Separation of carrier mediated and vesicular release of GABA from rat brain slices.

In this study the temperature dependence of [3H]GABA release from brain slices evoked by electrical field stimulation and the Na+/K+ ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S2/S1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 microM) inhibited stimulation-induced [3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 microM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H-labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine-treated mice. The overflow of [3H]DA and its 3H-metabolites exhibited similar distribution patterns in [3H]DA-preloaded striatum dissected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (-)-Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation-induced tritium efflux from striatum of untreated mice, whereas (+/-)-sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (-)-quinpirole and (+/-)-sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (+/-)-SKF-38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of endogenous GABA can occur through Ca-dependent and Ca-independent processes

Release of γ-aminobutyric acid (GABA) can be elicited by electrical field stimulation even in the absence of external Ca²⁺. Indeed, the release of GABA under such conditions is even higher than in the presence of Ca²⁺. To investigate the underlying mechanism of this phenomenon, the release of endogenous GABA from rat striatal slices was measured by high performance liquid chromatography with electro- chemical detection. Electrical stimulation at 2 Hz for 3 min elevated GABA efflux by 4.5-fold. Withdrawing external Ca²⁺ and adding 1 mM EGTA produced a small, transient increase in the basal efflux of GABA and increased electrically-evoked overflow 3-fold. Tetrodotoxin (5 μM) did not affect basal efflux in either normal or Ca²⁺-free conditions, but abolished electrically-evoked release. In the presence of normal Ca²⁺, nipecotic acid (1 mM) enhanced both spontaneous efflux and evoked overflow. Nipecotic acid also increased spontaneous release when external Ca²⁺ was removed. However, in the absence of Ca²⁺, nipecotic acid failed to increase electrically evoked GABA overflow. These results suggest that there exists a Ca²⁺-independent process for GABA release via the same carrier system that is utilized for high affinity GABA uptake.     

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.     

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Various putative striatal transmitters and related compounds were studied for their effects on the release of gamma-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal outflow whereas oxotremorine caused no consistent change; the effect of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, alpha 2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.     

N-type calcium channels are involved in the dopamine releasing effect of nicotine

Mouse striatum was incubated with [³H]dopamine ([³H]DA) and superfused with and the tritium efflux induced by nicotine, electrical stimulation, or simultaneous nicotine and electrical stimulation was measured, to characterize the role of different Ca²⁺ channels in the transmitter release. Nicotine stimulation and electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotine or electrical stimulation increases the release of [³H]DA and that the outflow of³H-labeled metabolites was similar with the two different stimulation procedures. Removal of Ca²⁺ from the superfusate resulted in a marked reduction in the tritium release evoked by nicotine, whereas the electrical stimulation-evoked tritium release was completely dependent on external Ca²⁺. The L-and N-type calcium channel blockers omega-conotoxin GVIA and Cd²⁺ inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the L-type and T-type channel blockers diltiazem and Ni²⁺ did not alter release of [³H]DA. We conclude that N-type voltage-sensitive Ca²⁺ channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as Ca²⁺ and N-type voltage-sensitive calcium channels may simultaneously open up, and they additively increase free intracellular Ca²⁺ concentration.     

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.     

THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Differential Effects of Bromocriptine on Dopamine and Acetylcholine Release Modulatory Receptors

Rabbit neostriatal slices were prelabeled with [3H]dopamine (DA) and [14C]choline and then superfused. The electrical stimulation-evoked release of DA and of acetylcholine (ACh) was abolished by 0.33 microM tetrodotoxin and by low calcium concentrations (0.13 mM). Bromocriptine, a selective D2-DA receptor agonist, inhibited in a concentration-dependent manner the evoked overflow of DA and ACh, without affecting the basal efflux of both transmitters. The effects of bromocriptine were antagonized by sulpiride, a specific antagonist of D2-DA receptors. With stimulation at 0.3 Hz and 120 pulses, bromocriptine was eight times more potent in inhibiting the evoked overflow of DA (IC50: 11 nM) than that of ACh (IC50: 83 nM). Stimulations at 3 Hz and 360 pulses markedly reduced the potency of bromocriptine in inhibiting DA and ACh release, and diminished its selectivity for presynaptic receptors. These results indicate that DA receptors that modulate the release of DA and ACh are of the D2 subtype. The greater potency of bromocriptine at pre- than at postsynaptic sites suggests that these receptors may be different in quantity and/or quality [D2-alpha (presynaptic) versus D2-beta (postsynaptic)]. Finally, marked differences in the potency and efficacy of DA agonist actions on DA and ACh release modulatory receptors are obtained, depending on the parameters of stimulation used.     

Effects of Creatine on Synthesis and Release of γ-[3H]Aminobutyric Acid

Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of gamma-[3H]aminobutyric acid ([3H]GABA) as synthesized from [3H]glutamine or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA-alpha-oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitter GABA.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.