Colocalization of (3H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas

Using combined autoradiography and immunohistochemistry, we have compared (3H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas. Colocalization of the two markers was observed in a subset of amacrine cells and in certain cell bodies in the ganglion cell layer in both species and in a few horizontal cells in the chicken retina. Cells that contained only (3H)-adenosine or GABA were also seen. The degree of colocalization differed greatly between the two species. The results demonstrate a morphological relationship between the adenosine and GABA systems and provides information on the possible anatomical substrates underlying at least some types of functional interactions.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Involvement of Bidirectional Adenosine Transporters in the Release of l-[3H]Adenosine from Rat Brain Synaptosomal Preparations

Abstract: Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l -[³H]adenosine, our objectives here were to determine (1) if l -[³H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d -adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l -[³H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l -[³H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l -[³H]adenosine alone or l -[³H]adenosine plus unlabeled l -adenosine, d -adenosine, or uridine, l -[³H]-adenosine release was inhibited 42% by l -adenosine, 69% by uridine, and 81% by d -adenosine. The inhibition of l -[³H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.     

Neuronal and Glial Release of [3H]GABA from the Rat Olfactory Bulb

Abstract: GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [³H]-γ-aminobutyric acid ([³H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [³H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine-evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine-evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca²⁺-free solution. β-Alanine was strongly exchanged with [³H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The β-alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine-stimulated release of [³H]GABA was not significantly reduced after the β-alanine heteroexchange. Stimulation of the [³H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [³H]GABA release when a high magnesium concentration was added to a calcium-free solution. When D-600 was used as a calcium-flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.     

Lack of a high affinity uptake system for the GABA agonists THIP and isoguvacine in neurons and astrocytes cultured from mouse brain

The possibility that the GABA-receptor agonists isoguvacine and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) might be taken up into brain cells via the high affinity GABA transport system was tested by incubation of cultured neurons and astrocytes in media containing either [³H]GABA, [³H]isoguvacine or [³H]THIP at different concentrations. While GABA was actively taken up into both cell types via high affinity transport mechanisms, no high affinity transport could be demonstrated for isoguvacine or THIP. Both compounds did, however, penetrate into the cells. It is concluded that isoguvacine and THIP interact with the high affinity GABA-carrier neither in neurons nor in astrocytes.     

FACTORS INFLUENCING THE EFFLUX OF [3H]GAMMAAMINOBUTYRIC ACID FROM SATELLITE GLIAL CELLS IN RAT SENSORY GANGLIA

The spontaneous efflux of [³H]GABA from the satellite glial cells of rat dorsal root ganglia and the efflux evoked by 64 mM-K⁺ were studied in the presence of 10^-5M-amino-oxyacetic acid and found not to be affected by 10^-4M-D 600 or by elevated (9.6mM) Ca²⁺ in the absence of Mg²⁺. [³H]GABA efflux was increased by replacing sodium ions in the washing medium by choline ions and 64 mM-K⁺ failed to increase the efflux further. The drugs veratridine (10^-6 and 10^-4M) and batrachotoxin (10^-8 and 10^-6 M) failed to alter the spontaneous efflux of [³H]GABA from the glial cells. A variety of compounds, including amino acids, a GABA analogue and a GABA antagonist were tested for their ability to affect [³H]GABA efflux. The results indicated that compounds which inhibit GABA uptake into glial cells were also able to stimulate [³H]GABA efflux from these cells. The results are discussed with reference to possible mechanisms involved in the release of GABA from glial cells.     

Compartmentation and release of exogenous GABA in sheep brain synaptosomes

Exogenous tritiated -aminobutiric acid ([³H]GABA) is retained in two compartments in sheep cortex synaptosomes, corresponding to cytoplasmic and vesicular spaces, assuming that freeze-thawing the synaptosomes loaded with [³H]GABA releases the cytoplasmic [³H]GABA (81±3.9%), and that subsequent solubilization of the synaptosomes with 1% sodium cholate releases the vesicular [³H]GABA (19±3.9%). Depolarization of synaptosomes with 40 mM K⁺ in a Na⁺-medium, in the absence of Ca²⁺, releases 20.3±2.7% of the [³H]GABA retained in the synaptosomes. The [³H]GABA released under these conditions comes predominantly from the cytoplasm. The presence of 1 mM Ca²⁺ during depolarization releases and additional 13% (a total of about 33.5±9.9%) of the releasable [³H]GABA, and the [³H]GABA release which is Ca²⁺-dependent also comes mostly from the cytoplasmic compartment. When choline replaces external Na⁺, the [³H]GABA release is absolutely Ca²⁺-dependent, and the [³H]GABA released also comes mostly from the cytoplasmic pool. Therefore, it appears that [³H]GABA taken up by synaptosomes is accumulated mostly in the cytoplasmic compartment from which it is released upon depolarization. The technique described permits distinguishing the effect of different factors on the two pools of accumulated [³H]GABA.     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Study of the Mechanism of Release of [3H]GABA from a Teleost Retina In Vitro

Abstract: γ-Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca²⁺ dependency of the process of release of [³H]GABA from the retina of the teleost Eugenes plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration-dependent release of [³H]GABA. The veratridine effect was inhibited in Na⁺-free solution, but was not affected by 1 μM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine-and potassium-stimulated release of [³H] GABA occurred in Ca²⁺-free medium. Inhibitors of the Ca²⁺ channel, such as Mg²⁺(20 mM), La³⁺ (0.1 mM), and methoxy-verapamil (4 μM-0.4 mM), inhibited the veratridine-and K⁺-stimulated release. However, Co²⁺ and Cd²⁺ caused a potentiation and no change of the K⁺-and veratridine-stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [³H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradio-graphic studies with [³H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Muller cells. β-Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [³H]GABA was found with β-alanine, and no inhibition of the subsequent veratridine-stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [³H]GABA and lacked the capacity to induce the veratridine-stimulated release of [³H]GABA. These results suggest a voltage-and Ca²⁺-dependent neuronal release of [³H]GABA from retina.     

Insulin effect on GABA uptake in astroglial primary cultures

Astroglial cultures from newborn mouse cerebral cortex contain [¹²⁵I]insulin binding sites. Binding was specific reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [¹²⁵I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time-and dose-dependent inhibition of the [³H]GABA high affinity uptake. A decrease in theV _max rather than, an effect on theK _m was observed. This effect was dose-dependent and effective at 10^–10 M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [³H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin of [³H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate, that insulin may interfere in neuromodulation through astroglial signals.     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

Release of 3H-gamma-aminobutyric acid (GABA) by inhibitory neurons of the crayfish

Inhibitory neurons innervating the muscle receptor organ (MRO) of crayfish were used to study the uptake and release of tritiated GABA. MROs that have been directly exposed to ³H GABA for 60–75 min release radioactivity during low-frequency electrical stimulation. When ganglia containing the inhibitory cell bodies are exposed to ³H GABA, the isotope travels along a pathway unique to the inhibitory axon, at rates that range between 160 and 240 mm per day. Electrical stimulation of inhibitory axons whose cell bodies have been exposed to ³H GABA for 4–5 hr produces release of isotope from isolated MROs. Low calcium, high magnesium exposure prevents the stimulus-dependent release of radioactivity. Thin layer chromatographic analyses indicate that GABA comprises at least a major fraction of the radioactivity collected from stimulated preparations. A number of unidentified radioactive compounds are usually present with GABA, and it is suggested that most of these are catabolites of GABA.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Neurotransmitter candidates in the retina of the mudpuppy,Necturus maculosus

Summary Neurons accumulating (³H)-glycine and (³H) GABA were demonstrated with the use of autoradiography. Both were accumulated by different types of amacrine cells, similar those of goldfish. (³H)-GABA was also accumulated by horizontal cells, again similar to the goldfish. These results and physiological studies from other laboratories suggest that GABA and glycine are neurotransmitter candidates in amacrine cells of the mudpuppy.Immunoreactive neuropeptide Y (NPY), glucagon, vasoactive intestinal peptide (VIP), somatostatin, substance P, and neurotensin were found in different types of stratified amacrine cells. Weakly immunoreactive enkephalin and bombesin processes were also seen in the inner plexiform layer. Gastrin-immunoreactive neurons were not detectable.Endogenous 5-hydroxytryptamine was visualized immunohistochemically in a population of diffuse amacrine cells and some cells in the ganglion cell layer. This suggests that 5-hydroxytryptamine may be a neurotransmitter in the retina of the mudpuppy.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Maximal GABA and muscimol binding to high-affinity sites differ in physiological and in non-physiological buffers

The sensitivity of [³H]GABA and [³H]muscimol high-affinity binding sites to physiological (Krebs-Ringer's bicarbonate) and non-physiological (Tris-citrate) buffers was examined using synaptosomal membranes from bovine retinas. The maximum number of sites (B_max) for [³H]GABA was present when the tissue was assayed in KRB. With only one exception, this effect was independent of the washing conditions used or a small change in pH. In contrast, [³H]muscimol binding sites were maximally present when the tissue was washed in Tris, regardless of the assaying conditions or the small change in pH. Neither [³H]GABA nor [³H]muscimol was displaced by ( - )baclofen. The apparent dissociation constants (K_d) of the ligands did not change under any of the conditions tested. These findings demonstrate a fundamental difference between GABA and muscimol binding sites.     

Turnover and release of GABA in rat cortical slices: Effect of a GABA-T inhibitor,gabaculine

The turnover and release of endogenous and labeled GABA were followed in rat cortical slices after incubation with [³H]GABA. High performance liquid chromatography was used to measure endogenous GABA and to separate [³H]GABA from its metabolites. During superfusion with 3 mM K⁺ the slices rapidly lost their [³H]GABA content while maintaining constant GABA levels. Exposure to 50 mM K⁺ for 25 min caused an initial rapid rise in the release of both endogenous and [³H]GABA followed by a more rapid decline in the release of the latter. The specific activity of released GABA was two to four times higher than that in the slices. Depolarization lead to a net synthesis of GABA. The GABA-T inhibitor, gabaculine, (5 M) in vitro arrested the metabolism of [³H]GABA and rapidly doubled the GABA content but did not significantly increase the high K⁺ evoked release of endogenous GABA. In vivo pretreatment with 0.5 mM/kg gabaculine quadrupled GABA content and increased both the spontaneous and evoked release of endogenous GABA but while its Ca²⁺-dependent release increased by 50%, the Ca²⁺-independent release was enhanced sevenfold. This large Ca²⁺-independent release of GABA is likely to have different functional significance from the normal Ca²⁺-dependent release.