Localization of high affinity [3H]glycine transport sites in the cerebellar cortex

A study was made of [3H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [3H]glycine at the low concentration used. Glial cells also took up [3H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [3H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [3H]GABA uptake (Ki over 600 microM vs the Kt of about 20 microM) and that GABA is an even weaker inhibitor of [3H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [3H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [3H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: Ionic influences

Ammonium salts of some anions decrease the potency of glycine in inhibiting [³H]strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC₅₀ of glycine in inhibiting the [³H]strychnine binding, their capacity to reduce the [³H] strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of [³H]strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting [³H]strychnine is not. Binding of [³H]strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li⁺, K⁺, and Na⁺ up to 150 mM decrease [³H]strychnine binding, while higher concentrations of the cations increase [³H]strychnine binding. Inhibition by glycine of [³H]strychnine binding is enhanced by low concentrations of these cations.     

Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography

Autoradiographic studies of rat cortical cultures were conducted with tritiated transmitters and related drugs. Autoradiographs prepared from cultures incubated in [³H]GABA showed selective labeling: dense accumulations of silver grains over the somas and all processes of approximately 30–50% of the neuronal population, few grains over the non-neuronal cells. This labeling was blocked by diaminobutyric acid (DABA) and sodium-free media but not by β-alanine and thus has the characteristics of GABA uptake in other neuronal systems. There were no obvious differences in the size, shape, number of processes or distribution in the culture between neurons which accumulated GABA and those which did not. Similar cultures incubated in either [³H]glycine or [³H]glutamate and processed for autoradiography resulted in a much different distribution of silver grains than that seen for [³H]GABA. Following incubation in [³H]glycine, silver grains were distributed uniformly over all cells in the culture, both neuronal and non-neuronal. This distribution suggests a metabolic and not a neurotransmitter role for glycine in the cultures, as would be expected of neuronal cells derived from cerebral cortex. Glutamate incubations resulted in the appearance of silver grains over only the non-neuronal cells with very few over the neuronal population. Autoradiograms were also prepared following incubation in the potent GABA receptor agonist [³H]muscimol. These autoradiograms were indistinguishable from those obtained following [³H]GABA incubation. Thus, a finite population of neurons was densely labeled, the labeling was blocked by the GABA uptake inhibitors DABA, nipecotic acid, guvacine and Na⁺-free media, while substances which interact with the GABA receptor, bicuculline methiodide, THIP, isoguvacine and the noncompetitive antagonist, picrotoxin, were without effect. These results demonstrate that the affinity of muscimol for the GABA uptake site far outweighs its affinity for the GABA receptor site in autoradiographic experiments where intact cells are employed, presumably because its binding to receptors is fleeting. Therefore, muscimol autoradiography may not be informative about GABA receptor localization.These autoradiographic studies suggest that nearly half the neurons in our culture system are GABA neurons but disclosed no morphological handle for GABA neurons.     

Transmitter histochemistry of the rat olfactory bulb. III. Autoradiographic localization of [H]GABA

The distribution of [³H]γ-aminobutyric acid (GABA) labeled elements in rat olfactory bulb was studied by light and electron microscopic autoradiography. [³H]GABA was strongly taken up into glial cells and pericytes in all layers of the bulb. The neuronal uptake of [³H]GABA was mainly seen in certain types of nerve terminals. About one-third of the granule dendritic terminals, some nerve endings of short axon cells, and certain nerve endings of extrabulbar origin showed a strong labeling. Labeling was seen in a small population of the periglomerular, short axon and granule cell bodies. Most cell bodies of these 3 types as well as the mitral cells did not, however, accumulate any appreciable amount of [³H]GABA. The labeling pattern seen after injection of [³H]glycine and [³H]leucine was clearly different from the pattern seen after [³H]GABA injection. The labeling was more uniformly distributed over the components of the neuropil with a considerably higher activity over certain cell somata such as the mitral cells. The present results demonstrate that neuronal uptake and accumulation of [³H]GABA occur into populations of olfactory bulb cells and processes, which from neurophysiological and/or immunohistochemical studies are supposed to use GABA as a neurotransmitter.     

Comparison of binding affinities and capacities of gamma-aminobutyric acid and glycine in synaptosome-enriched fractions of rat cerebral cortex and spinal cord.

The “binding” affinities and capacities of [³H]-γ-aminobutyric acid (³H-GABA) and [¹⁴C]-glycine to synaptosome-enriched fractions of rat cerebral cortex and spinal cord have been compared over a 10^?8 to 10^?3m concentration range in isosmotic sucrose solutions containing 32 mm NaCl. At 0 C, the binding of both GABA and glycine to particles of the cerebral cortex appeared to take place by “triple-affinity” processes. The respective binding affinities (reciprocals of K_B values) of GABA for particles of the cortex were generally greater than those for glycine, whereas maximal binding capacities (B_max values) for GABA were about twice as great as those for glycine. In fractions of spinal cord, at 0 C, the binding of both GABA and glycine occurred by “double-affinity” processes, and though the binding affinities of GABA were about twice those of glycine, B_max values for GABA were smaller than those for glycine. Striking changes occurred when particles were allowed to stand in contact with [³H]-GABA plus [¹⁴C]-glycine at 23 C for 45 min. Although double-affinity mechanisms persisted at the higher temperature for the binding of both amino acids to particles of spinal cord (GABA still being bound with greater affinities than glycine), the binding of both amino acids to particles of cerebral cortex was best resolved as “single-affinity” processes. The differences between B_max values for GABA and glycine in particles of cerebral cortex and spinal cord appeared to reflect their different endogenous concentrations.     

A new function for glycine GlyT2 transporters: Stimulation of γ‐aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca2+‐dependent anion channels

Glycine GlyT2 transporters are localized on glycine‐storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ‐aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule‐associated protein‐2‐positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [³H]GABA and exposed to glycine. Glycine stimulated [³H]GABA release in a concentration‐dependent manner. The glycine effect was insensitive to strychnine or to 5,7‐dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca²⁺ entered by reversal of plasmalemmal Na⁺/Ca²⁺exchangers. A significant portion of the GlyT2‐mediated release of [³H]GABA (about 50% of the external Ca²⁺‐independent release) occurred by reversal of GABA GAT1 transporters. Na⁺ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na⁺/Ca²⁺ exchangers, causing an increase in cytosolic Ca²⁺, which in turn triggered a Ca²⁺‐induced Ca²⁺ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca²⁺ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels. © 2013 Wiley Periodicals, Inc.     

Binding of [H]glycine, [H]β-alanine and [H]strychnine in cultured rat spinal cord and brain stem

Cultures of rat brain stem and spinal cord were used to visualize binding sites for [³H]glycine, [³H]β-alanine and their antagonist [³H]strychnine by light microscopic autoradiography. In spinal cord cultures, all radio-ligands were bound mainly to large neurones, probably motoneurones whereas in brain stem cultures, both medium-sized and large neurones were labelled. In contrast, glial cells did not show binding sites for any of the compounds studied, suggesting that glial elements may not possess receptors for glycine and β-alanine.     

Effects of glycine and GABA on the ganglion cells of the retina of the skate Raja erinacea

Application of glycine and GABA caused inhibition of light-evoked activity from different populations of ‘ON’ center ganglion cells in the retina of the skate (Raja erinacea). Cells affected by glycine were not affected by GABA and vice-versa. Addition of 100 μM strychnine to the glycine-containing perfusate caused a resumption in light-evoked responses of the cells. The action of GABA was antagonized by 100 μM bicuculline methochloride, but not by picrotoxin. Autoradiography revealed the presence of [³H]glycine accumulating amacrine cells.     

Biochemical evidence thatl-glutamate is a neurotransmitter of primary vagal afferent nerve fibers

To determine in rat if vagal afferent fibers projecting into the intermediate one third of the nucleus tractus solitarius (NTS), the site of termination of baroafferents, utilize glutamate as a neurotransmitter, the high-affinity uptake of [³H]l-glutamate and content of glutamate were analyzed in micropunches of rat brain stem. The intermediate NTS contains a high-affinity synaptosomal uptake system for [³H]l-glutamate that is greater in capacity than that in areas adjacent to the NTS; it is almost two-fold higher than uptake in medial septum and nucleus accumbens and equal to that of hippocampal regions purportedly containing a rich glutamatergic innervation. Unilateral ablation of the nodose ganglion (i.e. cells of origin of vagal afferents) resulted, within 24 h in a prolonged significant reduction, to 56% of control, of [³H]l-glutamate uptake, bilaterally in the NTS. The reduction of Na⁺-dependent synaptosomal uptake of [³H]l-glutamate, resulted from a decrease in V_max without change in theK_m of the process, was anatomically restricted to the intermediate NTS, and was not associated with changes in [³H]GABA uptake. The content of glutamate in the NTS was significantly (P < 0.01) decreased by 30% 7 days following unilateral extirpation of the nodose ganglion without changes in the concentrations of aspartate, glycine, glutamine, or GABA. A population of vagal afferent fibers projecting to NTS are glutamatergic. The results are consistent with the hypothesis obtained by physiological and pharmacological techniques that glutamate is a neurotransmitter of baroafferents.     

Taurine binding to membranes from rat brain regions

[³H]-taurine binding to membranes from different regions from rat brain was studied. Binding to membranes from cerebral cortex and its subcellular fractions, hypothalamus, olfactory bulb and cerebellum was measured. Binding to membranes from dorsal root ganglion was also determined. Na⁺-dependent taurine binding was consistently observed in all the membranes except those from dorsal root ganglion. A K_D = 4.06 μM was obtained for binding to membranes from cerebral cortex. Na⁺-dependent taurine binding was displaced by 20 μM strychnine or bicuculline. Na⁺-independent taurine binding with properties corresponding to a postsynaptic interaction could not be detected in any of the regions studied. The possibility of Na⁺-dependent taurine binding, representing binding to uptake sites or to postsynaptic receptors for GABA and glycine, is discussed.     

Uptake [H]GABA by oligodendrocytes in dissociated brain cell culture: A combined autoradiographic and immunocytochemical study

Uptake of [³H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [³H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galatocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [³H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by β-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [³H]β-alanine, which labeled only astrocytes. Oligodendroglial [³H]GABA uptake was Na⁺-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

Quantitative studies of enkephalin's coexistence with γ-aminobutyric acid, glycine and neurotensin in amacrine cells of the chicken retina

Previous double-label studies demonstrate that enkephalin coexists with γ-aminobutyric acid, glycine or neurotensin in amacrine cells of the chicken retina. The present study utilizes double- and triple-label paradigms to quantitatively analyze these coexisting relationships. Twenty-eight percent of enkephalin-like immunoreactive amacrine cells were found to exhibit high-affinity uptake of [³H]GABA, while 53% of enkephalin-amacrine cells specifically accumulate [³H]glycine. Moreover, the present study predicts that at least 26% of enkephalin-amacrine cells which accumulate [³H]glycine should also be immunoreactive for neurotensin.     

Reduction of [H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3–4 months, specific binding of [³H]muscimol and [³H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20–30% decrease of the number of [³H]muscimol binding sites was observed after capsaicin treatment. In contrast, [³H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

Biochemical correlates of GABA function in rat cortical neurons in culture

Serial biochemical studies of a rat cortical tissue culture system in which synapses regularly form showed that γ-aminobutyric acid (GABA) is present in the cultures and increases with their maturation. The tissue GABA concentration in mature cultures is similar to that of adult rat cortex in vivo. The synthetic enzyme, glutamate decar☐ylase, also increases with age as does high affinity GABA uptake. GABA uptake was blocked by l-2,4-diaminobutyrate (DABA) and had the properties of neuronal GABA uptake. Specific release by depolarizing media of both exogenous [³H]GABA and GABA synthesized from d-[U-¹⁴C]glucose was demonstrated. The GABA released by high potassium media had higher specific activity and a greater contribution from glucose (as compared to acetate) than GABA found in the medium in the absence of depolarization. Calcium dependency of evoked GABA release could be shown only after pretreatment of cultures with ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid or EGTA. Synaptosomes may exhibit greater calcium dependence of evoked transmitter release than intact cells in culture because their intracellular calcium stores are depleted during preparation. Glycine uptake by the cultures was much less in amount than was GABA uptake, and specific release of glycine could not be demonstrated.Specific binding of both a GABA agonist ([³H]muscimol) and an antagonist ([³H]bicuculline) was shown by membranes prepared from the cultures. By contrast, when [³H]muscimol binding to intact cells was studied, essentially all binding was sodium dependent and had the properties of GABA uptake binding. We conclude that the use of [³H]muscimol for receptor studies is valid only after the elimination of GABA uptake systems.Biochemical data from these studies support the concept that GABA is the transmitter for many cortical synapses. Glycine and taurine are not likely to be transmitters in these cortical cultures. When considered together with physiological data from the preceding paper, we have satisfied Werman's criteria (see ref. 36) for accepting GABA as the major inhibitory transmitter in the cortical culture system.     

Effect of pentobarbitone on the spontaneous efflux of γ-amino acids from rabbit retina

The effect of pentobarbitone on the spontaneous release of radioactivity from rabbit retinas preloaded with [³H]GABA, [³H]DABA, [³H]β-alanine and [³H]glycine was studied. In high concentration of pentobarbitone (10⁻³ M) the rate of spontaneous efflux of neuronal [³H]GABA, [³H]DABA and [³H]β-alanine was reduced but not that of [³H]glycine. Glial release of [³H]GABA was much less influenced. At lower concentrations (10⁻⁴-10⁻⁵ M) there was an initial increase in the release of [³H]GABA, [³H]DABA and [³H]β-alanine followed by a decrease, indicating several components in the release systems for the amino acids.     

Effects of chronic treatment with a cyclic AMP-selective phosphodiesterase inhibitor, rolipram, on excitatory amino acid neurotransmission systems in young and aged rat brains

Summary Rolipram selectively inhibits cyclic AMP-specific phosphodiesterase, and leads to an increase in cyclic AMP levels in the brain. In this study, we investigated the effects of chronic rolipram treatment on excitatory and inhibitory amino acid neurotransmission systems in young and aged Wistar rat brains. We used in vitro autoradiography with [³H]MK-801, [³H]glycine, D-[³H]aspartate, and [³H]muscimol to label N-methyl-D-aspartate (NMDA) receptors, glycine modulatory sites, glutamate transport sites, and -aminobutyric acid-A (GABA) receptors, respectively. Rolipram (0.01 or 0.1 mg/kg, per os) or its vehicle (distilled water) was administered once a day for 4 weeks. The highest binding of [³H]MK-801, [³H]glycine, and d-[³H]aspartate was seen in the hippocampus in vehicle-treated rats. No significant differences in these binding activities were seen between young and aged rat brains. [³H]Muscimol binding was the highest in the cerebellum, and decreased in many brain regions in aged rats. The chronic rolipram treatment resulted in (1) an increase in [³H]MK-801 binding in the dentate gyrus in both young and aged rats, (2) remarkable reductions in D-[³H]aspartate binding in many regions of both young and aged rats, and (3) no or minimal changes in [³H]glycine and [³H]muscimol binding. These results suggest that the chronic rolipram treatment modifies the excitatory amino acid neurotransmission system.     

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate,and [3H]γ-aminobutyric acid,in taste buds of the mudpuppy,Necturus maculosus

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and γ-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [³H]serotonin, [³H]glutamate, and [³H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4–6 μM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [³H]serotonin. When the tissue was treated with 40 mM K⁺ after incubating the tissue in [³H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [³H]serotonin was Ca-dependent: if Ca²⁺ was reduced to 0.4 mM and 20 mM Mg²⁺ added to the high K⁺ bathing solution, Merkel-like basal cells did not release [³H]serotonin. In contrast, [³H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [³H]glutamate was not released by depolarizing the tissue with 40 mM K⁺. [³H]GABA uptake was also widespread, but did not occur in taste bud cells. [³H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds. J. Comp. Neurol. 392:199–208, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Regional differences in the enhancement by GABA of [H]zolpidem binding to ω1 sites in rat brain membranes and sections

The potential heterogeneity in the allosteric coupling between GABA and ω₁ binding sites within the native GABA_A receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [³H]zolpidem binding to ω₁ site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 ± 47%) in cortical membranes than in cerebellar (165 ± 48%) or in hippocampal (118 ± 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than ω₁ since: (1) the pharmacological properties of the [³H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of ω₁ sites in each structure; and (3) GABA did not increase the B_max for [³H]zolpidem. Regional differences in the effect of 100 μM GABA on [³H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3–4-fold) effects of GABA in [³H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [³H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [³H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in the molecular (3.8-fold) than in the granular (2.2-fold) layer. In the hippocampus the effect of GABA was also heterogeneous with larger increases in CA1 and CA2 fields (3.5-fold) than in CA3 field (2.2-fold) and dentate gyrus (2.5-fold). Finally in the deep layers of the superior colliculus GABA stimulation of [³H]zolpidem binding was greater than the superficial layer. In the other structures examined the GABA-induced increase in [³H]zolpidem binding was less than 3-fold. The smallest stimulations were quantified in the entorhinal cortex (2.1-fold), amygdala (2.4-fold) and nucleus accumbens (1.7-fold). These results suggest that [³H]zolpidem sites are associated to, at least, two GABA_A receptor subtypes that can be differentiated by their allosteric interaction between GABA and [³H]zolpidem sites.