959). In the present study, we correlated the behavioural effects of 5-HTP with brain monoamine oxidase (MAO) activity. The correlation was made over a wide range of brain MA0 (9-100 per cent normal) by pretreating the birds with iproniazid (isopropyl-isonicotinyl hydrazide).

Free nucleotides of the brain in various mammals.

Abstract: INTEREST in the study of the free nucleotides of the tissues results from the fact that adenine nucleotides play an essential part in energy metabolism and in transphosphorylation (LIPMANN, 1941 ; KORNBERG, 1951). Cytosine nucleotides participate in the biosynthesis of lipids (KENNEDY and WEISS, 1956), and the nucleotides of uracil and guanine take part in the synthesis of polysaccharides (LELOIR, 1957); guanosine triphosphate appears to be concerned in the biosynthesis of proteins. Many authors have studied the easily hydrolysable phosphate esters of the acidsoluble fraction of the brain, either directly or by the method using the barium and calcium salts (KERR, 1935, 1941; STONE, 1943; DAWSON and RICHTER, 1950; ALBAUM, TEPPERMAN and BODANSKY, 1946). KRATZING and NARAYANASWAMI (1953) as well as THORN, PFLEIDERER, FROWEIN and Ross (1955) have carried out enzymic estimations. However, these methods gave no information about the content of the nucleoside polyphosphates, ADP, GTP, UTP, UDP, CTP and CDP. * Recently, by means of column chromatography, the distribution of free nucleotides was determined in rat brain (SCHMITZ et al., 1954a, b) and in mouse, rat and rabbit brain (MANDEL and HARTH, 1957; MANDEL, HARTH and REBEL, 1958). DOHRING and GERLACH (1957) used paper chromatography to estimate several of the free nucleotides in the brain and HEALD (1956) used electrophoresis for the same purpose. It has been maintained that excitation of the nervous system during decapitation could lead to a reduction in energy-rich compounds (STONE, 1940; KRATZING and NARAYANASWAMI, 1953 ; DOHNNG and GERLACH, 1957; WEIL-MALHERBE, 1953). COPER, KERKEN and KORANSKY (1954) reported satisfactory values for animals decapitated without anaesthesia. DOHRING and GERLACH (1957) obtained high values for ATP in animals killed under anaesthesia but found no appreciable difference in the values for GTP, UTP, ADP and AMP. One objection to killing animals under anaesthesia has been that ATP may accumulate because of lack of utilization (MCILWAIN, BUCHEL and CHESHIRE, 1951 ; STONE, 1940). Consideration of these facts prompted us to study the distribution of the nucleotides of the brain in various species of mammals, in order to determine the best way of obtaining brain material which would give reliable values.

The synthesis of creatine by the brain of the intact rat

Abstract: CREATINE synthesis was first demonstrated by BORSOOK and DUBNOFF (1940) in experiments in which rat liver slices were incubated with guanidoacetic acid and methionine. In subsequent studies, DUVIGNEAUD (1941, 1956) demonstrated that the methyl group of methionine is transferred as a unit to guanidoacetic acid to form creatine in tlico. BLOCK and SHOENHEIMER (194l), using [15N]-labelled compounds, and BORSOOK and DUBNOFF (1941) using kidney slices and cell free extracts, later demonstrated guanidoacetic acid synthesis from arginine and glycine. BORSOOK and DUBNOFF (1941) proposed a mechanism, accommodating all of these findings, whereby the total body requirement of creatine might be synthesized in animals. The synthesis of creatine was tentatively described as proceeding in two generalized steps :

Effect of convulsants on brain glycogen in the mouse.

Abstract: THE first reliable method for the measurement of glycogen in brain tissue was published by KERR (1936). He and his coworkers found that a profound insulin hypoglycemia reduced the brain glycogen level in dogs, cats, and rabbits (KERR and GHANTUS, 1936; KERR, HAMPEL and GHANTUS, 1937). This change was confirmed in dogs by CHESLER and HIMWICH (1944), in cats by OLSEN and KLEIN (1947~7, b), and recently in rats by SCHILLER (1958). KERR and ANTAKI (1937) studied effects of picrotoxin, pentylenetetrazol, and strychnine in rabbits. These convulsants did not induce measurable changes in the brain glycogen. In these experiments, however, the convulsive activity was suppressed by administration of an anaesthetic before the brain was frozen. LEPAGE (1946) reported that intense and prolonged pentylenetetrazol seizures reduced the brain glycogen in rats. KLEIN and OLSEN (1947) tested a series of convulsants on cats paralysed with dihydro-B-erythroidine and found a decrease in brain glycogen associated with cerebral stimulation. In a smd1 series of similar experiments on dogs under morphine, GURDJIAN, WEBSTER and STONE (1 947) found that pentylenetetrazol did not induce statistically significant changes in the glycogen level. CHANCE and YAXLEY (1950) reported findings in the mouse which are in sharp contrast with all of those quoted above. These workers studied the effects of insulin and a number of other convulsing agents, all of which appeared to increase the brain glycogen above the normal level. The conclusion was stated that an increase of glycogen in the mouse brain is specifically related to the convulsive discharge. Further data in support of this thesis were presented by CHANCE (1951, 1953) and by CHANCE and WALKER (1953-54). This discrepancy has never been resolved. Since the possibility of a species difference is apparent, further studies of brain glycogen in the mouse seemed desirable. The determination of glycogen has been improved and simplified by the application, after hydrolysis, of a specific enzymic method for glucose. The convulsants used were pentylenetetrazol, picrotoxin, and insulin. Pentobarbital was also tested in order to compare the effect of an anaesthetic with those of the excitatory agents.

Uptake of amino acids by mouse brain slices.

Abstract: AMINO acids injected into the bloodstream enter the brain only slowly (GREENBERG and WINNICK, 1948; S c m , BESWAN and WAELSCH, 1950; KAMIN and HANDLER, 1951 ; DINGMAN and Smm, 1959; RICHTER, 1959) although taken up readiy by many other tissues. Investigations using labelled methionhe (RICHTER, 1959) or glutamic acid (LAJTHA, BERL and WAELSCH, 1959) confirm that these amino acids pass from the blood into the brain slowly, but show that on entering the brain they are rapidly involved in metabolic activity. A blood-brain barrier might account for the observed facts. In metabolic experiments interference by a blood-brain barrier may be avoided by the use of brain slices. By this means the uptake of glutamic acid has been investigated in vitro by STERN, EGGLESTON, HEMS and KREBS (1949), who showed that the glutamic acid concentration in brain slices was raised above that of the medium in which the slices were suspended. More recently TSUKADA, NAGATA and HIRAMO (1960) have shown that this can also occur with y-aminobutyric acid. A number of free amino acids (MCILWAIN, 1959) including glutamic acid and y-aminobutyric acid (CRAVIOT~, MASSIEU and IZQUIERDO, 1951) are found in brain at concentrations greater than 1 m-mole/kg tissue weight, whereas in blood their concentrations are in most cases much less than that figure. However, many amino acids are present in brain at concentrations of 0.2 m-mole/kg or less (SCHURR, THOMPSON, HENDERSON, WILLUMS and ELVEHJEM, 1950), a figure not very different from their concentrations in the blood. It would be of interest to know whether the ability of brain to take up amino acids against a concentration gradient is confined only to those free amino acids present in brain at concentrations greater than 1 mmole/kg, or whether it also includes those amino acids which are present in brain at very low concentrations. Experiments have been carreid out in vitro on six of the amino acids which are present in brain in the free state at concentrations less than 0.2 m-mole/kg (including proline, lysine and methionine, whose concentrations in the brain appear to be slightly less than in the blood). They show that brain tissue is not unlike other tissues (WISEMAN, 1955; WISEMAN and GHADIALLY, 1955) in that it can take up against a concentration gradient in vitro all six amino acids investigated. The effect of different experimental conditions on the uptake of histidine by brain in vitro has also been demonstrated. Although experiments in vivo suggest that brain is different from other tissues as regards the uptake of amino acids, experiments in uitro indicate that it is basically similar and that the blood-brain bamer probably prevents the accumulation of a number of amino acids by brain in vivo.

Effect of drugs on amino acid levels in the rat brain: hypoglycemic agents

Abstract: THE use of insulin-induced hypoglycemic coma, though no longer as widespread as formerly (LAQUEUR and LA BURT, 1960) is nevertheless still successfully employed in the treatment of certain types of mental illness. The effect of this and of other hypoglycemic agents on the biochemistry of the brain is therefore of practical as well as theoretical interest. Several investigators have studied the influence of insulin on amino acid levels in the brain. DAWSON (1950) was the first to observe the fall in glutamic acid in rat brain during insulin hypoglycemia and CRAVIOTO, MASSIEU and IZQUIERO (1951) to demonstrate the considerable increase in brain aspartic acid levels that occurs under these conditions. Other workers have since confirmed these findings ( DAWSON, 1953; OKUMURA, OTSUKI and NASU, 1959; JACOBSON, 1959). It has also been shown (JACOBSON, 1959) that the increase in aspartic acid in insulin-treated rats is not due to its liberation from N-acetyl-L-aspartic acid, the presence of which in the brain was first demonstrated by TALLAN, MOORE and STEIN (1956). Other effects on brain amino acids produced by insulin are a decrease in the level of y-aminobutyric acid (GABA) and of glutamine (CRAVIOTO et al., 1951). The aim of the present study was to extend these observations. The levels of ten components in the brain, namely glutamic acid (Glu), aspartic acid (Asp), GABA, glutamine (Glu-NH,), alanine (Ala), taurine (Tau), glycine (Gly), ethanolamine (EtOH-NH,), phosphoethanolamine (P.eth), and glutathione (GSH), were determined in brains of rats treated with four different hypoglycemic agents: insulin, tolbutamide, hypoglycin A, and the ketoacid derived from hypoglycin A (2-methylenecylopropanepyruvic acid). Attention was directed to the relationship between blood sugar levels and the change in brain amino acid patterns. The role played by N-acetyl-L-aspartic acid in the rise of aspartic acid in insulin-treated rats was also studied.

Action of insulin on glucose uptake of rat brain slices and isolated rat cerebellum.

Abstract: RAT spinal cord can be isolated for incubation experiments, and insulin added in tjifro or injected in uiuo increases the glucose uptake of isolated rat spinal cord during incubation (RAFAELSEN, 1958, 1961). The spinal cord preparation was chosen as a sample of the central nervous system which could be isolated relatively intact and which, during incubation, would still be covered with natural organ membranes over the greater part of the surface. It was supposed that a direct effect of insulin in eifro was most easy to demonstrate on tissue samples which were as intact as possible and it was thought that the preservation of natural organ membranes was especially important. From these aspects the spinal cord was well suited for incubation studies, and after the demonstration of a direct effect of insulin on the uptake of glucose and some other sugars in the isolated rat spinal cord it was natural to try to demonstrate a direct effect of insulin on the glucose uptake of other isolated samples of the central nervous system. This paper consists of a description of the findings using rat brain slices and isolated rat cerebellar tissue.

The quantitative histochemical distribution of thiamine in deficient rat brain.

Abstract: THE dietary deprivation of thiamine in both humans and experimental animals results in a number of characteristic neurological manifestations, many of which can be promptly reversed by the administration of either the coenzyme (thiaminepyrophosphate) or the free vitamin (thiamine). The histopathological alterations resulting from the lack of thiamine characteristically involve certain parts of the neuraxis in a selective and bilaterally symmetrical fashion (PRICKJXT, 1934; KALM, LUCKNER and MAGUN, 1952; DREYFUS and VICTOR, 1960). Although the general histological appearance of these changes is very similar from species to species, different anatomical sites are usually affected (DREYFUS and VICTOR, 1960). In the rat, a bilaterally symmetrical area of pannecrosis is frequently observed in the vicinity of the lateral vestibular nucleus situated in the lateral pontine tegmentum. The afflicted portions of the brain thus exhibit what appears to be a selective vulnerability to the lack of the cofactor. The demonstration of a striking ‘biochemical lesion’ in avitaminous pigeon brain by PETERS (1936), the relatively restricted and localized nature of the biochemical changes (KINNERSLEY and PETERS, 1930; GAVRILESCU and PETERS, 1931; MEIKLEJOHN, PASSMORE and PETERS, 1932) and the relation of these abnormalities to the symptoms and signs of thiamine deficiency have provided the stimulus for further investigations on this subject. In an attempt to clarify further the problem of selective vulnerability, a series of investigations on various aspects of thiamine deficiency and its effects on the nervous system have been undertaken in this laboratory. As a first step, the distribution of thiamine was determined in various subdivisions of the nervous system of normal animals. It was hoped, thereby, to demonstrate differences in the vitamin content of the more vulnerable parts of the brain. Such differences would have partially explained the inherent selectivity of the pathological process. The results of the study (DREYFUS, 1959) revealed that total thiamine is evenly distributed throughout the brain with the exception of the cerebellum and the thalamus, the former containing more and the latter less vitamin than the remainder of the brain. Since no correlation could be made between tissue thiamine levels in the normal brain and the susceptibility of certain parts of the neuraxis to vitamin B, deprivation, it was decided to undertake a similar survey in animals at various stages of depletion. These experiments represent a pilot

Tryptophan metabolism in schizophrenic patients

Abstract: SEVERAL facts suggest the existence of a definite relation between the metabolism of aromatic amino acids and mental disease, and between indole compounds and schizophrenia. In recent years, much research has been carried out in the attempt to shed light on this problem. BOCOCH (1957) noted that the administration to 23 schizophrenic patients of a diet deficient in aromatic amino acids for three weeks had marked effects; in 60 per cent of the treated cases the clinical condition deteriorated, while no improvement was noted in the others; these results indicate that the diet tested not only had no therapeutic effect but was apparently detrimental. Concerning tryptophan metabolism (by the kynurenine pathway) ZELLER et al. (1957), ZELLER (1958) and LAUER et al. (1958) reported that tryptophan (50 mg/kg) administered to 24 schizophrenic patients caused an increase in xanthurenic acid excretion which was significantly higher than that in normal controls: the difference between patients and controls disappeared in 5 weeks when iproniazid (Marsilid) was administered together with tryptophan. While V. M. BUSCAINO (1957) maintained that indole derivatives play an important role in the causation of schizophrenia, G. A. BUSCAINO and STEFANACHI (1958) concluded from the analysis of the urines of schizophrenic and other patients that indole derivatives occur with varying frequencies in all cases examined and are not specifically related to schizophrenia. Tryptophan metabolism was investigated in neurological diseases by VERGA (1951), Tinelli and Calvario (1 955) and by Calvario (1958), who determined the xanthurenic acid excretion after administration of tryptophan (100 mg/kg). The greater amounts of xanthurenic acid eliminated by certain patients suggested an alteration of tryptophan metabolism, probably related to pyridoxine deficiency. Concerning the other metabolic pathway of trytophan, studies have been made of the fraction of ingested tryptophan which is metabolized via serotonin to give 5hydroxyindoleacetic acid. Although the excretion of this compound is generally normal in schizophrenic patients (BUSCAINO and STEFANACHI, 1958; FELDSTEIN et al., 1958), it can be increased by the administration of large doses of tryptophan: ZELLER el al. (1957), ZELLFX (1958) and LAUER et al. (1958) found that schizophrenic patients were unable to increase their elimination of 5-hydroxyindoleacetic acid after tryptophan ingestion while similarly treated non-psychotic controls doubled their excretion of this compound. BANERJEE and AGARWAL (1958), on the other hand, reported exactly the opposite: they found that schizophrenic patients doubled their excretion of this indole derivative while the controls did not. According to KOPIN (1959) schizophrenic patients do not differ from controls in their excretion of 5-hydroxyindoleacetic

Brain cholesterol: biosynthesis with selected precursors in vivo.

Abstract: OUR previous studies (KABARA et al., 1957,1958) on the biosynthesis and metabolism of cholesterol revealed that certain precursors were incorporated into the brain cholesterol of adult mice. Prior to these findings other workers reported low or almost undectable rates of lipid metabolism in the brain of adult animals (ROSSITER, 1957; MCILWAM et ul., 1959). More recent studies have suggested that the adult mouse brain can synthesize cholesterol in vivo when the route of precursor administration is either intracisternal (MCMILLAN et ul., 1957) or intracerebral (NICHOLAS and THOMAS, 1959). However, only a small amount of incorporation was detected when precursors were administered intraperitoneally. In our initial studies on the in vivo incorporation of precursors into brain sterol we employed the intraperitoneal route of injection. Since our data appeared to differ from the results obtained by others, a more detailed investigation of our initial findings (KABARA and OKITA, 1959) was carried out.