Showing papers in "Journal of Neurochemistry in 1972"

THE STRUCTURAL SPECIFICITY OF THE HIGH AFFINITY UPTAKE OF l‐GLUTAMATE AND l‐ASPARTATE BY RAT BRAIN SLICES

Abstract: — The high affinity uptake system for l-glutamate and l-aspartate in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo-3-hydroxy-dl-aspartate, l-cysteinesulphinate, l-cysteate and d-aspartate strongly inhibit the observed high affinity uptake of l-[3H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by l-glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated. l-Glutamate high affinity uptake was shown to be sodium-dependent, but under certain conditions appeared to be less sensitive than GABA uptake to changes in the external sodium ion concentration, and to drugs which modify sodium ion movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate. l-Glutamate high affinity uptake was inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.

Cerebral carbohydrate metabolism during acute hypoxia and recovery.

Abstract: — The levels of ATP, ADP, AMP and phosphocreatine, of four amino acids, and of 11 intermediates of carbohydrate metabolism in mouse brain were determined after: (1) various degrees of hypoxia; (2) hypoxia combined with anaesthesia; and (3) recovery from severe hypoxia. Glycogen decreased and lactate rose markedly in hypoxia, but levels of ATP and phosphocreatine were normal or near normal even when convulsions and respiratory collapse appeared imminent. During 30 s of complete ischaemia (decapitation) the decline in cerebral ATP and phosphocreatine and the increase in AMP was less in mice previously rendered hypoxic than in control mice. From the changes we calculated that the metabolic rate had decreased by 15 per cent or more during 30 min of hypoxia. Hypoxia was also associated with decreases of cerebral 6-phosphogluconate and aspartate, and increases in alanine, γ-aminobutyrate, α-ketoglutarate, malate, pyruvate, and the lactate :pyruvate ratio. Following recovery in air (10 min), increases were observed in glucose (200 per cent), glucose-6-phosphate, phosphocreatine and citrate, and there was a fall in fructose-1, 6-diphosphale. Similar measurements were made in samples from cerebral cortex, cerebellum, midbrain and medulla. Severe hypoxia produced significant increases in lactate and decreases in glycogen in all areas; γ-aminobutyrate levels increased in cerebral cortex and brain stem, but not in cerebellum. No significant changes occurred in ATP and only in cerebral cortex was there a significant fall in phosphocreatine. Phosphocreatine, ATP and glycogen were determined by quantitative histochemical methods in four areas of medulla oblongata, including the physiological respiratory centre of the ventromedial portion. After hypoxia, ATP was unchanged throughout and the changes (decreases) in phosphocreatine and glycogen were principally confined to dorsal medulla, notably the lateral zone. Thus there is no evidence that respiratory failure is caused by a ‘power’ failure in the respiratory centre. It is suggested that in extremis a protective mechanism may cause neurons to cease firing before high-energy phosphate stores have been exhausted.

Effects of immobilization and food deprivation on rat brain tryptophan metabolism.

Abstract: — Withdrawal of food or immobilization both led to changes in rat brain tryptophan metabolism. Brain tryptophan and 5-hydroxyindolylacetic acid concentrations both increased while changes in 5-hydroxytryptamine were much smaller. Changes were greater upon withdrawal of food. The brain tryptophan change did not appear merely to reflect an overall increase of brain amino acid concentrations, brain tyrosine concentration being only slightly increased by food withdrawal and significantly decreased upon immobilization. Plasma tryptophan did not increase. The changes in brain indole metabolism were not abolished by adrenalectomy. Results are discussed in relation to the regulation of brain serotonin metabolism.

Distribution and properties of angiotensin converting enzyme of rat brain

Abstract: — Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue.

Abstract: — Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [14C]- or [3H]-labelled S-adenosyl-l-methionine. In a double-isotopic form of the assay, a tracer of [3H]histamine is employed along with [14C]S-adenosyl-l-methionine, and the ratio [14C]:[3H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [3H]S-adenosyl-l-methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [14C]methylhistamine with [14C]S-adenosyl-l-methionine serving as the methyl donor.

The effect of moderate and marked hypercapnia upon the energy state and upon the cytoplasmic nadh/nad+ ratio of the rat brain

Abstract: — The energy state of brain tissue was evaluated from the tissue concentrations of ATP, ADP and AMP and the cytoplasmic NADH/NAD+ ratio from the tissue, CSF and blood concentrations of lactate and pyruvate, and from the intracellular pH′, in rats exposed to carbon dioxide concentrations of 640 per cent. The hypercapnia had no significant effect on the energy state of the tissue. Hypercapnia of increasing severity gave rise to a progressive decrease in the pyruvate concentration; the lactate concentration fell at low CO2 concentrations, but no further decrease was observed at CO2 concentrations greater than 20 per cent. There was a progressive rise in the intracellular lactate/pyruvate ratio at increasing CO2 concentrations, corresponding to the fall in intracellular pH, i.e. the calculated NADH/NAD+ ratios remained normal. It is therefore concluded that hypercapnia does not affect the cytoplasmic redox state.

Isolation and some chemical properties of oligodendroglia from calf brain.

Abstract: — The method of Norton and Poduslo (1970) for isolating brain cells has been adapted for the isolation of oligodendroglia from the white matter of calf brain. The cells were obtained in greater than 90 per cent purity, and in a yield of 11 × 106 cells/g of white matter. This number of cells represented a recovery of 11 per cent of the total cells in the tissue and therefore a considerably higher recovery of the original number of oligodendroglia. The average cell contained 5, 2 pg of DNA, 2–0 pg of RNA and 6, 7 pg of lipid. The lipid comprised cholesterol, galactolipid (both cerebroside and sulphatide) and phospholipid in the molar ratio of 1:0, 45:2, 3. Gangliosides were present in a concentration similar to that found in isolated rat neurons, The myelin-specific enzyme, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, was present at a level nearly equal to that in myelin, and eight-fold higher than the levels in rat neurons or astrocytes. The isolated oligodendroglia differed considerably from isolated astrocytes in size, morphology and chemical composition.

Tyrosine hydroxylase in rat brain: developmental characteristics.

Abstract: — The development of tyrosine hydroxylase (tyrosine 3-hydroxylase, EC 1.14.3.a) activity has been examined in whole rat brain and in various regions and subcellular fractions thereof. The specific activity of tyrosine hydroxylase increased almost 15-fold from 15 days of gestation to adulthood. With maturation, those regions of the brain that contain only terminals of the catecholaminergic neurons showed the greatest increases in enzyme activity. There was a shift in the subcellular distribution of tyrosine hydroxylase from the soluble fraction in the fetal brain to the synaptosomal fraction in the adult brain. Tyrosine hydroxylase, dopamine hydroxylase (EC 1.14.2.1) and the specific uptake mechanism for norepinephrine appear to develop in a coordinated fashion.

THE EFFECT OF HYPERCAPNIA UPON INTRACELLULAR pH IN THE BRAIN, EVALUATED BY THE BICARBONATE‐ CARBONIC ACID METHOD AND FROM THE CREATINE PHOSPHOKINASE EQUILIBRIUM

Abstract: — Abstract-Intracellular pH in the brain was evaluated by the bicarbonate-carbonic acid method and from the creatine phosphokinase equilibrium, in rats exposed to 6–40 % CO2 for 45 min. There was a very good agreement between the two methods, indicating that the creatine phosphokinase equilibrium in vivo shows the pH dependence predicted from previous in vitro studies. The stepwise increase in the tissue CO2 tension from 45 to 265 mm Hg resulted in a lowering of the intracellular pH from 7.04 to 6.68. The regulation of intracellular pH in hypercapnia was far better than that which can be predicted from physicochemical buffering alone, and calculations indicate that the intracellular buffer base concentration increased by more than 10 mequiv./kg at the maximal Pco2 values encountered.

N-acetyl-L-aspartic acid content of human neural tumours and bovine peripheral nervous tissues.

Abstract: — Abstract-Tumors of the human nervous system were utilized to investigate the cellular distribution of N-acetyl-L-aspartic acid (NAA). Astroglial tumours contained about 0.144 μmol/g. Oligodendrogliomas and medulloblastomas contained somewhat larger amounts. However, the level of NAA in all gliomas studied was less than that of normal human white matter. NAA was undetectable in meningiomas and acoustic neurinomas. If these results may be taken as representative of normal tissue, they imply a predominantly neuronal localization for NAA. Substantial amounts of NAA were found in peripheral nervous tissues and retina. Neurons seem to vary widely in NAA content.

Dopamine- -hydroxylase in the rat brain: developmental characteristics.

Abstract: — A sensitive and specific assay for dopamine-8-hydroxylase (DBH) in the rat brain has been developed. The enzyme in the brain has requirements for cofactors and affinity for substrate similar to DBH in the adrenal medulla. DBH activity was demonstrable in the brain of the fetal rat at 15 days of gestation; there was an increase in DBH activity with maturation that preceded and paralleled the rise in levels of endogenous norepinephrine until 3 weeks after birth. There was a shift in the distribution of total DBH activity from the caudal to the rostral regions of the brain during development. In the adult brain, DBH was highly localized in the nerve terminals. Between 17 days of gestation and adult-hood, there was 2300-fold increase in the DBH activity that sedimented with sheared-off nerve terminals.

Metabolism of beds of mammalian cortical synaptosomes: response to depolarizing influences.

Abstract: — The metabolic properties of synaptosome beds (deposits positioned between nylon gauzes) were studied. They respired, glycolysed, produced ATP and phosphocreatine, and metabolized [U-14C]glucose to glutamate, aspartate, alanine and GABA at similar rates to synaptosome suspensions. Metabolic inhibitors caused massive loss of amino acids from the beds. Synaptosome beds also responded metabolically to electrical pulses; respiration and lactate production increasing by 40 per cent. Differential release of glutamate, aspartate and GABA occurred during electrical stimulation, maximum release being after 10–15 min of stimulation. This differential release also occurred when medium potassium was increased. Omitting and chelating calcium reduced or abolished this response with both forms of stimulation. Including amino acid analogues (β-aminobutyric acid, α, γ-diaminobutyric acid and N-acetyl glutamic acid) in the incubation medium changed the patterns of amino acids present in the medium, indicating that under normal conditions active amino acid uptake processes are occurring in synaptosomes. Tetrodotoxin and ouabain also interfered with amino acid release without greatly affecting the response to stimulation. Cerebral cortex slices incubated between gauzes also showed a glycolytic response to electrical stimulation. GABA was the only amino acid showing a significant increase in the amount released with both potassium and electrical stimulation of the slices.

Partial characterization of a new myelin protein component.

Abstract: A new protein component has been demonstrated in myelin isolated from rat whole brain and from white matter dissected from bovine, dog and rabbit brain. It is also present in rabbit optic nerve. It does not appear to be present in other subcellular fractions of rat brain. It has a molecular weight of 20,540 ± 490(S.D.), as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. A rapid procedure for the isolation of myelin is also described.

The accumulation of arachidonate and docosahexaenoate in the developing rat brain.

Abstract: —Fatty acids typical of grey matter lipids (C20:4 and C22:6) and of myelin lipids (C20:1 and C24:1) were estimated in developing rat brains The polyenoic fatty acids (C20:4 and C22:6) are synthesized from the essential fatty acids (C18:2 and C18:3) The results showed that more than 50 per cent of the adult content of the brain polyenoic acids were laid down by day 15 In contrast, the fatty acids characteristic of myelin lipids did not appear in significant quantities until after this time These findings distinguish biochemically the different periods of brain development associated firstly with cell division (formation of neurons and glial cells) and secondly with myelination It is of special interest that the period of cell proliferation is accompanied by the appearance in brain lipids of long-chain polyenoic acids derived from the essential fatty acids

Effects of corticosteroids on the biochemical maturation of rat brain: postnatal cell formation.

Abstract: (1) Treatment with cortisol acetate (0.2 mg daily during the first 4 days after birth) reduced the rate of growth in the rat: at 35 days of age the body weight was reduced by 50 per cent and the brain weight, depending on the region, by up to 30 per cent. (2) In the brain the normal increase in cell number was severely inhibited during the period of cortisol treatment; this resulted in a final deficit in cell number of about 20 per cent in the cerebrum and 30 per cent in the cerebellum. (3) To determine whether cortisol affected primarily cell formation or cell destruction the labelling of brain DNA was studied 1 h after a subcutaneous injection of 20 Ci/100 g [2-14C]thymidine. In the controls the amount of labelled DNA increased by a factor of two in the cerebrum and seven in the cerebellum during the period 2-13 days, and it decreased to 40 and 27 per cent of the peak values in the cerebrum and cerebellum respectively in the following 7 days. The results indicated that mitotic activity is higher in the cerebellum than in the cerebrum in the 2nd week of life. It would appear that in the cerebrum appreciable cell death accompanies new cell formation, especially during the period 13-35 days of age. (4) Cortisol treatment affected cell division rather than cell destruction in the brain since it strongly inhibited the incorporation of [2-14C]thymidine into DNA. The inhibition was severe during the period of treatment but it did not result in a lasting fall in mitotic activity. At the age of 13 days the amount of labelled DNA formed approached the normal level and it was twice that in controls at 20 days, indicating a tendency for compensating cell deficit by an accelerated mitotic activity. Nevertheless, massive cell proliferation ceased at about the same age as in normals; the labelling of DNA decreased markedly between 13 and 20 days after birth, and the DNA content did not increase after the age of 20 days. (5) In contrast to the marked effect on cell number, cortisol treatment did not influence significantly the maturational changes related to average cell size (DNA concentration) or the chemical composition of cells (RNA/DNA and protein/DNA).

The effect of hypercapnic acidosis upon some glycolytic and Krebs cycle-associated intermediates in the rat brain.

Abstract: — In order to study the influence of intracellular pH on the carbohydrate metabolism of brain tissue, the concentrations of glucose, glucose-6-phosphate, pyruvate, lactate, citrate, α-oxoglutarate, malate, glutamate, aspartate and ammonia were measured in rats exposed to 6–40% CO2, for 45 min. Hypercapnia of increasing severity gave rise to progressive increases in the concentrations of glucose, glucose-6-phosphate and ammonium ion and to progressive decreases in the concentrations of all metabolic acids measured. The results fit with aH+ inhibition of a rate-limiting step between glucose-6-phosphate and pyruvate, and by inference from the results published by others it may be assumed that this step is the phosphofructokinase reaction. Since the proportionally largest decrease occurred in a α-oxoglutarate, the results might be compatible either with an inhibition of a second rate-limiting step such as isocitrate dehydrogenase, or with a loss of α-oxoglutarate through carboxylation to citrate.

Changes in the protein composition of mouse brain myelin during development.

Abstract: — Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.

Uptake and release of taurine from rat brain slices.

Abstract: — Rapid efflux of [35S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca2+ content of the perfusion medium was replaced with Mg2+. Uptake of [35S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [35S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed.

Histamine formation in rat brain in vivo: effects of histidine loads.

Abstract: — Administration of l-histidine at the rate of 500 mg/kg induced an increase of nearly 50 per cent in the level of histamine in rat brain which lasted several hours. The augmentation of histamine level was not significant 3 h after lower doses or after d-histidine α-methyl DOPA and Ro 4-4602 neither affected the cerebral level of histamine nor its elevation induced by l-histidine. Brocresine, a known histidine decarboxylase inhibitor not only prevented the effect of histidine load but also induced a prompt fall in the amine level. These results confirm those from earlier experiments in vitro indicating that histamine synthesis in rat brain depends on a specific decarboxylase (EC 4, 1.1.22) which is not normally saturated by the endogenous level of its substrate. When histamine levels were enhanced by histidine treatment, histidine decarboxylase activity, as evaluated on hypothalamus homogenates, was significantly reduced; intracisternal administration of cycloheximide, an inhibitor of protein synthesis, had similar effects. On the other hand, enzyme activity was not altered by the addition of histamine to hypothalamus homogenates. These results are compatible with the existence of a regulation mechanism of histidine decarboxylase involving repression by its end-product.

Effects of 5,6-dihydroxytryptamine on monoaminergic neurones in the central nervous system of the rat.

Abstract: —The injection of 50 μg of 5,6-dihydroxytryptamine (5,6-HT) into a lateral ventricle of the rat depleted the spinal cord and various regions of the brain of indoleamines (presumably 5-HT) and 5-hydroxyindole acetic acid. The concentrations of 5-HT were measured by two different methods: the formation of a fluorescent derivative with o-phthalaldehyde, and the native fluorescence in hydrochloric acid. When the results of both methods were compared on the pons and medulla 4 days after injecting 5,6-HT, the loss in indoleamine appeared to be greater when o-phthalaldehyde was used. This suggests that the two methods may be measuring different compounds. According to both methods, the loss of 5-HT persisted for several days after the injection of 5,6-HT, but by 2 months 5-HT concentrations (measured only by the native fluorescence procedure), had recovered to near-normal values. The depletion of 5-HT was most pronounced in regions adjacent to the ventricular system and in the spinal cord. Initially, caudate and septum were more affected on the side of the injection, and later showed some permanent atrophy. The injection of up to 50 μg of 5,6-HT did not lead to any significant loss of noradrenaline or dopamine from the brain, or to any reduction in the activity of the enzyme tyrosine hydroxylase. The drug was a potent inhibitor of the uptake of [3H]5-HT by brain slices, but was less effective in inhibiting catecholamine uptake systems. These observations suggest a preferential action on tryptaminergic neurones. Larger doses of 5,6-HT caused a loss of catecholamines and tyrosine hydroxylase from the brain, and were severely toxic.

Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: combined biochemical and eiistochemical observations

Abstract: — This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically-defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of ‘normal’ white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter-white matter distant from plaqueclose white matter-edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base-line of thin-layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

Development of a circadian rhythm in the activity of pineal serotonin N-acetyltransferase.

Abstract: — Pineal serotonin N-acetyltransferase (EC 2.3.1.5) is a neurally regulated enzyme. It is detectable in the rat as early as 4 days prior to birth. A circadian rhythm in enzyme activity appears on the fourth day after birth. It develops most rapidly during the second week and achieves an adult magnitude by the end of the third week at which time nocturnal values are more than 30-fold greater than daytime values. Norepinephrine, which appears to be the neurotransmitter regulating this enzyme, can cause a 2- to 3-fold stimulation of N-acetyltransferase in organ cultures of pineal glands from 4-day-old animals and a 17-fold increase in the activity of glands from 15-day-old animals. Apparently the norepinephrinesensitive system controlling pineal N-acetyltransferase activity also develops most rapidly during the first few weeks of life. The circadian rhythm in the activity of serotonin N-acetyltransferase develops in the pineal glands of both male and female rats at the same rate. A similar rhythm for the enzyme was not observed in twelve other tissues of the rat.

Zinc in maturing rat brain: hippocampal concentration and localization.

Abstract: —Alterations in the localization and concentration of zinc in the hippocampus and in other regions of rat brain were studied during postnatal maturation. Histochemical observations indicated increasing levels of zinc in the hippocampal mossy fibre layer at about 20 days of age. Between 18 and 22 days, hippocampal levels of zinc increased by 35 per cent to reach adult levels (121 ng/mg of protein). After subcellular fractionation, more than one-half of the hippocampal zinc was found in the first pellet (P1; 1085 g– 10 min), where large boutons would be expected. Autoradiographic evidence, revealing the sequential location of positron tracks first in the granular cell layer and later in the mossy fibre layer, raises the possibility of axoplasmic transport of zinc from granule cell perikarya to their terminal boutons. Our data suggest that zinc may be involved in the maturation and function of the mossy fibre pathway.

Acetylcholine compartments in stimulated electric organ of Torpedo marmorata

Abstract: Resume— L'acetylcholine ‘libre’ et ‘liee’ a ete mesuree avant et apres stimulation de l'organe electrique de la Torpille in vivo ou in vitro, sur des tranches de tissu incubees dans une solution physiologique. Des potentiels de plaque miniatures peuvent etre enregistres. Des deharges d'une amplitude atteignant 30 V ont ete obtenues apres stimulation du tissu. La reponse electrique decroit rapidement lors de stimulations repetitives. L'acetylcholine ‘libre’ est celle qui est hydrolysee en cours d'homogeneisation par les esterases du tissu, l'acetyl-choline ‘liee’ ne l'est pas. Dans l'homogenat, cette derniere est associee aux vesicules synaptiques. L'acetylcholine ‘libre’ est mesuree par la difference entre l'acetylcholine totale et l'acetylcholine ‘liee’. Une baisse d'acetylcholine ‘libre’ est mise en evidence lorsque la reponse electrophysiologique est epuisee par la stimulation. La chute d'acetylcholine ‘libre’ est, comme l'amplitude de la deharge, dependante du rapport Ca2+/Mg2+. Dans d'autres experiences une diminution des deux compartiments a ete notee a la fin de la periode de stimulation. Cependant nous avons aussi observe successivement la chute de l'acetylcholine ‘libre’ puis celle de l'acetylcholine ‘liee’ lorsque la stimulation est poursuivie. En presence d'inhibiteurs d'esterase on peut recueillir de l'acetylcholine liberee par stimulation dans la solution physiologique. Une preincubation des tissus avec de la [14C]choline permet de marquer les compartiments d'acetylcholine. La radioactivite speifique varie dans le compartiment ‘libre’ apres une serie de stimulations, alors qu'elle reste stable dans le compartiment ‘lie’. Ces resultats indiquent que l'acetylcholine ‘libre’ represente un compartiment immediatement disponible, libere quand on stimule le tissu. Abstract— Changes in ‘free’ and ‘bound’ acetylcholine before and after stimulation have been investigated in vivo and in slices of electric organ of Torpedo marmorata incubated or superfused with physiological saline solutions. Spontaneous miniature end-plate potentials could be recorded and on electrical stimulation discharges of up to 30 V could be elicited. The electrical response fell off rapidly on repetitive stimulation. ‘Bound’ acetylcholine is that which relhains after the tissue has been homogenized since any ‘free’ acetylcholine is hydrolysed by the esterases when the tissue is disrupted. ‘Free’ acetylcholine can therefore be determined as the difference between the total acetylcholine found when the tissue is extracted with trichloroacetic acid and that which remains when the tissue is homogenized. Most of the ‘bound’ acetylcholine is present in synaptic vesicles. Stimulation of the tissue until the electrical response had fallen was accompanied by a drop in the level of ‘free’ acetylcholine. Lowered calcium and increased magnesium concentrations in the medium caused a decrease in the electrical response to stimulation and a decrease in the fall of ‘free’ acetylcholine. In other experiments, a decrease of both compartments was noticed at the end of the stimulation period. However the drop in ‘bound’ acetylcholine could also be elicited after the ‘free’ had fallen, by continuing the stimulation. When anticholinesterases were put in the medium, acetylcholine released on stimulation could be collected. On pre-incubation of the slice with [14C]choline, the acetylcholine stores became labelled. The specific radioactivity of the ‘free’ acetylcholine fluctuated on serial stimulations, whereas the specific radioactivity of the ‘bound’ acetylcholine remained stable under these experimental conditions. It is concluded that the ‘free’ compartment of acetylcholine is the most immediately available for release on stimulation.

Occurrence and Distribution of Aromatic L-amino Acid (L-DOPA) Decarboxylase in the Human Brain

Abstract: —The enzymatic decarboxylation of l-DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating 14CO2 evolved from tracer amounts of d l-DOPA[carboxy1-14C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO2 as a reaction product. The enzyme preparation behaved like an aromatic l-amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d-DOPA[carboxy1-14C]. Within the human brain, l-DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l-DOPA decarboxylase activity was not significantly above blank values. The activity of l-DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.

The presence of 2',3'-cyclic AMP 3'-phosphohydrolase in glial cells in tissue culture.

Abstract: — The Enzyme 2′, 3′-cyclic AMP 3′-phosphohydrolase (CNP) is regarded as a marker for myelin (KURI- HARA and MANDEL, 1970) on the basis of its regional and subcellular distribution (Kurihara and Tsukada, 1967), its ontogenetic characteristics (KURIHARA and TSUKADA, 1968), and its behaviour in two strains of myelin-deficient mutant mice (Kurihara, Nussbaum and Mandel, 1969). However we have isolated highly-purified preparations of neuronal plasma membrane from rat brain synaptosomes which contain this enzyme activity (Morgan, Wolfe, Mandel and Gombos, 1971). Two explanations of this finding are possible. The activity could be due to the presence of myelin, but this explanation is ruled out by electron microscopy and by the low level of cerebrosides in the synaptosomal plasma membrane preparations. Myelin is extremely rich in cerebrosides (norton and Autilio, 1966). The second possibility is that the enzyme, 2′, 3′-cyclic AMP 3′-phosphohyrolase, may also be found in the glial cells from which myelin is derived (Bunge, Bunge and Pappas, 1962). To test our hypothesis that 2′, 3′-cyclic AMP 3′-phosphohydrolase is not a specific marker for myelin, but is also found, in glial cells, we have examined a tumoral glial cell line maintained in myelin-free tissue culture.

Ornithine decarboxylase activity in developing rat brain.

Abstract: —Total ornithine decarboxylase (ODC) (EC 4.1.1.17) activity per rat brain was elevated markedly from 14 days after conception to 12 days postnatum. ODC activity in the brainstem was very low and changed little during postnatal development. Activity in the cerebral hemispheres declined from a high level at birth to the low adult level by 8 days postnatum. Conversely activity in the cerebellum increased markedly from 3 days until 11 days postnatum, then suddenly decreased. Hence, the periods of greatest ODC activity paralleled those of maximal cell proliferation in each brain region. During perinatal brain development ODC activity changed considerably; it declined at about one day prior to term, and then increased rapidly to its highest level of activity at 4 h postnatum. Premature birth by caesarian section or lack of maternal care and nutrition did not affect this early postnatal response. The postnatal burst in ODC activity appears to be unique for brain tissue, since this response did not occur in heart, skeletal muscle or liver. Data from studies in which portions of fractions characterized by high or low enzymatic activity, respectively, were mixed or in which the supernatant enzyme fraction was dialysed are not consistent with the presence of direct inhibitors or activators of the enzyme. In addition, administration of cycloheximide to newborn rats abolished the 4-h postnatal burst in ODC activity. Our results suggest that the increase in ODC activity reflects enzyme synthesis de novo.

Fast axonal transport in vitro in the sciatic system of the frog

Abstract: — An in vitro system from the frog has been used to study fast axonal protein transport. The preparation, which was incubated in a specially made chamber, consisted of the gastrocnemius muscle, the sciatic nerve, the dorsal ganglia and part of the spinal cord. The parts were separated from each other by silicone grease barriers, which made it possible to follow the migration of labelled proteins from the spinal cord and ganglia, along the sciatic nerve, towards the muscle. About 80 per cent of transported proteins in the sciatic nerve originated from the dorsal spinal ganglia and moved antidromically at a rate of 60–90 mm per day at 18°C. The rapidly transported proteins were 90 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. The effects of cyclohexirnide showed that the synthesis of rapidly moving proteins and their transport were separate processes. A low concentration of colchicine inhibited the transport when it was present in the medium surrounding the ganglia, but had no effect even at a higher concentration, when it was added to the nerve compartment. The presence of vinblastine at a low concentration in either of the two compartments completely arrested the protein transport. Likewise N-ethylmaleimide or p-chloromercuribenzene sulphonic acid in the nerve medium effectively blocked the fast transport. Results from experiments performed to test the possibility of disto-proximal flow and of transfer of proteins from the muscle to the nerve are discussed.

A technique for measuring brain protein synthesis.

Abstract: — Mice were infused intravenously for varying periods of time with L-[U-14C]- tyrosine. The specific activity of free tyrosine in the blood and the brain, and of protein-bound tyrosine in the brain, was measured and the mean rate of protein synthesis calculated. The half-life of mixed brain proteins was found to be close to 4 days with infusions lasting 0.5–2 h. The origin of the intracellular tyrosine pool was investigated and it was shown that 60 per cent of this was derived directly from the plasma tyrosine and 40 per cent from protein breakdown within the tissue.

Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibres.

Abstract: — Acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) is carried down mammalian nerve fibres by the fast axoplasmic transport system. This conclusion was derived from experiments involving the ligation of cat sciatic nerves at two sites placed 83.5 mm apart. The enzyme accumulated in segments of nerve proximal to the upper ligation in a linear fashion over a period of at least 20 h. At approximately 5 h the accumulation of enzyme ceased in the nerve segment proximal to the distal ligation within the isolated length of nerve, an observation indicating that the portion of AChE free to move within the isolated nerve had been depleted during this period of time. The freely moving fraction of AChE was estimated to be 15% of the total enzyme activity present in the nerve (10% in the proximo-distal direction and 5% in the retrograde direction). The rate of AChE downflow (as estimated from the intercept of the curve plotting accumulation with the line denoting when depletion started) was 431 mm/day within a 95% confidence interval of 357–543 mm/day. In view of the variability, our results demonstrated that AChE was being carried by the fast axoplasmic transport system, which in earlier studies was estimated to have a characteristic rate close to 410 mm/day. An accumulation of AChE was also found on the distal side of the ligations that represented a movement of AChE in the distal-proximal direction in the fibres. This retrograde transport was smaller in amount (about one-half) than the proximo-distal rate of transport, or close to 220 mm/day. The rate of AChE transport was discussed in relation to the ‘transport filament’ hypothesis of fast axoplasmic transport.