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.     

Effects of gabaculine,a new potent inhibitor of gamma-aminobutyrate transaminase,on the brain gamma-aminobutyrate content and convulsions in mice

Effects of gabaculine (5-amino cyclohexa-1,3-dienyl carboxylic acid), a new potent inhibitor of gamma-aminobutyrate (GABA) transaminase, on the brain GABA content and convulsions were studied in mice. Gabaculine (0.12–2.0 ug) injected intraventricularly elevated brain GABA content for a long period. Three hrs after treatment with gabaculine, picrotoxin- and thiosemicarbazide-induced convulsions were prevented, but not electroschock-induced convulsions.Intraperitoneal injection of gabaculine also increased brain GABA content.Thus gabaculine seems to be a useful tool for investigating the GABA function in the brain.     

The Use of Inhibitors of GABA-Transaminase for the Determination of GABA Turnover in Mouse Brain Regions: An Evaluation of Aminooxyacetic Acid and Gabaculine

Abstract: The accumulation of γ -aminobutyric acid (GABA) after inhibition of GABA-T (4-aminobutyrate: 2-oxoglutamate aminotransferase, EC 2.6.1.19) by various doses of aminooxyacetic acid (AOAA) and gabaculine was studied in four different regions of the mouse brain. The dose-response curve for GABA accumulation after treatment with AOAA was linear up to 10 mg/kg i.p., and then leveled off. The increase in GABA accumulation after gabaculine treatment was linear up to 100 mg/kg i.p. No further increase was observed with doses up to 300 mg/kg i.p. The selectivity of both GABA-T inhibitors was assessed by measuring their effects on the content of free amino acids in mouse brain. Apart from the substantial increase in the GABA concentration, there were significant decreases in the content of glutamic acid, aspartic acid, alanine and glutamine, and an increase in ornithine content after administration of gabaculine. The same changes in amino acid content were observed after treatment with AOAA, but the level of lysine was also increased and the change in alanine level was biphasic. All these changes, however, were very small compared with the large increase in GABA level. A method for estimating the rate of the GABA turnover in vivo by measuring the initial rate of GABA accumulation after administration of AOAA or gabaculine is proposed, and the validity of the two techniques is discussed. The effect of diazepam on GABA levels and on the gabaculine-induced accumulation of GABA was studied. The results obtained with diazepam show that this method can provide valuable insight into the effects of drugs on GABAergic mechanisms in vivo.     

γ-Aminobutyric Acid Turnover in Rat Striatum: Effects of Glutamate and Kainic Acid

The turnover rate of gamma-aminobutyric acid (GABA) in the rat striatum was estimated by measuring its accumulation after inhibition of GABA-transaminase (GABA-T) with gabaculine. Intrastriatal injections of 100 micrograms gabaculine induced a rapid and complete inhibition of GABA-T. GABA accumulation was linear with time for at least 60 min (estimated turnover rate = 25 nmol/mg protein/h). The accumulation of GABA after gabaculine administration in animals that had been treated with kainic acid (5 nmol intrastriatally, 7 days) was only 40% of the control value, indicating that a major fraction of the net increase in GABA content induced by gabaculine originates in kainic acid-sensitive neurons. Intrastriatal injection of a mixture of kainic acid (5 nmol) and gabaculine caused a net increase in striatal GABA content significantly greater than that observed in controls, suggesting that neuronal death induced by kainic acid is preceded by a period of increased neuronal activity. Glutamic acid, the putative neurotransmitter for the excitatory corticostriatal pathway, also produced a significant increase in striatal GABA accumulation when injected together with gabaculine. This effect was blocked by the administration of the glutamate receptor antagonist glutamic acid diethyl ester. The interactions between GABAergic neurons and other neurotransmitters present in the striatum were also analyzed.     

Correlation between alterations in brain GABA metabolism and seizure excitability following administration of GABA aminotransferase inhibitors and valproic acid—a re-evaluation

The time course of the effects of aminooxyacetic acid, γ-vinyl GABA, γ-acetylenic GABA, gabaculine, ethanolamine-O-sulphate (EOS) and valproic acid (VPA) on brain GABA content and the activities of glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T), the enzymes involved in biosynthesis and degradation of GABA, was re-determined and compared with the action on the electroconvulsive threshold in mice. All drugs caused significant increases in the seizure threshold, and the temporal pattern of this effect correlated rather well with the induced elevation of brain GABA. However, no clear relationship was found between the extent of GABA increase and the relative increase of seizure threshold. Except for VPA, the time course of the increment in brain GABA followed closely the inhibition of GABA-T. The activity of GAD was gradually decreased by γ-acetylenic GABA and a slow decline of GAD activity was also observed after γ-vinyl GABA. EOS and gabaculine suggesting a feedback repression of GAD synthesis by highly elevated GABA concentrations. Concomitant with significant reduction of GAD activity, a decrease in seizure threshold occurred though brain GABA levels remained markedly elevated. On the other hand, following administration of VPA the effect of GABA levels was paralleled by an increase in GAD activity indicating that the GABA-elevating action of this drug can be attributed at least in part to an activation of GABA synthesis. The data suggest that reduction of GAD activity may be an inevitable consequence of increasing brain GABA concentrations over a certain extent and this effect seems to limit the anticonvulsant efficacy of GABA-T inhibitors.     

Regulation of the γ-Aminobutyric AcidA Receptor by γ-Aminobutyric Acid Levels Within the Postsynaptic Cell

Synaptosomes and synaptoneurosomes were prepared from rat cerebral cortex. Comparison of the amino acid levels in the two types of organelles and of the effects of gabaculine thereon indicated that the neurosome portion of synaptoneurosomes constituted the major influencing component of the organelles. Administration to rats of inhibitors of gamma-aminobutyric acid (GABA) degradation, such as gabaculine and L-cycloserine, resulted in elevated GABA levels in synaptoneurosomes and a decrease in muscimol-stimulated Cl- up-take by the organelles. Addition of gabaculine directly to the incubation medium for the uptake assay had no effect on the Cl- transport. In contrast, administration to rats of isonicotinic acid hydrazide, an inhibitor of GABA synthesis, decreased the GABA level in synaptoneurosomes and increased the muscimol-stimulated Cl- uptake by the organelles. Although the evidence is not unequivocal, it does support the concept of GABA released from nerve endings being taken up by the postsynaptic cell, from where it exerts a regulatory influence on the functioning of the GABA receptor/ion channel complex.     

Response of the cockroach brain gamma-aminobutyric acid system to isonicotinic acid hydrazide and mercaptopropionic acid

The presence of gamma-aminobutyric acid (GABA) as well as glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) enzymes was demonstrated in the cockroach (Periplaneta americana) brain. Isonicotinic acid hydrazide (INH) in vivo (2.19 mumol/g) inhibited brain GAD activity, the inhibition lasted for about 2 hours and the normal activity levels reappeared at 4 h after INH administration. Brain GABA levels increased initially but then declined and were restored to normal levels at 4 h after INH administration. GABA-T activity was strongly inhibited by INH and a total 100% inhibition was observed at 2-3 h following INH treatment. The GABA-T activity, however, began to recover after 3 h but only 37% of the total enzyme activity was released from inhibition. Mercaptopropionic acid (MPA) in vivo (32 micrograms/g) inhibited brain GAD activity and depleted GABA level also. Results indicate that INH response of the cockroach brain GABA system is similar to that reported for the chick brain but differs from that of the mammalian brain.     

The Differential Effects of GABA-Transaminase Inactivation in the Chick Retina and Brain

Abstract: The inactivation of γ-aminobutyrate (GABA)-transaminase by the highly specific and potent neurotoxin gabaculine leads to different neurochemical consequences in the chick brain as opposed to the chick retina. In the brain, GABA levels continually climb, reaching approximately eightfold increases over control values after 24 h. The elevation in GABA levels leads to a time-dependent and coincident fall in glutamate decarboxylase and cysteine- sulfinatc decarboxylase activities, to approximately 50% of control values. On the other hand, in the retina GABA levels only increase to a plateau level two- to threcfold that of control after inactivation of GABA-transaminase. Further- more, although the glutamate decarboxylase activity decreases to about 50% of control values, cysteinesulfinate decarboxylase activity is not affected. These studies show that the processing of GABA in the retina differs from that in the brain, and that cysteinesulfinate and glutamate decarboxylase activity probably reside in different enzyme molecules in the retina, although they may reside in the same enzyme in the brain.     

The Inactivation of γ-Aminobutyric Acid Transaminase in Dissociated Neuronal Cultures from Spinal Cord

Abstract: It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter γ-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10⁻⁶ M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.     

Gabuculine and isogabaculine: in vivo biochemistry and pharmacology in mice.

Two irreversible enzyme-activated GABA-transaminase inhibitors, gabaculine (5-amino cyclohex-1, 3-dienyl carboxylic acid) and an isomer, isogabaculine (3-amino cyclohex-1, 5-dienyl carboxylic acid), were investigated in mice for their effects on brain GABA metabolism and on seizures induced by a variety of stimuli. Biochemical and pharmacological activities of the two inhibitors were very similar. Both produce dose- and time-related, sustained inhibition of GABA-T activity and, to a lesser extent, of GAD activity and long-lasting increases in brain GABA concentrations. Both protect mice against audiogenic seizures and significantly decrease the frequency of seizures induced by isoniazid, thiosemicarbazide and pentylenetetrazol. They do not affect the frequency of seizures induced by strychnine, bicuculline or picrotoxin and do not alter the threshold to electroconvulsive shock. Although the effects of gabaculine and isogabaculine on brain GABA metabolism resemble those of other GABA-T inhibitors, important differences in pharmacological activities exist.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

Interrelationships between ornithine,glutamate, and GABA. II. Consequences of inhibition of GABA-T and ornithine aminotransferase in brain

The objective of the present study was to compare the effects of elevation of GABA concentration and those of inactivation ofl-ornithine: 2-oxoacid aminotransferase (OAT) on the in vivo metabolism ofl-ornithine (Orn) in brain. Vigabatrin (4-aminohex-5-enoic acid) and gabaculine (5-amino-1,3-cyclohexadienyl carboxylic acid), two well known inactivators of GABA-T, were used to elevate brain GABA concentrations. The latter inactivates OAT also. Transamination of Orn is, from a quantitative point of view, a significant reaction in mouse brain. GABA is a feed-back regulator of OAT. Within GABAergic neurons Orn concentration may be regulated by endogenous GABA. Extensive inactivation of OAT causes a considerable increase of Orn concentration, both in synaptosomes and in non-synaptosomal compartments. The results are compatible with a role of Orn as precursor of glutamate and/or GABA in certain neurons.     

Combined Effects of a Metabolic Inhibitor (Gabaculine) and an Uptake Inhibitor (Ketamine) on the γ-Aminobutyrate System in Mouse Brain

Abstract: The intramuscular administration of a γ-aminobutyrate-α-oxoglutarate aminotransferase (GABA-T) inhibitor, gabaculine, to mice resulted in significant increases in GABA content and decreases in the content of aspartate, glutamate, and glutamine in the nerve endings (synaptosomes). These effects were ameliorated by the concurrent administration of the GABA uptake inhibitor ketamine. A major cause of these effects was the gabaculine-induced inhibition of GABA-T activity and the lessening of this inhibition by ketamine. The latter phenomenon was not due to a direct action of ketamine on the enzyme, nor to an interaction between gabaculine and ketamine. Rather, it appeared that ketamine might be interfering with the transport of gabaculine into the cellular structures. The anticonvulsant action of the GABA-T inhibitor and the GABA uptake inhibitor together was little different from that of the GABA-T inhibitor alone.     

Invivo inactivation of γ-aminobutyric acid-α-ketoglutarate transaminase by 4-amino-5-fluoropentanoic acid

Intraperitoneal injection into mice of varying concentrations of (S)-4-amino-5-fluoropentanoic acid ((S)-AFPA) produces a dose-dependent irreversible decrease in brain γ-aminobutyric acid-α-ketoglutaric acid aminotransferase (E.C. 2.6.1.19) activity. Concomitant with this inactivation is an increase in whole brain γ-aminobutyric acid (GABA) levels. Four hours after a dose of 100 mg/kg body weight of (S)-AFPA to mice, endogenous brain GABA concentrations increase to 16 times that of the untreated animals and the enzyme activity decreases to 20% that of the controls. The binding of (S)-AFPA to GABA receptors was more than three orders of magnitude poorer than for GABA itself.     

REDUCTION OF LEVEL OF L-GLUTAMIC ACID DECARBOXYLASE BY γ-AMINOBUTYRIC ACID IN MOUSE BRAIN1

Abstract— The effects of accumulated endogenous GABA on the activity of L-glutamic acid decarboxylase (GAD) were studied in mouse brain. When the content of GABA in the brain was increased after administration in vivo of aminooxyacetic acid (AOAA), there was a reduction of GAD activity which could not be reversed by the addition of pyridoxal-5′-phosphate (PLP). Since inhibition of GAD activity by AOAA could be readily reversed by PLP, the reduction of GAD activity measured in the presence of added PLP indicated a decrease in the level of GAD apoenzyme. Similarly, increase of GABA content by hydrazine was also accompanied by a reduction in the level of GAD. Thiosemicarbazide and hydroxylamine did not affect the content of GABA appreciably, and in both cases levels of GAD remained unchanged when measured in the presence of added PLP. The correlation of the reduction in the levels of GAD with the increases in content of GABA suggests that GABA may regulate its own synthesizing enzyme by feedback repression.     

EFFECT OF DRUGS ON RAT BRAIN, CEREBROSPINAL FLUID AND BLOOD GABA CONTENT

Acute administration of GABA transaminase inhibitors to rats results in a dose-dependent increase in both brain and blood GABA content and administration of isonicotinic acid hydrazide (INH), at a dose which decreases the amount of brain GABA, also lowers blood levels of this amino acid. Chronic treatment (10 days) with INH (20mg/kg), y-acetylenic-GABA (10 mg/kg) or aminooxyacetic acid (AOAA) (10 mg/kg) results in a significant elevation in both rat brain and blood GABA concentrations. At the doses studied, only AOAA caused a significant elevation in CSF GABA content. Co-administration of pyridoxal phosphate (2 mg/kg) blocks the chronic INH-induced rise in blood GABA but does not affect the increase in brain content of this amino acid. Chronic administration of di-n-propylacetate (20 mg/kg) did not significantly alter brain, blood or CSF GABA levels. The results suggest that, under the proper conditions, changes in blood GABA levels after administration of inhibitors of GABA synthesis or degradation may be an indirect indicator of changes in the brain content of this amino acid. Blood GABA determinations may be useful for studying the biochemical effectiveness of GABA transaminase inhibitors in man.     

Effects of Creatine on Synthesis and Release of γ-[3H]Aminobutyric Acid

Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of gamma-[3H]aminobutyric acid ([3H]GABA) as synthesized from [3H]glutamine or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA-alpha-oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitter GABA.     

Serotonin and γ-Aminobutyric Acid Turnover After Injection into the Median Raphe of Substance P and D-Ala-Met-Enkephalin Amide

The raphe nuclei [which contain serotonin (5-HT) cell bodies] are also known to contain axons that store substance P, met-enkephalin, and gamma-aminobutyric acid (GABA). We have previously shown that GABA has a tonic inhibitory action on 5-HT turnover. To examine other possible interactions of these neuronal systems, we assessed the effect on 5-HT turnover of injecting substance P and 2-D-ala-met-enkephalin into the median raphe nucleus, and the effects of substance P on GABA turnover. Serotonin turnover was increased by 30% in the hippocampus after the injection of substance P (4 micrograms) into the median raphe, indicating an excitatory effect of substance P on the raphe-hippocampal system. Local injection of the metabolically stable metenkephalin analog 2-D-ala-met-enkephalin amide (10 micrograms) increased the hippocampal steady state content of 5-hydroxyindoleacetic acid (5-HIAA) by 60%. The data suggest an excitatory effect of met-enkephalin within the raphe nucleus. We attempted to estimate GABA turnover from the rate of disappearance of GABA after inhibition of glutamic acid decarboxylase by isoniazid and by the rate of accumulation of GABA after inhibition of GABA transaminase by gabaculine. Isoniazid, which is a competitive inhibitor, had too short and incomplete an action to be of use when injected intranuclearly. Gabaculine, which is an irreversible inhibitor, induced a rapid-onset increase in GABA content. This accumulation was linear up to 90 min. The injection fo gabaculine (80 ng) into the raphe increased GABA content by five times the control values, but hippocampal 5-HT and 5-HIAA contents were not significantly changed. Substance P injection increased the GABA turnover by 30%. Gabaculine seems a promising tool for detecting changes in GABA turnover.     

Effects of the Antidepressant/Antipanic Drug Phenelzine on Alanine and Alanine Transaminase in Rat Brain

1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T).2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase.3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner).4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine.5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.     

The γ-Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ-Aminobutyrate-Elevating Agents: A Possible Role in Anticonvulsant Activity

Abstract: The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the γ-aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.