Dependent GABA Release Research Articles

Ca 2+-dependent release of [ 3H]GABA in cultured chick retina cells

Depolarization by K + (50 mM) of cultured chick retina cells released 1.14 ± 0.28% of the accumulated [ 3H]γ-aminobutyric acid (GABA) in the absence of Ca 2+, but when 1.0 mM Ca 2+ was present, the internal free calcium ion concentration [Ca 2+] i rose by about 750 nM and the [ 3H]GABA release about doubled to a value of 2.22 ± 0.2% of the total [ 3H]GABA. Nitrendipine (0.1 μM), a blocker of the L-type Ca 2+ channels, blocked the [Ca 2+] i response to K + depolarization by about 65%, and the ω-Conotoxin GVIA (ω-CgTx) (0.5 μM), a blocker of the N-type of Ca 2+ channels, inhibited by 27% the [Ca 2+] i rise due to K + depolarization. Parallel experiments showed that nitrendipine inhibits [ 3H]GABA release to the level observed in the absence of Ca 2+, whereas ω-CgTx did not inhibit significantly the release of [ 3H]GABA. The results also show that the release of [ 3H]GABA due to K +-depolarization in the absence of Ca 2+ can be totally blocked by 1-(2-(((Diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca 2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca 2+-independent release. Thus, it is concluded that [ 3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca 2+-dependent, and by reversal of the carrier, which is Ca 2+-independent, in much the same way as has been found for other GABAergic neurons. Previous reports that cultured chick retina cells show no Ca 2+-dependent release of GABA should be re-examined in light of our results obtained with a superfusion system.

Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog d-Ala 2-Met 5-enkeph-alinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential—independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential—evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non—action potential—dependent GABA release reveals a mechanism that contributes to reducing action potential—evoked GABA release, thereby decreasing synaptic inhibition.

Calcium dependent release of γ-aminobutyric acid (GABA) from human cerebral cortex

The release of the amino acids GABA, taurine, glycine, glutamine and leucine from human neocortex was investigated in vitro by utilizing brain tissue removed during 8 standard temporal lobectomies for epilepsy or tumor. Slices (0.5 mm thick) were cut from each biopsy and randomly placed in three different chambers. After 90 min preincubation, the three sets of slices were incubated for 60 s in wells containing, respectively, (A) regular ACSF (control), (B) ACSF with 50 mM K+ (to depolarize the cell membrane) and (C) ACSF with 50 mM K+, 0 mM Ca2+ and 4 mM Mg2+ (depolarization during blocked synaptic transmission). The content of amino acids in the wells was determined by high-performance liquid chromatography after pre-column derivatization of the amino acids with o-phthalaldehyde. Membrane depolarization (well B) increased the GABA release to 650% (620 pmol/mg) of control (well A, 95 pmol/mg). Blocking synaptic transmission (well C) reduced the evoked release by 50% (360 pmol/mg). The release of glycine, taurine, glutamine and leucine during membrane depolarization was not significantly different from the control values. The data provide evidence for a Ca2+-dependent release of GABA, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Relationship between the elevation of internal free [Ca 2+], membrane potential and the Ca 2+-dependent release of GABA in synaptosomes

Relationship between the elevation of internal free [Ca 2+], membrane potential and the Ca 2+-dependent release of GABA in synaptosomes

Release of γ‐Amino[3H]butyric Acid from Cultured Amacrine‐Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.

The effect of verapamil on GABA and dopamine release does not involve voltage-sensitive calcium channels

The effect of verapamil, which belongs to the group of drugs collectively reffered to as ‘organic Ca 2+ channel blockers’, was investigated on the basal and stimulated release of the neurotransmitters dopamine and GABA in rat striatum synaptosomes. Verapamil inhibits the Na +-dependent release of GABA in response to depolarization with an IC 50 of 25 μM, whereas it is unable to modify the Na 2+-independent, Ca 2+-dependent fraction of GABA release induced by high K + depolarization. Verapamil does not modify the basal release of GABA but stimulates the basal release of dopamine in a dose-dependent manner (ED 50 5μM). This verapamil-induced outflow of dopamine is independent of Ca 2+ and occurs in the presence of tetrodotoxin, indicating that it is not mediated by voltage-sensitive Ca 2+ or Na + channels of the presynaptic membrane. Dopamine release induced by verapamil is cumulative with that induced by depolarizing agents (high K + or veratridine). As verapamil, pimozide, a neuroleptic of the diphenylbutylpiperidine type, increases the basal and stimulated release of dopamine. We conclude that the opposite effects of verapamil of GABA and dopamine release are due to differences in the releasable fractions of these 2 types of neurotransmitters. Besides, none of these effects are directly linked with the blockade of voltage-operated Ca 2+ channels of the presynaptic membrane.

Is GABA release modulated by presynaptic excitatory amino acid receptors?

The purely GABAergic nature of spontaneous synaptic activity in cultures from the neonatal rat superior colliculus (SC) is of great advantage in investigations aimed at characterizing presynaptic factors regulating GABAergic synaptic transmission. Using SC-derived cultures it was confirmed that excitatory amino acids (EAA) can induce a marked increase in the frequency of spontaneous synaptic Cl − currents (I CI(GABA)SYN). However, this tetrodotoxin-resistant facilitation of Ca 2+-dependent GABA release required application of EEA to several neurons (multiple cell superfusion). In contrast, no frequency increase of I CI(GABA)SYN was seen with restricted access of EAA to only one neuron and the presynaptic axonal terminals (single cell superfusion). It is therefore concluded that the strong facilitatory effect of glutamate (Glu) and kainate (KA) on GABAergic synaptic activity, as observed under the condition of multiple cell superfusion, is mediated via somatodendritic excitatory amino acid receptors (EAARs).

Kindling increases the K + -evoked Ca 2+ -dependent release of endogenous GABA in area CA1 of rat hippocampus

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K +) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K + was significantly increased (40–46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45–49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.

ω-Conotoxin GVIA and dihydropyridines discriminate two types of Ca 2+ channels involved in GABA release from striatal neurons in culture

We previously reported that the opening of the L-type Ca 2+ channel was only partly involved in the K +-evoked Ca 2+-dependent γ-aminobutyric acid (GABA) release from striatal neurons, suggesting that probably different types of voltage-sensitive Ca 2+ channels were implicated in this physiological process. Here we demonstrate that ω-contoxin GVIA, which has been reported to block L- and N-type neuronal Ca 2+ channels, also partly inhibits the Ca 2+-dependent GABA release. The maximal effects of ω-conotoxin GVIA and nifedipine, a highly specific antagonist of the L-type channels, were additive, a total inhibition of the Ca 2+-dependent GABA release being obtained in the presence of both drugs. We therefore propose that ω-conotoxin GVIA and nifedipine block two different types of Ca 2+ channels, both involved in the GABA release process.

Evidence for a chronic loss of inhibition in the hippocampus after kindling: biochemical studies

Brain tissue from kindled animals prepared 1 month after their last seizure was compared to tissue from matched surgical control animals. Quantitative film autoradiography was used to study muscimol binding in the CA1 region and 3 other brain areas (dentate gyrus, cerebral cortex, and thalamus). The K d values so obtained were constant from region to region and comparable to those published by others. B max values varied; of the 4 regions studied CA1 had the lowest value of B max There were no differences in either K d or B max values in any region studied between kindled and surgical control rats. The release of GABA from nerve terminals was assessed with hippocampal tissue maintained in vitro and perfused with different solutions in which the concentrations of K + and Ca 2+ were varied. This allowed the examination of K +-induced depolarization release and the Ca 2+ dependence of this process. K +-induced, Ca 2+-dependent release of GABA from hippocampus derived from kindled animals was significantly less than that from hippocampus derived from controls. The biochemical studies reported here provide additional support for the hypothesis that there is a chronic decrease in GABA-mediated inhibition in the hippocampus associated with kindling. The data point to a dysfunction at the presynaptic level, within the GABAergic interneuron, but do not exclude changes at a level postsynaptic to the GABAergic interneuron not detected with the methods employed.

Involvement of protein kinase C in the Ca 2+-dependent vesicular release of GABA from central and enteric neurons of the guinea pig

The involvement of protein kinase C (PKC) in the release of endogenous γ-aminobutyric acid (GABA) was studied using slices of deep cerebellar nucleus and strips of small intestine from the guinea pig. 12-O-tetradecanoylphorbol 13-acetate (TPA), but not 4α-phorbol-12,13-dedecanoate (4α-PDD), potentiated the high K +-evoked release of GABA from both preparations in the presence of tetrodotoxin. Ouabain evoked the release of GABA from both preparations, and this release was not altered by TPA. Therefore, the activation of protein kinase C potentiates the Ca 2+-dependent vesicular release of GABA from nerve terminals of the central and enteric GABAergic neurons of the guinea pig.

Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture

The mechanisms leading to Ca2+-dependent and Ca2+-independent GABA release were studied on highly purified striatal neurons developed in primary culture. Ca2+-dependent GABA release, which represents about 75% of the 56 mM K+ effect was totally inhibited when striatal neurons were first exposed to tetanus toxin (TnTx) (10 micrograms/ml) for 24 hr. The K+ effect was potentiated when 1 mM nipecotic acid (an inhibitor of the GABA uptake system) was added during the stimulation period or when Na+ was replaced by Li+. However, no difference in the GABA release measured under high-K+ conditions was observed after a 22 min preincubation of the neurons in a medium containing nipecotic acid or Li+. Replacement of Cl- ions by SO4(2-) did not modify K+-evoked GABA release. Ca2+-independent GABA release was stimulated by veratridine (20 microM), ouabain (3 mM), and monensin (20 microM), as well as the excitatory amino acids glutamate (100 microM), N-methyl-D-aspartate (100 microM), quisqualate (10 microM), and kainate (1 mM), drugs known to increase intracellular Na+ concentration. The veratridine- or glutamate-evoked GABA release was neither inhibited when intracellular Ca2+ content was reduced by more than 90% nor by treatment of the neurons to TnTx. However, the Ca2+-independent GABA release elicited by veratridine was inhibited by preincubation of the neurons in a medium containing 1 mM nipectotic acid and in a medium containing Li+ instead of Na+ or SO4(2-) instead of Cl-. These results strongly suggest that 2 different GABA release mechanisms exist in striatal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamines appear to be second messengers in mediating Ca 2+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes

High potassium (50 mM) depolarization induces a rapid (<15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor α-difluoromethylornithine blocked the K +-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca 2+ influx and efflux and the Ca 2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of α-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.

Dopaminergic regulation of GABA release from the intact goldfish retina

The rate of release of [ 3H]GABA from intact goldfish retinas was studied using a modified superfusion technique. Small, significant increases in the rate of GABA release were observed when the retinas were exposed to dopamine (DA) (100–1000 μM); however, when free Ca 2+ was removed from the medium, the basal rate of GABA release was increased and DA became inhibitory. Forskolin, a non-specific stimulator of adenylate cyclase in intact cells, also inhibited GABA release in the absence of Ca 2+. There was no significant effect of forskolin in the presence of Ca 2+; however, (+)-butaclamol, a dopamine antagonist, increased basal GABA release under these conditios. l-glutamic acid ( l-Glu) (1–10 mM) causes up to a 10-fold increase in GABA release. In the presence of Ca 2+, DA did not significantly alter the effects of l-Glu; however, in the absence of Ca 2+ a significant inhibition of the effects of l-Glu by DA was observed. Forskolin, on the other hand, inhibited the effects of l-Glu both in the presence and absence of Ca 2+. Finally, EGTA (0.3–1 mM) produced a large release of GABA: this release was inhibited by DA, forskolin, theophylline and 8-bromo cyclic AMP. These results suggest a model wherein DA stimulates Ca 2+-dependent GABA release from one site and inhibits Ca 2+-independent GABA release from another site via cyclic AMP-mediated event.

Monoamine, amino acid and cholinergic interactions in slices of rat cerebral cortex

Interactions of monoamine, amino acid and cholinergic transmitter systems were studied in slices of rat cerebral cortex using a superfusion procedure and measuring release of endogenous dopamine (DA), norepinephrine (NE), serotonin (5-HT), GABA, glutamate (GLU) and aspartate (ASP). Depolarizing concentrations of K + were used to induce a Ca 2+-dependent, Mg 2+-inhibited release of the monoamines and amino acids. Submaximal release of the monoamines and amino acids was observed at 35 mM K +, which permitted studies of possible excitatory or inhibitory actions of the added agents. The 35 mM K +-stimulated, Ca 2+-dependent release of GABA was inhibited 40, 30 and 25% by 100 μM NE, DA and 5-HT, respectively. The release of GLU was potentiated by NE and reduced by DA. Both DA and 5-HT inhibited the release of ASP. The Ca 2+-dependent, K +-stimulated release of endogenous NE, DA and 5-HT was not altered by 100 μM GABA, GLU or ASP. However, 100 μM GLU did enhance the stimulated release of GABA. The cholinergic agonist, carbachol, enhanced the stimulated release of NE, 5-HT and GLU 10, 60 and 40%, respectively. On the other hand, carbachol attenuated the release of DA and GABA approximately 20%. One interpretation of the data is that the amino acid transmitter pathways in slices of the cerebral cortex of the rat can be controlled by monoaminergic and cholinergic systems while the monoamine afferents appear to have a cholinergic regulation but not a major direct amino acid transmitter influence.

In vitro release of endogenous monoamines and amino acids from several CNS regions of the rat.

The in vitro release of endogenous norepinephrine (NE), dopamine (DA), serotonin (5-HT), GABA, glutamate (GLU), aspartate (ASP), glycine (GLY), taurine (TAU) and alanine (ALA) from superfused slices of cerebral cortex (CTX), striatum (STR), hippocampus (HIP), hypothalamus (HYPO), midbrain (MB), thalamus (THAL), nucleus accumbens (ACC), pons-medulla (PM) and spinal cord (SC) was studied. Under resting conditions or with 60 mM K+ in the absence of Ca2+, there was little or no release of NE, DA, 5-HT, GABA, GLU or ASP from any region. In most regions, there was a measurable resting release of ALA, GLY and TAU; of these three amino acids, only GLY in the PM and SC showed an increased release in the 60 mM K+ plus 2.5 mM Ca2+ medium. In 8 of the regions studied, the release of both GABA and GLU were stimulated by 60 mM K+ in the presence of 2.5 mM Ca2+. For the amino acids, no reliable data were obtained for release from the ACC because of its small size. The highest amount of K+-stimulated, Ca2+-dependent release of GABA was found with slices from the HYPO, THAL and MB while the highest amount of GLU was released from slices of STR, HIP and CTX. In those regions where reliable levels of K+-stimulated, Ca2+-dependent release of ASP were observed (STR, CTX, THAL), the amount of ASP was at least 5-fold lower than the values for GLU. A K+-stimulated, Ca2+-dependent release of NE, DA and 5-HT was observed for all 9 CNS regions studied. The highest release of (a) DA occurred from slices of CTX, STR and ACC; (b) NE was found in the HYPO and ACC; and (c) 5-HT occurred in the HYPO. The data (a) do not support a transmitter role for ALA and TAU in the CNS; (b) support a major transmitter function for GLY only in the PM and SC; and (c) support a transmitter role for GABA, GLU, NE, DA and 5-HT in the CNS regions examined (with the exception of GABA and GLU in the ACC where no data were obtained).

Evidence of a neurotransmitter role for aspartate and γ‐aminobutyric acid in the rat olfactory cortex

1. Using a cortical cup technique, the release of seven endogenous amino acids from the isolated rat olfactory cortex slice has been monitored.2. Electrical stimulation of the lateral olfactory tract at a frequency of 4 min(-1) was accompanied by a significant increase in the release of aspartate, GABA and taurine; the release of GABA and aspartate but not that of taurine was Ca(2+)-dependent.3. Chronic unilateral bulbectomy was accompanied by a specific, significant fall in the aspartate content of the olfactory cortex which reached a maximum 5 days after surgery and persisted for at least a further 5 days. Electrical stimulation of the lateral olfactory tract of such preparations did not release any of the amino acids under investigation.4. When slices from the unoperated side were exposed to solutions containing 25 mM-K+ or stimulated with electrodes placed directly on the cortex enclosed by the cup there was a Ca(2+)-dependent release of aspartate and GABA accompanied by a Ca(2+)-independent release of taurine. Following chronic bulbectomy, these procedures failed to evoke significant release of aspartate whereas the characteristics of GABA and taurine release were unaltered.5. It is concluded that aspartate may be the excitatory transmitter of some of the terminals of the lateral olfactory tract fibres and that GABA may be a transmitter at some inhibitory synapses of the rat olfactory cortex.

RELATIONSHIP BETWEEN Ca2+‐DEPENDENT AND INDEPENDENT RELEASE OF [3H]GABA EVOKED BY HIGH K+, VERATRIDINE OR ELECTRICAL STIMULATION FROM RAT CORTICAL SLICES

Abstract— It has been reported that the release of GABA by high K+ is Ca2+‐dependent while release induced by veratridine or electrical stimulation has been frequently found to be independent of Ca2+. To see the source of Ca2+‐dependent and independent release of GABA, cortical slices which had accumulated [3H]GABA were exposed to 50 mm‐K+ or 50 μm‐veratridine for 48min. In the presence of Ca2+ the 2 agents released approx the same amount of [3H]GABA but tetrodotoxin (TTX) abolished release induced only by veratridine, while omission of Ca2+ reduced release induced only by 50mm‐K+. Pre‐exposure of the slices for 48min to 50mm‐K+ in the presence of Ca2+ reduced the second release by 50mm‐K+ by 77% and that by veratridine by 74%, suggesting that in the presence of Ca2+ the 2 depolarizing agents release [3H]GABA from the same pool. Pre‐exposure to 50mm‐K+ in the absence of Ca2+ reduced the second release by 50mm‐K+ or by veratridine only by 37 and 27% respectively, indicating that most of the reduction in release was the result of a depletion of releasable [3H]GABA stores. The second exposure to 50mm‐K+ in the absence of Ca2+ reduced the evoked release further, while exposure to veratridine in the absence of Ca2+, after depletion of the stores, enhanced release 2.7 times. Electrical stimulation (64 Hz, 2 ms, 40 mA, alternating polarity) during 24min in the presence of Ca” caused an initial 5‐fold increase in efflux, which declined subsequently. In the absence of Ca2+, instead of a rapid increase, a slow but smaller increase in the efflux of [3H]GABA was found. TTX almost completely abolished the electrically evoked increase in release. Pre‐treatment with 50mm‐K+ reduced the electrically evoked release by 94% but electrical stimulation in the absence of Ca2+ after depletion of releasable stores doubled this release. Results suggest that in the presence of Ca2+, high K+, veratridine and electrical stimulation release [3H]GABA from the same Ca2+‐dependent store, but in the absence of Ca2+ veratridine and electrical stimulation enhance the release from a Ca2+‐independent store, probably as a result of an increased influx of Na+.

Effects of drugs on γ-aminobutyric acid receptors, uptake, release and synthesis in vitro

The effects of various convulsant and anticonvulsant drugs have been studied using in vitro assays for the postsynaptic action of the neurotransmitter γ-aminobutyric acid (GABA). GABA caused a receptor-ionophore mediated increase in chloride permeability in crayfish muscle. At 100 μ M concentrations, benzyl penicillin, bicuculline, diethyl barbiturate, diazepam, imipramine, and haloperidol partially inhibited this response while picrotoxinin inhibited it 100% (I 50 = 3 μ M). Muscimol (potently) andβ-( p-chlorphenyl) GABA (weakly) mimic GABA action in this assay. Muscimol and bicuculline (potent), and benzyl penicillin andβ-( p-chlorphenyl) GABA (weak), but not the other drugs, probably exerted their effects at the GABA receptor level, because only these four drugs among those tested were inhibitors of GABA receptor binding sites in mouse brain homogenates (sodium-independent sites having the specificity expected of receptor sites). Pentylenetetrazol weakly inhibited GABA receptor binding and GABA-induced chloride flux. Several other convulsants suspected of GABA antagonist activity, such as trimethyl caprolactam, isopropyl bicyclophosphate, tetramethylene disulfotetramine, and D-tubocurarine, showed no direct inhibition of either GABA receptor sites in mouse brain or of GABA-stimulated chloride flux in crayfish muscle. The drugs were also examined for effects on in vitro assays of GABA uptake, glutamate decar☐ylase activity (GAD), and Ca 2+-stimulated GABA release with mouse brain homogenates. GABA uptake by mouse brain particulate fractions was inhibited 50% by approximately 100μ M haloperidol, imipramine, chlorpromazine, diazepam, benzyl penicillin, bicuculline, andβ-( p-chlorophenyl) GABA, but by muscimol only at concentrations near1m M. Diazepam and benzyl penicillin also stimulated Ca 2+-independent efflux of GABA from mouse brain fractions enriched in synaptosomes. Diazepam, chlorpromazine, imipramine, and haloperidol inhibited Ca 2+-dependent release of GABA from brain particles enriched in synaptosomes. No drugs tested were found to inhibit L-glutamate decar☐ylase (GAD) activity at concentrations under100 μ M. The drugs diphenylhydantoin and diethyl barbiturate showed no effects on these GABA neurochemical assays which could be related to anticonvulsant action. Convulsant activity of benzyl penicillin appears to be related to competitive antogonism of GABA receptors, although the drugs is not very potent or specific. Diazepam at concentrations of50–100 μ M affected GABA transport in a manner consistent with anti-convulsant activity, although at similar concentrations it also could inhibit GABA release and postsynaptic action. Lioresal seems to be a definite GABA agonist, although weak and non-specific, while muscimol is a very potent and specific GABA agonist. Analogues of both these compounds are promising candidates for treatment of neurological disorders involving GABA dysfunction.