Acute and short term effects of ethanol on the metabolism of glutamic acid and GABA in rat brain

Although alcoholic intoxication is attributed to its pharmacological effects on the cell membranes in brain, the rapid metabolic utilisation of the same alters the metabolism of brain affecting the metabolism of glutamate and GABA which have varied metabolic roles besides serving a major proportion of synaptic activity. A study on the effects of ethanol, both acute and short-term, on glutamate (glu) and GABA metabolism in various regions of rat brain was carried out. Increased activities of glutamic acid decarboxylase (GAD) and aspartic acid aminotransferase (AST) in all brain regions, but decreased activity of glutamic acid dehydrogenase (GDH) in cerebral cortex (CC) and cerebellum (CB) following ethanol administration in brain was observed. Differential effects of ethanol were also obtained on the contents of glu and aspartate (asp), which were increased in CC, CB, and brain stem (BS) regions, as opposed to GABA content, which, although found to increase in acute toxicity, showed a decrease in all of the above brain regions in short-term toxicity. It is concluded that the above changes in glu, asp and GABA represent the consequences of metabolic utilization of alcohol in the brain, probably more a state of cerebral excitation than depression, and the changes may be a compensatory phenomenon.     

Regional distribution of homocarnosine and other ninhydrin-positive substances in brains of malnourished monkeys

—Eight male monkeys (Macaca nemestrina) aged 6–9 months were divided into two groups and fed either an adequate protein diet (20% casein) or a protein deficient diet (2% casein). After 3- 5 months of receiving the low protein diet, the malnourished monkeys showed extensive fatty metamorphosis of the liver cells, distorted patterns of plasma and hepatic free amino acid pools, and other features consistent with the diagnosis of protein-calorie malnutrition. Examination of the cerebrum, cerebellum and brain stem in the malnourished animals revealed profound accumulation of 3-methylhistidine, histidine and homocarnosine in all three regions. For histidine, the cerebral, cerebellar and brain stem levels in the protein deficient animals increased by 145, 104 and 101 per cent over levels observed in corresponding regions of the brain in well-fed monkeys. Similarly, there were significant elevations in homocarnosine contents of the cerebrum (+ 99 per cent), cerebellum (+ 140 per cent) and brain stem (+ 146 per cent) in comparison to levels in control animals. In contrast, the levels of valine, serine and aspartic acid were markedly reduced in all three brain areas in the malnourished animals. Protein-calorie deficiency also produced reductions in the brain levels of taurine, glutamic acid, isoleucine, leucine and threonine which varied in magnitude in the three major regions of the brain examined. These biochemical alterations which may in part underlie some of the psychomotor changes often observed in protein-calorie malnutrition, were discussed not only in relation to the role of amino acids as precursors for the synthesis of neuroregulatory substances but also with due regard to the possibility that some of these ninhydrin-positive substances such as GABA, homocarnosine, glycine and the dicarboxylic amino acids may possess neuroexcitatory or inhibitory properties in various parts of the central nervous system.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.     

Free brain amino acids in rats variously tolerant to alcohol

Free amino acid concentrations were studied in the right and left hemispheres, cerebellum and brain stem of rat strains with different tolerance to ethanol (AT and ANT rats). The differences found may be significant in mechanisms of metabolic and neurogenic tolerance to alcohol. Within each of the rat strains, the distribution of some amino acids and their relationships markedly differed in the brain regions investigated.     

Immuno-detection of aluminium and aluminium induced conformational changes in calmodulin - implications in Alzheimer's disease

Studies were conducted to ascertain any involvement of free radical mediated prooxidative processes in different brain regions following diazopam administration. A significant decrease in TBA reactive substance formation was observed in cerebral cortex, cerebellum and brain stem regions after single doses of 1.5, 3 and 6 mg/kg b.wt. For further studies rats were given diazepam (i.p.) at 3 mg/kg body weight dose and sacrificed after 1 h to follow changes in the pro/antioxidant status. An enhancement in the TBARS formation was found in the mitochondrial fractions from cerebral cortex and brain stem. This effect was highest in brain stem being 107% as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a decrease in activity which was region dependent. Even though, total thiols were not significantly altered, free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Glutathione reductase activity was also decreased in cerebellum and brain stem. The results indicate that a single dose of diazepam causes free radical mediated changes and the modulatory response of antioxidant defences appears to be region specific.     

Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell

Free amino acids and cholinergic enzymes were investigated in the cerebellum of reeler and weaver mice in an attempt to identify the neurotransmitter characteristic of the granule cell population and to clarify any neurotransmitter abnormalities of their pre- and postsynaptic neurons induced by their depletion. The data indicate that glutamic acid may be the neurotransmitter of the granule cells. Pre- and postsynaptic neurotransmitter activity seemed not to be markedly altered in cerebellar granule cell dysgenesis.     

The effect of caffeine on some mouse brain free amino acid levels

Changes in free amino acids were examined in the central nervous system of mice treated with caffeine for three weeks. Caffeine was administered in the drinking water, and at the end of three weeks the level of caffeine in the cerebral cortex was 113±19 g/g. When amino acid levels in cerebral hemispheres, midbrain, pons and medulla, and cerebellum were measured a significant increase in glutamine levels was found in all four regions. Glycine, alanine, serine, threonine, and GABA were significantly reduced in some regions. Caffeine appears to alter some of the metabolic or transport processes regulating amino acid pools in the brain. The decrease of GABA found in pons and medulla may contribute to the observed increase in reflex excitability after caffeine.Special issue dedicated to Dr. Elling Kvamme     

POSTNATAL CHANGES IN FREE AMINO ACID,DNA, RNA AND PROTEIN CONCENTRATIONS OF MINIATURE SWINE BRAIN1

Postnatal developmental characteristics of miniature swine brain were evaluated through the first 9 weeks of age. Differential growth rates of cerebrum, cerebellum and brain stem were defined in terms of DNA, RNA, protein and free amino acid concentrations at ages 5, 21, 35 and 63 days. Within the experimental conditions provided, hyperplasia ceased just prior to ages 21, 35 and 63 days for cerebellum, brain stem and cerebrum, respectively. An additional cerebral growth spurt, observed between weaning at age 35 days and sacrifice at age 63 days, may be indicative of impaired brain development due to inadequate nutrition provided by the dam's milk. Developmental changes in mean concentrations of brain free amino acids varied with anatomical area and differed somewhat from those of other species previously reported. For example, mean cerebral concentrations of aspartic acid, γ-aminobutyric acid and asparagine + glutamine decreased significantly (P < 0·05) with age and mean glutamic acid concentration was 5 times that of taurine.     

Regional and subcellular distribution of enzymes of branched-chain amino acid metabolism in brains of normal and diabetic rats

Branched-chain-amino-acid:alpha-ketoglutarate transaminase and branched-chain alpha-ketoacid dehydrogenase have been assayed in brains of control and of streptozotocin-induced diabetic rats. Enzyme activities were measured in five distinct regions of the brain: cerebellum, pons + medulla, midbrain, thalamus + hypothalamus, and telencephalon. Subcellular distribution of these enzymes in whole brain was assessed by fractionating brain homogenate into cytoplasm, free mitochondria, and synaptosomes. The following enzymes were used as markers: lactate dehydrogenase for cytoplasm, glutamate dehydrogenase for mitochondria, and glutamate decarboxylase for synaptosomes. The activity of the branched-chain amino acid transaminase in all brain regions was considerably higher than that of the branched-chain alpha-ketoacid dehydrogenase. While the highest activity of the transaminase occurred in brain-stem regions, the highest activity of the dehydrogenase was present in cerebellum and telencephalon. Diabetes did not affect the activity of the transaminase, but it caused a decrease in the total activity of the dehydrogenase in midbrain and in thalamus + hypothalamus. The transaminase was localized in the cytoplasmic fraction of whole brain, while the dehydrogenase was enriched in the free mitochondria.     

Changes in Free Amino Acid Levels in Developing Human Foetal Brain Regions

The levels of free amino acids were determined in human foetal brain regions during prenatal development. Variation in the distribution of amino acids and their rate of change in five segments of the CNS at different stages of ontogeny was observed. Striking developmental changes were found in the levels of aspartic acid in medulla-pons and spinal cord, glycine in the spinal cord, gamma-aminobutyric acid in the cerebral cortex, glutamic acid in the cerebral cortex, midbrain, and spinal cord, and taurine in the medulla-pons and spinal cord. At a late gestational period, glutamic acid was found most abundantly over all the brain regions, whereas the level of taurine was highest at an early gestational stage but not in spinal cord.     

Characterization and subcellular distribution of specific thyrotropin-releasing hormone binding sites in rat cerebellum

The specific binding of thyrotropin-releasing hormone (TRH) by 30,000g pellet fraction was ubiquitously distributed throughout various rat brain regions including cerebellum. Although the cerebellum had the lowest apparent density of specific TRH binding sites found in any of the brain regions studied, it represented a single class of high afinity receptor (K _D=37.73±4.88 nM,B _max=156.0±5.7 fmol/mg protein,n=4). Furthermore, the cerebellar synaptic plasma membrane fractions were richly endowed with TRH-binding, two other membrane fractions (light-synaptic plasma membrane and microsomal) exhibited high TRH-binding whereas nuclear, mitochondrial or myelin fractions were devoid of significant binding activity. These data show for the first time the existence of specific TRH-binding in cerebellum, and thus suggest that TRH may modulate cerebellar synaptic functions by acting through a specific high affinity-receptor.     

Transmembrane topography and evolutionary conservation of synaptophysin

Synaptophysin is the major integral membrane protein of small synaptic vesicles. Its primary structure deduced from rat and human complementary DNA sequences predicts that synaptophysin contains four transmembrane regions and a carboxyl-terminal domain having a novel repetitive structure. To elucidate the transmembrane organization of this protein in the synaptic vesicle, five antipeptide antibodies were raised. The site-specific antibodies were used to map the cognate sequences to the cytoplasmic or intravesicular side of the synaptic vesicle membrane by determining the susceptibility of the epitopes to proteolysis. The results confirm a topographic model for synaptophysin in which the protein spans the vesicle membrane four times, with both the amino and carboxyl terminus being cytoplasmic. In addition, the evolutionary conservation of the synaptophysin domains was addressed as a function of their membrane localization. To this end the primary structure of bovine synaptophysin was determined. Sequence comparisons between bovine, rat, and human synaptophysin revealed that only the intravesicular loops showed a significant number of amino acid substitutions (22%), while the transmembrane regions and cytoplasmic sequences were highly conserved (3% substitutions). These results depict synaptophysin as a protein with multiple membrane spanning regions whose functional site is likely to reside in highly conserved intramembranous and cytoplasmic sequences.     

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.     

Changes in Regional Brain Levels of Amino Acid Putative Neurotransmitters After Prolonged Treatment with t he Anticonvulsant Drugs Diphenylhydantoin, Phenobarbitone, Sodium Valproate, Ethosuximide, and Sulthiame in the Rat

The effect of prolonged treatment (10 days) with the anticonvulsant drugs diphenylhydantoin (DPH), phenobarbitone, sodium valproate, ethosuximide and sulthiame, both singly and in combination, on regional rat brain amino acid neurotransmitter concentrations (GABA, glutamate, aspartate and taurine) were assessed. DPH had a major effect in the cerebellum and hypothalamus in that it significantly reduced cerebellar GABA, taurine and aspartate and hypothalamic GABA and aspartate. Sodium valproate significantly elevated GABA and taurine in most regions. Aspartate and glutamate were less affected. Phenobarbitone significantly elevated GABA concentrations in all brain regions, while taurine concentration was only elevated in the cerebral cortex. Ethosuximide induced changes were small compared to the other anticonvulsants while sulthiame produced complex changes. Anticonvulsant drugs administered in combination resulted in complex changes, suggesting that their mode of action is different.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Expression of neuronal plasticity markers in hypoglycemia induced brain injury

The expression of neuroplasticity markers was analyzed in four brain regions, namely cerebral hemispheres (CH), cerebellum (CB), brain stem (BS) and diencephalon (DC) from insulin-induced hypoglycemic young adult rats. Significant decrease in neural cell adhesion molecule (NCAM) isoforms and growth-associated protein-43 (GAP-43) was observed following hypoglycemic injury from majority of brain regions studied. The glial fibrillary acidic protein (GFAP) level increased significantly in cerebral hemispheres and diencephalon regions, whereas, synaptophysin level increased in cerebellum, brain stem and diencephalon regions. The selective downregulation of the neuronal plasticity marker proteins (GAP-43 and NCAM), and enhanced expression of GFAP and synaptophysin suggests that in acute hypoglycemia, mechanisms other than energy failure may also contribute to neuronal cell damage in the brain.     

PHYSICO-CHEMICAL CHARACTERISTICS AND REGIONAL DISTRIBUTION STUDIES OF GP-350, A SOLUBLE SIALOGLYCOPROTEIN FROM BRAIN

The apparent molecular weight of GP-350, a sialoglycoprotein from calf and rat brain, has been determined by SDS-polyacrylamide gel electrophoresis. The electrophoretic mobility corresponds to the mobility of a polypeptide with a molecular weight of 11,600 ± 200. On this basis it can be calculated that only one sialic acid residue is present/GP-350 molecule. From isoelectric focusing experiments it appeared that the isoelectric point of GP-350 is about 2. The determination of the amide content of the polypeptide chain showed that out of 22.0 acidic amino acid residues of glutamic acid and aspartic acid, only 4.9 residues are amidated. The total amount of the basic amino acid residues lysine and arginine, is 6.5. So, per molecule GP-350 10.6 acidic amino acid residues are not counteracted by basic amino acid residues. The surplus of the acidic amino acid residues as well as sialic acid result in the pronounced acidic character of GP-350. This fact is supported by the electrophoretic experiments. The carbohydrate-polypeptide linkage type has been studied by alkaline sodium borohydride treatment. Two thirds of all the galactosamine was destroyed, whereas the amount of glucosamine remained the same. Amino acid analysis indicated a decrease in serine and threonine with a concomitant small increase in alanine. These data point to the occurrence of linkages between the carbohydrate chain and the polypeptide core of the galactosamineserine or –threonine type. Per molecule GP-350 about two residues of galactosamine are destroyed, indicating that two carbohydrate chains of this binding type are present. Only one of these chains can be terminated by a sialic acid residue. The other carbohydrate chain may be terminated by fucose. Regional distribution studies showed the presence of GP-350 in all brain areas studied; in relatively large amounts in the regions rich in ganglia such as caudate nucleus, cerebellar grey matter, pons and medulla oblongata, and in relatively small amounts in the regions poor in ganglia such as corpus callosum, cerebral white, cerebral grey and cerebellar white matter. GP-350 is also present in the pituitary gland. In the cerebrospinal fluid a glycoprotein is present with the same electrophoretic mobility as GP-350. However, this glycoprotein gave no precipitin reaction with GP-350 specific antiserum. Moreover, the amino acid composition was quite different from that of GP-350. Subcellular distribution study revealed that GP-350 is present in the soluble cell fraction and in the synaptosomal membrane fraction, whereas it is absent from the purified nuclei, mitochondria, myelin, and also from the microsomal fraction.     

σ-Receptor Regulation of [3H]Arachidonic Acid Release from Rat Neonatal Cerebellar Granule Cells in Culture

Abstract: σ receptors have been identified in many brain areas and are especially abundant in those regions known to be involved in control of movement. σ receptors have been located autoradiographically in the granule cell layer of cerebellum in adult rat brain. In the current study, we identified σ receptors in rat neonatal granule cells in culture using radioligand binding. The tritium labeled form of the putative σ antagonist haloperidol bound with high affinity to membranes prepared from these cells, and ligands selective for σ receptors competed well against [³H]haloperidol binding. The excitatory amino acid N -methyl- d -aspartate and the direct phospholipase A₂ activator melittin stimulated the release of [³H]arachidonic acid from cerebellar granule cells. The N -methyl- d -aspartate-stimulated, but not the melittin-stimulated, release was inhibited in a concentration-dependent manner by the σ-selective agonist (+)-pentazocine. In addition, the novel σ₁ agonist BD737 inhibited N -methyl- d -aspartate-stimulated release. Pentazocine inhibition was almost completely reversed by the σ antagonists NPC-16377 and opipramol. A 1 µ M concentration of the phencyclidine receptor-selective ligand MK-801 inhibited ∼65% of N -methyl- d -aspartate-stimulated release. These results suggest that σ receptors may play a role in modulating arachidonic acid release in cerebellar granule cells.     

Regulation of Five Tubulin Isotypes by Thyroid Hormone During Brain Development

Nucleic acid probes derived from the 3' noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2)- and three beta (beta 2, beta 4, and beta 5)-tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of alpha 1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The alpha 2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the beta 2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The beta 5 level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain-specific beta 4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development. This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency.     

Localization of glucose-6-phosphatase and fructose-1-6-diphosphaiase in subcellular fractions from different regions of the rat brain

Two key enzymes of gluconeogenesis, glucose-6-phosphatase and fructosp-1-6-diphosphatase, were present in the cerebral hemispheres, the cerebellum and the brain stem of the rat brain. Significant activities of these-enzymes were associated with the particulate fraction.