THE GAMMA-AMINOBUTYRIC ACID (GABA) SYSTEM IN BRAIN DURING ACUTE AND CHRONIC ETHANOL INTOXICATION

Abstract— The effects of acute and chronic ethanol intoxication on the GAGA system of rats have been investigated. Under the terminal conditions provoked. by ethanol (6–8 g/kg, i.p.) the brain GABA content sharply increased. There was a simultaneous decrease of 35–40% in the glutamate decarboxylase (GAD) activity of the cerebellum and cerebral hemispheres. In contrast, the transaminase, GABA-T was either unchanged, or it increased: by 28% only in cerebellum and by 1.5–2.0–fold in liver and kidney. It is suggested that effects of acute ethanol intoxication at different doses (2–8 g/kg) on the brain GABA system is connected with the phases of the functional condition of the CNS and a disturbance of homeostatic function. Chronic ethanol consumption caused a decrease in brain GABA. an increase of GAD activity in cerebellum and cerebral hemispheres, and no change in GABA-T activity. The activity of this last enzyme was increased 1.5–2.0-fold in liver and kidneys of rats consuming a diet containing 10% ethanol daily. A 50-fold purified preparation of GABA-T obtained from pig brain was inhibited by butanol-l and propanol-1 (0.03–0.6m) with no effect of ethanol. It is suggested that the mechanisms involved in the ethanol effect on nervous cells are linked with the GABA system and the phases of the functional condition of the CNS.     

Antibodies to catecholamines and serotonin during alcoholic intoxication in animals with different predispositions to the development of experimental alcoholism

Experiments on C57Bl/6, CBA and DBA/2 mice characterized by different preferences for ethanol have shown that during chronic administration of alcohol to animals with natural ethanol motivation (strain C57Bl/6) the level of antibodies to catecholamines and serotonin was increased on the 3rd month of ethanol intoxication, with the voluntary alcohol consumption in mice decreased by this time. On the contrary in mice rejecting alcohol (strains DBA/2, CBA) no antibodies to catecholamines and serotonin have been found.     

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.     

THE PROPORTIONATE LOSS OF l-3,4-DIHYDROXYPHENYLALANINE AND l-5-HYDROXYTRYPTOPHAN DECARBOXYLATING ACTIVITY IN RAT CENTRAL NERVOUS SYSTEM FOLLOWING INTRACISTERNAL ADMINISTRATION OF 5,6-DIHYDROXYTRYPTAMINE OR 6-HYDROXYDOPAMINE

—5,6-Dihydroxytryptamine or 6-hydroxydopamine was administered intracisternally to rats to effect a selective destruction of serotonin or catecholamine-containing neurons. The l -DOPA and l -5-hydroxytryptophan decarboxylating activities of the spinal cord and brain were then determined at several time intervals following this treatment. In both cases the relative loss of l -DOPA decarboxylating activity was the same as the relative loss of l -5-hydroxytryptophan decarboxylating activity. 5,6-Dihydroxytryptamine treatment had little or no effect on catecholamine-containing neurons and 6-hydroxydopamine did not effect serotonin-containing neurons. These data support the idea that only one decarboxylase is involved in the biosynthesis of both serotonin and catecholamines in the rat CNS.     

CHEMICAL SPECIFICITY OF DOPAMINE TRANSPORT IN THE NIGRO-NEOSTRIATAL PROJECTION

Axoplasmic transport of dopamine in the nigro-neostriatal system has previously been shown by the specific accumulation of labelled dopamine in the striatum following injections of labelled DOPA or dopamine into the substantia nigra. To test the specificity, 17 different labelled materials (pipecolic acid, inulin, taurine, GABA, glycine, histidine, histamine, serotonin, 5-HTP, D-amphetamine, 3-methoxytyramine, dopamine, tyramine, norepinephrine, octopamine and high and low specificity activity DOPA) were injected into the substantia nigra and the distribution of radioactivity in the brain studied after 6 and 24 h. Only the catecholamines and octopamine gave evidence of specific accumulation in the ipsilateral striatum although some of the other compounds caused diffuse labelling of the striatum along with other brain areas.     

Participation of cholinergic mechanisms in realizing the effects of bicuculline]

Screening and electrophysiological methods were applied to the verification of the hypothesis on a possibility of participation of cholinergic structures in the realization of bicucullin effects. M- and N-cholinolytics (benactizine, atropine, aprophen, and pediphen) failed to arrest the convulsions induced in mice by bicucullin adminstration. At the same time substances inducing accumulation of gamma-aminobutyric acid (GABA) in the brain, i.e. aminooxycetic acid and depakin produced a manifest protective action in convulsions caused by bicucullin administration. In electrophysiological experiments there was also revealed an incapacity of M-cholinolytic benaltizine to arrest the bicucullin effects. Bicucullin proved to diminish depression of the test response in the restoration cycle of the primary response of the rat sensory motor cortex at the intervals of 40--125ms between the stimuli, whereas benactizine decreased the late facilitation of the test response at the intervals of 150--300ms between the stimuli. There was also noted no interaction between benactizine and bicucullin by this test. On the basis of these data a conclusion was drawn that bicucullin effects were caused by the block of postsynaptic GABA receptors, and were not connected with the cholinergic structures activity.     

Biphasic effect of acute ethanol administration on rat liver tyrosine-2-oxoglutarate aminotransferase activity.

1. Acute ethanol administration causes a biphasic change in rat liver tyrosine aminotransferase activity. 2. The initial decrease is significant with a 200 mg/kg dose of ethanol, is prevented by adrenoceptor-blocking agnets and by reserpine, but not by inhibitors of ethanol metabolism, and exhibits many of the characteristics of the inhibition caused by noradrenaline. 3. The subsequent enhancement of the enzyme activity by ethanol is not associated with stabilization of the enzyme, but is sensitive to actinomycin D and cycloheximide. 4. It is suggested that the initial decrease in aminotransferase activity is caused by the release of catecholamines, whereas the subsequent enhancement may be related to the release of glucocorticoids.     

Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.     

Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism.

The localization of alcohol dehydrogenase (ADH) in brain regions would demonstrate active ethanol metabolism in brain during alcohol consumption, which would be a new basis to explain the effects of ethanol in the central nervous system. Tissue sections from several regions of adult rat brain were examined by in situ hybridization to detect the expression of genes encoding ADH1 and ADH4, enzymes highly active with ethanol and retinol. ADH1 mRNA was found in the granular and Purkinje cell layers of cerebellum, in the pyramidal and granule cells of the hippocampal formation and in some cell types of cerebral cortex. ADH4 expression was detected in the Purkinje cells, in the pyramidal and granule cells of the hippocampal formation and in the pyramidal cells of cerebral cortex. High levels of ADH1 and ADH4 mRNAs were detected in the CNS epithelial and vascular tissues: leptomeninges, choroid plexus, ependymocytes of ventricle walls, and endothelium of brain vessels. Histochemical methods detected ADH activity in rodent cerebellar slices, while Western-blot analysis showed ADH4 protein in homogenates from several brain regions. In consequence, small but significant levels of ethanol metabolism can take place in distinct areas of the CNS following alcohol consumption, which could be related to brain damage caused by a local accumulation of acetaldehyde. Moreover, the involvement of ADH in the synthesis of retinoic acid suggests a role for the enzyme in the regulation of adult brain functions. The impairment of retinol oxidation by competitive inhibition of ADH in the presence of ethanol may be an additional origin of CNS abnormalities caused by ethanol.     

Ethanol and other CNS depressants decrease GABA synthesis in mouse cerebral cortex and cerebellum in vivo

GABA synthesis in mouse brain $\text{in vivo}$ was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation using the selective and irreversible antagonism of GABA-transaminase by gabaculine. Using this method we found that acute and repeated ethanol administration lead to a potent depression of gabaculine induced enhancement of GABA levels in mouse brain cerebellum and cerebral cortex. Alcohol, in the absence of gabaculine had no effect on steady state GABA levels. These results demonstrate potent effects of ethanol on the dynamics of GABA metabolism which are compatible with a GABA like effect of ethanol.     

Different effects of monoamine oxidase inhibition on MPTP depletion of heart and brain catecholamines in mice

Pargyline, an inhibitor of monoamine oxidase type B (MAO-B), did not prevent the depletion of heart norepinephrine 24 hr after a single dose of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice. In mice killed 24 hr after the last of 4 daily doses of MPTP, the depletion of dopamine in the striatum and of norepinephrine in the frontal cortex was completely prevented by pargyline, but the depletion of heart norepinephrine was not prevented. These results with pargyline are the same as results obtained earlier with deprenyl, another selective inhibitor of MAO-B. The doses of pargyline and of deprenyl that were used resulted in almost complete inhibition of MAO-B activity (phenylethylamine as substrate) in brain, heart and liver of mice. Deprenyl did not inhibit MAO-A activity (serotonin as substrate) in brain, but pargyline caused some inhibition of MAO-A in brain. In heart and liver, serotonin was oxidized only at about 1/10 the rate of phenylethylamine oxidation, suggesting that MAO-B predominates in these tissues. Both pargyline and deprenyl caused some inhibition of serotonin deamination in heart and liver, suggesting that the oxidation may have been due partly to MAO-B. Experiments with selective MAO inhibitors in vitro showed that only about 20% of the oxidation of serotonin was occurring via MAO-B in heart and liver. The in vitro oxidation of MPTP by MAO in mouse brain, heart and liver was almost completely inhibited by pretreatment with either pargyline or deprenyl. Neither pargyline nor deprenyl had any significant effect on the concentrations of MPTP in brain or heart one-half hr after injection of MPTP into mice. The concentrations of the metabolite, MPP+ (1-methyl-4-phenyl-pyridinium), were markedly reduced in brain and in heart by pretreatment with either pargyline or deprenyl. The data suggest that MPP+ formation, which is necessary for the depletion of brain catecholamines after MPTP injection, may not be necessary for depletion of norepinephrine in heart. Since the oxidation of MPTP in vitro was inhibited more by pargyline or deprenyl pretreatment than was the appearance of MPP+ in vivo, the possibility exists that some MPP+ formation might occur by an enzyme other than MAO.     

Neurotransmitter Metabolism in Rat Brain Synaptosomes: Effect of Anoxia and pH

Synaptosomes isolated from the rat cerebral cortex by means of a discontinuous Ficoll gradient carry out net, sodium-dependent, veratridine-sensitive accumulation of gamma-aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine. The intrasynaptosomal contents of the four neurotransmitters are: 30.4 nmol/mg protein, 17.4 pmol/mg protein, 13.5 pmol/mg protein, and 21.2 pmol/mg protein, respectively. Anaerobic preincubation of synaptosomes causes an irreversible decrease in the rates of neurotransmitter accumulation but does not affect the rates of their release. The inhibitory effect of anaerobiosis is enhanced by increased concentration of [H+] (decreased pH) in the medium. The most sensitive is the uptake of dopamine, the least that of serotonin. The rates of neurotransmitter efflux are unaffected by anaerobiosis. Synaptosomes leak catecholamines, GABA, and serotonin into the medium when subjected to anaerobiosis, and reintroduction of oxygen is accompanied by a rapid reaccumulation of all four neurotransmitters. It is concluded that: (1) Responses of synaptosomes to anaerobiosis are remarkably similar to the behavior of intact brain in hypoxia and ischemia. (2) Neurotransmitter uptake systems are more sensitive to short periods of anaerobiosis than either the energy metabolism or ion transport. (3) Some neurotransmitter uptake systems are more easily damaged by anaerobiosis than others.     

Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication

The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O₂ ^– radicals; this may account for some nervous system disorders during alcohol intoxication.     

Modulation of insulin receptors and catecholamines in rat brain in hyperglycemia and hyperinsulinemia

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat whole brain membranes, synaptosomes and choroid plexus in alloxan induced short term and long term hyperglycemia and hyperinsulinemia. Insulin receptor activity was measured by [125I]insulin binding and catecholamines by high performance liquid chromatography with electrochemical detection. While choroid plexus insulin receptors modulate along with norepinephrine, dopamine and serotonin with the changes in insulin and/or plasma glucose levels, insulin receptor activity in synaptosomes and total membranes is not affected to a great extent except in long term hyperglycemia.     

Methyl parathion induced alterations in monoaminergic system of developing rat pups

Methyl parathion induced alterations in the level of monoamines, viz. norepinephrine, dopamine and serotonin were studied in discrete regions of developing central nervous system of rat pups. A significant decrease in the level of monoamines noticed in methyl parathion toxicosis may be related to the altered neuronal activity and inefficiency, leading to depression and impairment in various behavioural activities. In contrast to AChE inhibition, monoamine oxidase (MAO) activity showed an increasing trend and it could cause deamination of catecholamines and accumulation of its metabolites. This suggests that an increased AChE inhibition may indirectly stimulate MAO activity in developing rat pups exposed to methyl parathion.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

Modulation of insulin receptors and catecholamines in rat brain in hyperthyroidism and hypothyroidism

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat brain using Triton X-100 extracts from total membranes, synaptosomes and choroid plexus in experimental hypothyroidism and hyperthyroidism. Insulin receptor activity was assessed by binding to [125I]insulin and catecholamines by high performance liquid chromatography. In choroid plexus thyronines effects are well pronounced and there is modulation vis a vis plasma hormone concentrations. Triiodothyronine levels increase in brain in all experimental groups. This suggests that rat may serve as a useful model for thyronine homeostasis in brain and there may be involvement of very complex regulatory mechanisms in glucose tolerance.     

Effect of serotonin on smooth muscle]

Serotonin caused contracion of the smooth muscles of the deferent duct and of the strip of rat stomach, acting upon the D-serotonin receptors. Only a small portion of its contractile effect (about 10--14%) was caused by the release of endogenous catecholamines. The action of serotonin on the strip of the rat stomach was accompanied by an increase of its entrance into the cells of Ca45 and Na22 isotopes. The concentration turn of the concentration-effect curve of serotonin on the stomach strip was connected with the accumulation of sodium ions in the cells.     

Effect of ethanol on the concentration of serotonin in the brain of the rat and the excretion of 5-hydroxyindoleacetic acid

It is found that serotonin content in the brain areas and heart of rats with low alcohol motivation decreases after 5 months of chronic consumption of 48% ethanol solution in a dose of 4 g/kg; in animals with high alcohol motivation serotonin content decreases only in the hypothalamus. Under chronic alcoholization for 1 and 12 months no considerable changes were found in serotonin level of the studied tissues. 60 min after intraperitoneal administration of 20% ethanol solution in a dose of 3 g/kg in intact animals there occurs an increase of serotonin content in the brain hemispheres and heart and its decrease in the hypothalamus; in rat with low alcohol motivation after taking ethanol for 5 months this administration evokes a decrease of serotonin content in the hypothalamus and truncus cerebri; in rats with high alcohol motivation--its decrease in the hypothalamus. Excretion of 5-oxyindoleacetic acid with urine decreases 10 months after alcohol intoxication. When rats were not given ethanol after its chronic taking for 3 months serotonin oxidation was intensified for the first day, which was not observed after 7-month alcoholization of animals.     

The effects of quipazine on serotonin metabolism in rat brain.

Quipazine, 2-(1-piperazinyl)-quinoline, is a drug that has been reported to stimulate serotonin receptors in brain. We therefore studied the effect of quipazine on several parameters of serotonin metabolism in rat brain. Quipazine caused a slight, dose-related elevation of serotonin levels and decrease in 5-hydroxyindoleacetic acid levels for 2–4 hrs after it was administered. The decrease in 5-hydroxyindoleacetic acid levels was probably due primarily to a depression of 5-hydroxyindole synthesis, since quipazine also decreased the rate of 5-hydroxytryptophan accumulation after NSD 1015, the rate of serotonin decline after α-propyldopacetamide, and the rate of 5-hydroxyindoleacetic acid accumulation after probenecid. The elevation of serotonin was probably due to weak inhibition of monoamine oxidase. Quipazine reversibly inhibited the oxidation of serotonin by rat brain monoamine oxidase $\text{in}$$\text{vitro}$ and protected against the irreversible inactivation of the enzyme $\text{in}$$\text{vivo}$. Quipazine also was a potent inhibitor of serotonin uptake into brain synaptosomes $\text{in}$$\text{vitro}$ and attained concentrations in brain higher than the $\text{in}$$\text{vitro}$ IC₅₀. However, quipazine did not prevent the depletion of brain serotonin by p-chloroamphetamine $\text{in}$$\text{vivo}$. In addition to stimulating serotonin receptors in brain, quipazine may inhibit monoamine oxidase and serotonin reuptake $\text{in}$$\text{vivo}$.