γ-Aminobutyric Acid Stimulates the Release of Endogenous Ascorbic Acid from Rat Striatal Tissue

Abstract: γ-Aminobutyric acid (GABA) was found to induce the release of ascorbic acid from rat striatal homogenates and minces. This release was studied with the use of a rapid supervision system with an on-line amperometric detector that monitors for the presence of easily oxidized substances (i.e., ascorbate, 3,4-dihydroxyphenylethylamine). The release was found to be calcium-independent and depolarization-dependent. This releasable pool of ascorbate could be replenished through nonstereospecific uptake. The releasing action of GABA was mimicked by the GABA agonist, muscimol, and was completely inhibited by the GABA antagonist, picrotoxin. The structural analogues of GABA, β-alanine and γ-hydroxybutyric acid, had no effect. These data indicate that ascorbate release is GABA-receptor mediated and syn-aptically localized.     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

N-Methyl-d-Aspartate Releases γ-Aminobutyric Acid from Rat Striatum In Vivo: A Microdialysis Study Using a Novel Preloading Method

Abstract: In vivo microdialysis was used in conjunction with a novel dual-label preloading method to monitor changes in extracellular levels of γ-aminobutyric acid (GABA) and glutamate due to N -methyl- d -aspartate (NMDA) infusion in the striatum of conscious, unrestrained rats. [¹⁴C]GABA and [³H]glutamate were applied in the dialysis stream for a preloading period of 30 min, after which dialysis perfusion was continued for up to 6 h and dialysate samples were collected for analysis by liquid scintillation spectrometry. NMDA (300 μ M in the dialysate) caused significant rises in both ¹⁴C and ³H content measured in the dialysates, the majority of which remained associated with the preloaded GABA and glutamate, respectively. The NMDA-evoked release of both GABA and glutamate was blocked by the specific NMDA receptor antagonist 3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP), indicating that the response was receptor mediated. The NMDA-stimulated release of glutamate was also totally abolished by concomitant application of the adenosine agonist 2-chloroadenosine or by prior frontal decortication. However, these two treatments caused little change in NMDA-evoked GABA release. These results show that NMDA causes release of GABA from the striatum in vivo by an NMDA receptor-mediated mechanism and that the majority of this release is not secondary to glutamate release from terminals of the corticostriate pathway. In addition, they confirm the results of previous studies investigating the effect of NMDA on endogenous glutamate release.     

Preloading In Vivo: A Rapid and Reliable Method for Measuring γ-Aminobutyric Acid and Glutamate Fluxes by Microdialysis

In vivo microdialysis was used in conjunction with a novel dual-label preloading method, to monitor changes in extracellular levels of gamma-aminobutyric acid (GABA) and glutamate in the striatum of conscious, unrestrained rats. [3H]GABA and [14C]glutamate were applied in the dialysis stream for a preloading period of 30 min, after which dialysis perfusion was continued for up to 6 h, and dialysate samples were collected for scintillation counting. Veratridine (Vtd: 100 microM in the dialysate) caused significant rises in both 3H and 14C content measured in the dialysates, the majority of which remained associated with the preload GABA and glutamate, respectively. The Vtd-stimulated release of GABA and glutamate measured in this way was blocked by tetrodotoxin and was Ca2+ dependent. Thus, by reproducing results obtained using other techniques, we have shown that the preloading method provides a quick and reliable method for measuring the effects of drugs on the release of neurotransmitter GABA and glutamate in vivo by dyalisis. It should enable sample times as low as 1 min to be used, thus allowing resolution of transient stimulated responses taking place over a time course of minutes.     

Modulation of Extracellular γ-Aminobutyric Acid in the Ventral Pallidum Using In Vivo Microdialysis

Intracranial microdialysis was used to investigate the origin of extracellular gamma-aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion-channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca(2+)-free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage-dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K(+)-induced release was predominantly Ca(2+)-dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K(+)- and veratridine-induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine-induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca(2+)- or Na(+)-dependent mechanisms. They also suggest that Na(+)-dependent release of GABA via reversal of the uptake carrier can be shown in vivo.     

Uptake of γ-Aminobutyric Acid by a Synaptic Vesicle Fraction Isolated from Rat Brain

Gamma-Aminobutyric acid (GABA) was taken up by a MgATP-dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and beta-alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+-ATPase.     

Release of Endogenous Taurine and γ-Aminobutyric Acid from Brain Slices from the Adult and Developing Mouse

The spontaneous and potassium-stimulated release of endogenous taurine and gamma-aminobutyric acid (GABA) from cerebral cortex and cerebellum slices from adult and developing mice was studied in a superfusion system. The spontaneous release of GABA was of the same magnitude in slices from adult and developing mice, but the spontaneous release of taurine was considerably greater in the adults. The potassium-stimulated release of GABA from cerebral cortex slices was about five times greater in adult than in 3-day-old mice, but the potassium-stimulated release of taurine was more than six times greater in 3-day-old than in adult mice. In cerebellar slices from 7-day-old mice, potassium stimulation also evoked a massive release of taurine, whereas the evoked release from slices from adult mice was rather negligible. Also in cerebellar slices the potassium-stimulated release of GABA exhibited the opposite quantitative pattern. The stimulated release of both GABA and taurine was partially calcium dependent. The results suggest that taurine may be an important regulator of excitability in the developing brain.     

Fast HPLC Estimation of γ-Aminobutyric Acid in Microdialysis Perfusates: Effect of Nipecotic and 3-Mercaptopropionic Acids

A method for rapid, automated (less than 5 min), and sensitive (detection limit 50 fmol/10 microliter) determination of gamma-aminobutyric acid (GABA) is described. The method is based on precolumn derivatization with o-phthaldialdehyde/t-butylthiol reagent and separation by reverse-phase HPLC with electrochemical detection under isocratic conditions. A 100 X 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3-mercaptopropionic acid is injected at a dose of 100 mg/kg i.p. There was a 15-fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.     

γ-Aminobutyric Acid: Temperature Dependence in Benzodiazepine Receptor Binding

The effects of muscimol and/or incubation temperature on the inhibition of [3H]flunitrazepam receptor binding by benzodiazepine receptor ligands were investigated. At 0 degree C muscimol decreased the Ki values for some ligands as displacers of [3H]flunitrazepam binding to brain-specific sites while increasing or having no effect on the Ki values for other ligands. The Ki values for some ligands are higher at 37 degrees C than at 0 degree C but are reduced by muscimol at both 0 degrees and 37 degrees C. In contrast, the ligands whose Ki values are increased by muscimol either decreased or did not alter the Ki values at 37 degrees C as compared to those at 0 degree C. Incubation of membranes at 37 degrees C for 30 min accelerated gamma-aminobutyric acid (GABA) release by 221% over that at 0 degree C. These results indicate that changes in incubation temperature alter benzodiazepine receptor affinity for ligands via GABA.     

GABAergic Growth Cones: Release of Endogenous γ-Aminobutyric Acid Precedes the Expression of Synaptic Vesicle Antigens

Growth cone fractions isolated from neonatal [postnatal day 3 (P3)] rat forebrain contain GABAergic growth cones as demonstrated by immunofluorescence staining with monospecific antibodies to gamma-aminobutyric acid (GABA). HPLC analysis shows that GABAergic growth cones release this endogenous GABA when stimulated with high K+. Endogenous GABA release is Ca2(+)-independent and, in this respect, similar to that seen previously with [3H]GABA. Isolated growth cone fractions also exhibit a K(+)-stimulated, Ca2(+)-independent release of endogenous taurine. None of the other amino acids shown to be present in isolated growth cone fractions were released, including glutamate, aspartate, and glycine. A population of dissociated cerebral cortical neurones prepared from P1 rat forebrain were GABA-immunoreactive after 1 day in culture. The cell body, neurites, and growth cones of these neurones were all stained with GABA antibodies. At this time in culture, neurones did not stain with either of two antibodies to synaptic vesicle antigens, i.e., p65 and synaptophysin. Growth cones isolated from P3 rat forebrain were also not immunoreactive with these antibodies. After about 8 days in culture, when neurones had established extensive networks of long, varicose axons and elaborately branched dendrites, many neurones and their neurites were immunoreactive for GABA antibodies. At this time in culture, p65 and synaptophysin antibodies did stain neuronal cell bodies and particularly their varicose axons. Dendrites were not stained with synaptic vesicle antibodies. These results suggest that GABAergic neurones synthesize GABA during neurite outgrowth and that GABA is present in, and can be released from, the growth cones of these neurones. The presence of GABA in GABAergic growth cones is not associated with synaptic vesicles, which explains the Ca2+ independency of both endogenous and [3H]GABA release from these growth cones.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.     

γ-Aminobutyric Acid Release from Synaptosomes Prepared from Rats Treated with Isonicotinic Acid Hydrazide and Gabaculine

The potassium-stimulated release of gamma-aminobutyric acid (GABA) from synaptosomes was determined in preparations from control rats and from rats treated with a convulsant agent [isonicotinic acid hydrazide (INH)] and an anticonvulsant agent (gabaculine). INH treatment brought about a significant decrease in Ca2+-dependent release of GABA with no effect on Ca2+-independent release, whereas gabaculine caused an increase in Ca2+-independent release with no effect on Ca2+-dependent release of GABA. Thus, the anticonvulsant action of gabaculine was not a simple reversal of the effects of INH on GABA release. The results indicate that there are at least two pools of GABA in nerve endings and support the hypothesis that exogenous GABA is taken up first into a pool that supplies GABA for Ca2+-independent release and then is transferred to a second pool (Ca2+-dependent releasable), where it mixes with newly synthesized GABA.     

Enhanced Potassium-Stimulated γ-Aminobutyric Acid Release by Astrocytes Derived from Rats with Early Hepatogenic Encephalopathy

Bulk-isolated astrocytes from rats with early hepatogenic encephalopathy (HE) induced with thioacetamide responded to the increase of potassium in the incubation medium from 5 mM to 75 mM with a markedly enhanced release of previously taken up [14C]gamma-aminobutyric acid ([14C]GABA). The process was not affected by omission of calcium and/or addition of EGTA to the incubation medium. Only a slight stimulation of GABA release by high potassium was observed in astrocytes from control rats. In contrast, histamine and histidine were vigorously released from control astrocytes in high-potassium medium, and their release was not enhanced by HE, indicating that the observed phenomenon is specific for GABA.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

γ-Aminobutyric Acid System in Developing and Degenerating Mouse Retina

Freeze-dried sections (14 microns thick) of retinal layers were prepared from mice with retinal degeneration (C3H strain) and control mice (C57BL strain). The weighed sections (2-30 ng dry weight) were analyzed using our microassay methods. In the control retina, gamma-aminobutyric acid (GABA) concentration and glutamate decarboxylase (GAD) activity, on a dry weight basis, increased from birth to 9 weeks of age and decreased slightly at 20 weeks. In the degenerated retina, the levels of GABA and GAD activity were higher at birth than in the control retina, and continued to increase until 20 weeks of age, at which time the GAD activity reached a markedly high level. This increase was found when the total GABA and GAD levels per retina were determined. In the normal retinal layers, GABA and GAD were confined primarily to the inner plexiform layer. In the degenerated retina, GAD activity gradually increased in the inner layers during postnatal development, but by 20 weeks the increase was most prominent in the inner part of inner nuclear layer and in the outer part of inner plexiform layer. GABA transaminase activity and its distribution were not much different in both normal and degenerated retinas during development.     

γ-Aminobutyric Acid Concentration in Cerebrospinal Fluid in Schizophrenia

Abstract: γ-Aminobutyric acid (GABA) concentration was determined in cerebrospinal fluid (CSF) of acute and chronic schizophrenic patients, in persons with psycho-organic or personality disorders, and in nonpsychiatric controls. The mean CSF GABA level in the chronic schizophrenic patients was found to be significantly higher than in any of the other groups. No other statistically significant differences were found. Statistical analysis revealed that the elevated CSF GABA concentration in the chronic schizophrenic patients was unlikely to be caused by medication. These results are interpreted as evidence for possible primary or secondary GABAergic overactivity in the brain in chronic schizophrenia.     

Triethyllead Inhibits γ-Aminobutyric Acid Binding to Uptake Sites in Synaptosomal Membranes

Triethyllead (TEL), the active metabolite of tetraethyllead, was shown previously to inhibit selectively high-affinity Na+-dependent uptake of gamma-aminobutyric acid (GABA) into cerebrocortical synaptosomes. Such inhibition was not related to the Na+ gradient, Na+,K+-ATPase activity, [Cl-], or energy charge. We report here that TEL inhibits GABA binding to the presynaptic transporter involved in Na+-dependent uptake. Scatchard plot analysis of Na+-dependent [3H]GABA binding to a highly purified synaptic plasma membrane preparation revealed that 25 microM TEL reduced the Bmax by 44%, leaving the KD unchanged. This binding was reversible and predominantly involved membrane uptake sites, as characterized by pharmacological specificity to GABA ligands. Approximately 85% of specific GABA binding was considered membrane uptake site binding, as indicated by sensitivity to nipecotic acid and diaminobutyric acid, with relative insensitivity to muscimol, bicuculline methiodide, baclofen, and beta-alanine. With respect to previous data, these finding suggest that TEL inhibits Na+-sensitive high-affinity GABA uptake by interfering with GABA binding to its presynaptic transporter.     

Effect of Early Iron Deficiency in Rat on the γ-Aminobutyric Acid Shunt in Brain

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in gamma-aminobutyric acid (GABA) (13%, p less than 0.01) and glutamic acid (20%, p less than 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA-transaminase, and of the NAD+-linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+-linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron-supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.