Developing oligodendrocytes express functional GABA(B) receptors that stimulate cell proliferation and migration

GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.     

Interactions between rho and gamma2 subunits of the GABA receptor

The gamma-aminobutyric acid type C (GABA(C)) receptor is a ligand-gated chloride channel with distinct physiological and pharmacological properties. Although the exact subunit composition of native GABA(C) receptors has yet to be firmly established, there is general agreement that GABA rho subunits participate in their formation. Recent studies on white perch suggest that certain GABA rho subunits can co-assemble with the GABA(A) receptor gamma2 subunit to form a heteromeric receptor with electrophysiological properties that correspond more closely to the native GABA(C) receptor on retinal neurons than any of the homomeric rho receptors. In the present study we examined the interactions among various perch GABA rho and gamma2 subunits. When co-expressed in Xenopus oocytes, the gamma2 subunit co-immunoprecipitated with Flag-tagged perch rho1A, rho1B, and rho2B subunits, but not with the Flag-tagged perch rho2A subunit. Immunocytochemical studies indicated that the membrane surface expression of the gamma2 subunit was detected only when it was co-expressed with perch rho1A, rho1B, or rho2B subunit, but not with the perch rho2A subunit or when expressed alone. In addition, co-immunoprecipitation of perch rho1B and gamma2 subunits was also detected in protein samples of the teleost retina. Taken together, these findings suggest that a heteromeric rho(gamma2) receptor could represent one form of GABA(C) receptor on retinal neurons.     

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.     

Increased Intracellular γ-Aminobutyric Acid Selectively Lowers the Level of the Larger of Two Glutamate Decarboxylase Proteins in Cultured GABAergic Neurons from Rat Cerebral Cortex

The regulation of glutamate decarboxylase (GAD; EC 4.1.1.15) was studied by using cultures of cerebral cortical neurons from rat brain grown in serum-free medium. About 50% of the neurons in the cultures were gamma-aminobutyric acid (GABA)ergic as determined by two double-staining procedures. Immunoblotting experiments with four anti-GAD sera that recognize the two forms to varying degrees, demonstrated that the cultures contained the two forms of GAD that are present in rat brain (apparent molecular masses = 63 and 66 kDa). GAD activity was reduced by 60-70% when intracellular GABA levels were increased by incubating the cultures with the GABA-transaminase inhibitor gamma-vinyl-GABA for greater than 5-10 h or with 1 mM GABA itself. Neither baclofen nor muscimol (100 microM) affected GAD activity. Immunoblotting experiments showed that only the larger of the two forms of GAD (66 kDa) was decreased by elevated GABA levels. These results, together with previous results indicating that the smaller form of GAD is more strongly regulated by pyridoxal 5'-phosphate (the cofactor for GAD), suggest that the two forms of GAD are regulated by different mechanisms.     

Endogenous Release of γ-Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.     

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.     

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.     

Elevated γ-Aminobutyric Acid Levels Attenuate the Metabolic Response to Biateral Ischemia

Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocreatine, glucose, and glycogen. The amino acid neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor gamma-vinyl-GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents.     

Bicuculline- and Baclofen-Insensitive γ-Aminobutyric Acid Binding to Rat Cerebellar Membranes

Up to 60% of gamma-[3H]aminobutyric acid ([3H]GABA) bound specifically to rat cerebellar membranes in the absence of Ca2+ was insensitive to the GABAA antagonist bicuculline and to the GABAB agonist baclofen. This indicates that a significant component of specifically bound [3H]GABA is associated with non-GABAA, non-GABAB binding sites. The presence of this binding component appeared seasonal, peaking in the month of September (early spring) each year over a 4-year period. The calcium independence and bicuculline and baclofen insensitivity of the binding indicate that this binding is not to the classical GABAA and GABAB binding sites. High concentrations of muscimol and isoguvacine inhibited non-GABAA, non-GABAB binding. Scatchard analysis of the non-GABAA, non-GABAB binding sites indicated two kinetic components: KD1 = 42 nM and KD2 = 9.2 microM; Bmax1 = 1.6 pmol/mg of protein and Bmax2 = 28 pmol/mg of protein.     

Evidence for a G protein-coupled gamma-hydroxybutyric acid receptor

gamma-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABA(B) receptor (GABA(B)R)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABA(B)R was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABA(B)R agonist (-)-baclofen. Also, using guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and low-K:(m) GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABA(B)R antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABA(B)R antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [(3)H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [(35)S]GTPgammaS binding and low-K:(m) GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABA(B)R antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABA(B)R antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABA(B)R.     

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.     

Stromal cell-derived factor-1alpha modulation of the excitability of rat substantia nigra dopaminergic neurones: presynaptic mechanisms

In rat substantia nigra (SN), Chemokine (CXC motif) receptor 4 (CXCR4) for the chemokine stromal cell-derived factor (SDF)-1alpha is expressed on dopaminergic (DA) neurones, but also on non-DA cells, suggesting presynaptic actions. Using whole-cell patch-clamp recordings in DA neurones of rat SN slices at a holding potential of -60 mV, we showed here that SDF-1alpha exerts multiple presynaptic effects. First, SDF-1alpha (10 nm) induced an increase in the frequency of spontaneous and miniature GABA(A) postsynaptic currents by presynaptic mechanisms, consistent with the presence of CXCR4 on GABAergic neurones of the SN, as revealed by immunocytochemistry. Second, SDF-1alpha (0.1-1 nm) induced a glutamatergic inward current resistant to tetrodotoxin (TTX), most probably the result of glutamate release from non-neuronal cells. This inward current was not blocked by the CXCR4 antagonist AMD 3100 (1 microm), consistent with the lack of CXCR4 on astrocytes as shown by immunocytochemistry under basal conditions. Finally, SDF-1alpha (10 nm) induced, via CXCR4, an outward G protein-activated inward rectifier (GIRK) current, which was TTX sensitive and prevented by application of the GABA(B) antagonist CGP55845A, suggesting GABA spillover on to GABA(B) receptors. Our results show that SDF-1alpha induces, via presynaptic mechanisms, alterations in the excitability of DA neurones as confirmed by current-clamp experiments.     

Pergolide Presynaptically Inhibits Calcium-Stimulated Release of γ-Aminobutyric Acid

The release of gamma-aminobutyric acid (GABA) in rat dorsolateral striatum was studied using in vivo microdialysis. Dialysis was conducted 2 days after probe implantation in awake, freely moving rats using a modified Ringer solution. Calcium induced a reversible increase in GABA release that was abolished by tetrodotoxin but was only slightly attenuated by a maximally effective dose of pergolide, a D2 receptor agonist. It was thus concluded that pergolide inhibits calcium-stimulated release of GABA presynaptically by a mechanism distinct from that of tetrodotoxin.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

GABAergic Modulation of Striatal Cholinergic Interneurons: An In Vivo Microdialysis Study

Abstract: Striatal cholinergic interneurons have been shown to receive input from Striatal γ-aminobutyric acid (GABA)-containing cell elements. GABA is known to act on two different types of receptors, the GABA_A and the GABA₆ receptor. Using in vivo microdialysis, we have studied the effect of intrastriatal application of the GABA_A-selective compounds muscimol and bicuculline and the GA- BA_B-selective compounds baclofen and 2-hydroxysaclofen, agonists and antagonists, respectively, at GABA receptors, on the output of Striatal acetylcholine (ACh). Intrastriatal infusion of 1 and 10 μmol/L concentrations of the GABA_A antagonist bicuculline resulted in a significant increase in Striatal ACh output, whereas infusion of 1 and 10 /μmol/L concentrations of the GABA_A agonist muscimol significantly decreased the output of Striatal ACh. Both compounds were ineffective in changing the output of Striatal ACh at lower concentrations. Infusion of concentrations up to 100 μmol/L of the GABA_B-selective antagonist 2-hydroxy-saclofen failed to affect Striatal ACh output, whereas infusion of 10 and 100 μmol/L baclofen, but not 0.1 and 1 μmol/L baclofen, significantly decreased the output of Striatal ACh. Thus, agonist-stimulation of GABAA and GABA_B receptors decreases the output of striatal ACh in a dose-dependent fashion, whereas the GABAergic system appears to inhibit tonically the output of striatal ACh via GABA_A receptors, but not via GABA_B receptors. We hypothesize that although GABA_A mediated regulation of striatal ACh occurs via GABA receptors on the cholinergic neuron, the GABA_B mediated effects may be explained by presynaptic inhibition of the glutamatergic input of the striatal cholinergic neuron.     

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.     

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.     

Role of Dendritic Dopamine of the Substantia Nigra in the Modulation of Nigrocollicular γ-Aminobutyric Acid Release: In Vivo Studies in the Rat

The release of gamma-[3H]aminobutyric acid ([3H]GABA) newly synthesized from [3H]glutamine was estimated in the superior colliculus of ketamine-anesthetized rats superfused via a push-pull cannula. A significant amount of [3H]GABA was spontaneously released in the superior colliculus (582 +/- 49 pCi/10 min). A major part of the large K(+)-evoked increase of the [3H]GABA release was Ca2+ dependent. When neuronal activity of the substantia nigra was enhanced by nigral application of K+ (30 mM) or bicuculline (10(-4) M), a persistent increase of the collicular [3H]GABA release was observed (60 and 80%, respectively). Conversely, when nigral activity was reduced by nigral application of GABA (10(-4) M) or superfusion with a Ca(2+)-free medium, a sustained decrease of the collicular [3H]GABA release was observed (-30 and -40%, respectively). Following the nigral application of a selective D2-receptor agonist. RU 24926 (10(-6) M), for 30 min in 6-hydroxydopamine-lesioned rats, a phasic increase (60%) of the collicular [3H]GABA release was detected. This effect could result from an activation of nigrocollicular GABAergic neurons by D2-receptor stimulation, because nigral activity and collicular release of [3H]GABA changed in a parallel direction.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

Coexistence of γ-Aminobutyric Acid Type A and Type B Receptors in Testicular Interstitial Cells

The existence of specific gamma-aminobutyric acid (GABA)ergic receptors in testicular interstitial cells was investigated in the present study. Specific binding of [3H]GABA to interstitial cell membranes was found to be time- and temperature-dependent and varied according to Ca2+ concentration present in the incubation medium. We analyzed the ability of different GABAergic agonists and antagonists to displace the bound radioactivity. In the absence of Ca2+ (1 mM EDTA), GABA and the GABAergic agonist isoguvacine displaced the bound radioactivity. When the radioligand assay was performed in the presence of 2.5 mM CaCl2, the [3H]GABA specifically bound increased twofold. Under such conditions, the specific GABAergic agonist baclofen, as well as GABA and isoguvacine, displaced the [3H]GABA bound. Saturation analysis revealed the presence of a population of GABAA binding sites with a KD value of 45.2 nM and a maximal number of binding sites of 57.4 fmol/mg of protein. The maximal binding increased on addition of 2.5 mM CaCl2 to 102 fmol/mg of protein, indicating the existence of a second population of GABAergic receptors, i.e., type B, with essentially the same affinity. In addition, the incubation of testicular interstitial cells with GABA and baclofen resulted in an increase in androgen production. These results support a functional role of GABA in the neuroendocrine control of the male gonad.