Stress-Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor

The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (gamma-aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of delta-aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas delta-aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicuculline and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexamethonium and d-tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, beta-alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and delta-aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

ELECTRICALLY INDUCED RELEASE OF [3H]GABA FROM NEOCORTICAL THIN SLICES. EFFECTS OF STIMULUS WAVEFORM AND OF AMINO-OXYACETIC ACID

The efflux of [³H]GABA or [¹⁴C]GABA from superfused neocortical thin slices, held on quick transfer electrodes, has been compared with that of the non-transmitter amino acid model [¹⁴C]α- amino-isobutyrate (AIB), and, to a lesser extent, with [³H]norepinephrine. Electrical stimulation of the slices with sine-wave current (50 Hz); rectangular, biphasic pulses, (80/s, 3 ms); or rectangular, monophasic pulses (100/s, 5 ms), was unable to release GABA at stimulating potentials that are able to release known transmitter substances. Release of GABA and AIB was only seen with higher applied potentials, when also non-transmitter amino acids were released. It was also found that amino-oxyacetic acid(10^-5 M and 5 × 10^-5 M) increased the excitability of the slices, and allowed the release of both GABA and AIB to occur with weaker stimuli. This effect was independent of extracellular calcium.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

Mechanisms for Facilitation of Nitric Oxide-Evoked [3H]GABA Release by Removal of Hydroxyl Radical

Abstract: We have investigated the mechanisms for enhancement of nitric oxide (NO)-evoked γ-[³H]aminobutyric acid ([³H]GABA) release from mouse cerebro-cortical neurons by hydroxyl radical (^?OH) scavengers. ^?OH scavengers, such as N,N′-dimethylthiourea (DMTU), uric acid, and mannitol, dose-dependently facilitated NO-evoked [³H]GABA release evoked by NO liberated from S-nitroso-N-acetylpenicillamine. Ionomycin-evoked [³H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, N^G-methyl-l -arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by ^?OH scavengers. These enhancing actions of ^?OH scavengers were completely abolished by Ca²⁺ removal from incubation buffer and by an l -type voltage-dependent Ca²⁺ channel (VDCC) inhibitor, nifedipine, whereas each ^?OH scavenger showed no effects on [³H]GABA release in the absence of NO. Inhibitors for P/Q- and N-type VDCCs had no effects on the enhancement. NO-induced ⁴⁵Ca²⁺ influx was also dose-dependently enhanced by ^?OH scavengers, although ⁴⁵Ca²⁺ influx was not altered by ^?OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO-induced ⁴⁵Ca²⁺ influx by ^?OH scavengers. These results indicate that the removal of ^?OH by its scavengers facilitates the NO-evoked [³H]GABA release dependent on Ca²⁺ and that this enhancement is due to the increase in Ca²⁺ influx via L-type VDCCs.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

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.     

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.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

RELEASE OF [3H]GABA FROM IN VITRO PREPARATIONS: COMPARISON OF THE EFFECT OF DABA AND β-ALANINE ON THE K+ AND PROTOVERATRINE STIMULATED RELEASE OF [3H]GABA FROM BRAIN SLICES AND SYNAPTOSOMES1

It has been proposed that the major portion of [³H]GABA released from rat cortical slices upon exposure to high K⁺ comes from a neuronal pool. Using carrier mediated exchange diffusion of DABA or β-alanine in the superfusion medium for GABA in the slice as a technique for manipulating neuronal and glial pools of GABA, it was found that DABA but not β-alanine substantially reduced the K⁺ stimulated release of [³H]GABA. The present study using synaptosomes as an in vitro model of the nerve ending was undertaken to ascertain whether this neuronal pool of releasable [³H]GABA was associated with a specific transmitter pool in nerve endings. A continuous superfusion system employing a Ca²⁺ pulse to produce a calcium coupled release (Levy et al, 1973) was used to study the effect of two concentrations (20 μm , 1 mm ) of DABA and β-alanine on the release of [³H]GABA from synaptosomes. In contrast to the results in slices, DABA at both concentrations had no effect on the release of [³H]GABA from synaptosomes in spite of evidence that exchange diffusion was occurring. With protoveratrine as the releasing agent there was no effect of DABA on the release of [³H]GABA from either slices or synaptosomes. The results suggest that the major portion of [³H]GABA released from cortical slices by high K⁺ comes from a non-transmitter pool in the neuron. Use of K⁺ stimulated release of amino acids from cortical slices as a criterion for neurotransmitter function must be viewed with caution.     

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

Studies on the mechanism of modulation of [3H]noradrenaline release from rat hippocampal synaptosomes by GABA and benzodiazepine receptors

Release of [³H]noradrenaline from rat hippocampal synaptosomes was triggered by pulses of 25 mM K⁺, 5 μM veratridine or superfusion with the Ca²⁺ ionophore A23187. GABA with bicuculline or chlordiazepoxide depressed the release of [³H]noradrenaline evoked by depolarisation but not by the Ca²⁺ ionophore. 8 Br-cAMP with [Ca²⁺]₀ 0.3 mM had no effect on spontaneous or K⁺-evoked release of [³H]noradrenaline and completely blocked the effect of chlordiazepoxide and GABA with bicuculline. With [Ca²⁺]₀ 1 mM 8 Br-cAMP enhanced spontaneous and K⁺-evoked release of [³H]noradrenaline, and reversed the depression caused by GABA with bicuculline. GABA alone evoked Ca²⁺-dependent release of [³H]noradrenaline which was sensitive to [Cl^?]₀. The results suggest that the GABA_A-receptor mediated release of [³H]noradrenaline is due to depolarisation resulting from increased Cl^? conductance whereas the depression of depolarisation-dependent release of [³H]noradrenaline by GABA_B or benzodiazepine receptors is mediated by a cAMP-dependent decrease in the voltage-dependent Ca²⁺ conductance.     

THE EFFECT OF BICUCULLINE, METRAZOL, PICROTOXIN AND STRYCHNINE ON THE RELEASE OF [3H]GABA FROM RAT BRAIN SLICES

Each of the four convulsants used significantly influenced the release of [³H]-GABA from brain slices, without affecting [³H]GABA uptake. Bicuculline (10^?5M, but not 10-fold higher or lower concentrations) potentiated the electrically evoked release of [³H]GABA but not the resting release, whereas metrazol (10^?4 to 10^?6 M) was found to inhibit resting but not electrically evoked release. Strychnine (10^?4 and 10^?5 M) and picro-toxin (10^?4 M) inhibited electrically evoked release.     

Release of Previously Incorporated γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

The release of gamma-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10-1,000 microM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.     

Modulation of striatal [3H]-glutamic acid release by dopaminergic drugs

Potassium induced release of [³H]-glutamic acid which had been recently taken up into striatal tissue in vitro was demonstrated and shown to be predominantly Ca²⁺-dependent. Dopamine and several DA agonists produced marked inhibition of this K⁺ -induced release which could be antagonised by butyrophenones. The effect of DA could not be mimicked by high concentrations of dibutyryl cAMP. These results are consistent with the existence of a D2-like DA receptor on striatal glutamatergic terminals capable of modulating the transmitter release.     

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

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

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.