Negative allosteric modulators of AMPA-preferring receptors inhibit [(3)H]GABA release in rat striatum

The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective glutamate receptor agonist, on the release of previously incorporated [(3)H]GABA was examined in superfused striatal slices of the rat. The slices were loaded with [(3)H]GABA in the presence of beta-alanine (1 mM) and superfused with Krebs-bicarbonate buffer containing nipecotic acid (0.1 mM) and aminooxyacetic acid (0.1 mM) to inhibit GABA uptake and metabolism. AMPA (0.01 to 3 mM) increased basal [(3)H]GABA outflow and nipecotic acid potentiated this effect. The [(3)H]GABA releasing effect of AMPA was an external Ca(2+)-dependent process in the absence but not in the presence of nipecotic acid. Cyclothiazide (0.03 mM), a positive modulator of AMPA receptors, failed to evoke [(3)H]GABA release by itself, but it dose-dependently potentiated the [(3)H]GABA releasing effect of AMPA. The AMPA (0.3 mM)-induced [(3)H]GABA release was antagonized by NBQX (0.01 mM) in a competitive fashion (pA(2) 5.08). The negative modulator of AMPA receptors, GYKI-53784 (0.01 mM) reversed the AMPA-induced [(3)H]GABA release by a non-competitive manner (pD'(2) 5.44). GYKI-53784 (0. 01-0.1 mM) also decreased striatal [(3)H]GABA outflow on its own right, this effect was stereoselective and was not influenced by concomitant administration of 0.03 mM cyclothiazide. GYKI-52466 (0. 03-0.3 mM), another negative modulator at AMPA receptors, also inhibited basal [(3)H]GABA efflux whereas NBQX (0.1 mM) by itself was ineffective in alteration of [(3)H]GABA outflow.The present data indicate that AMPA evokes GABA release from the vesicular pool in neostriatal GABAergic neurons. They also confirm that multiple interactions may exist between the agonist binding sites and the positive and negative modulatory sites but no such interaction was detected between the positive and negative allosteric modulators. Since GYKI-53784, but not NBQX, inhibited [(3)H]GABA release by itself, AMPA receptors located on striatal GABAergic neurons may be in sensitized state and phasically controlled by endogenous glutamate. It is also postulated that these AMPA receptors are located extrasynaptically on GABAergic striatal neurons.     

Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons.

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog D-Ala2-Met5-enkephalinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential-independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential-evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non-action potential-dependent GABA release reveals a mechanism that contributes to reducing action potential-evoked GABA release, thereby decreasing synaptic inhibition.     

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.     

Okadaic acid and cyclosporin A modulate [(3)H]GABA release from rat brain synaptosomes

Rat brain synaptosomes were used to investigate the effect of okadaic acid, an inhibitor of protein phosphatase 1 and 2A, and cyclosporin A, an inhibitor of protein phosphatase 2B (calcineurin), on [(3)H]GABA release. Release of [(3)H]GABA was evoked by 4-aminopyridine in the presence of calcium and by alpha-latrotoxin in the presence and absence of calcium. Pretreatment of synaptosomes with 1 microM okadaic acid reduced [(3)H]GABA release evoked by 4-aminopyridine by about 40%. The effect of alpha-latrotoxin on [(3)H]GABA release was stimulated by okadaic acid. This stimulation was equal in both media. The stimulating effect of 4-aminopyridine and alpha-latrotoxin on [(3)H]GABA release was activated when synaptosomes were pretreated with cyclosporin A. Activation of 4-aminopyridine-evoked [(3)H]GABA release was observed at 1 microM cyclosporin A, but the toxin effect was enhanced only when concentration of cyclosporin A was increased to 10 microM. The level of cyclosporin A activation depended on alpha-latrotoxin concentrations used - a higher stimulating effect of cyclosporin A was observed with lower toxin concentration. These results suggest that in calcium medium 4-aminopyridine- and alpha-latrotoxin-evoked [(3)H]GABA release was realized by different mechanisms.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons

We report that kainate receptors are present on presynaptic GABAergic terminals contacting interneurons and that their activation increases GABA release. Application of kainate increased the frequency of miniature inhibitory postsynaptic currents recorded in CA1 interneurons. Local applications of glutamate but not of AMPA or NMDA also increased GABA quantal release. Application of kainate as well as synaptically released glutamate reduced the number of failures of GABAergic neurotransmission between interneurons. Thus, activation of presynaptic kainate receptors increases the probability of GABA release at interneuron-interneuron synapses. Glutamate may selectively control the communication between interneurons by increasing their mutual inhibition.     

Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses

Kainate receptors (KARs) are a poorly understood family of ionotropic glutamate receptors. A role for these receptors in the presynaptic control of transmitter release has been proposed but remains controversial. Here, KAR agonists are shown to enhance fiber excitability, and a number of experiments show that this is a direct effect of KARs on the presynaptic fibers. In addition, KAR activation inhibits evoked transmitter release from mossy fiber synapses. Synaptic release of glutamate from either neighboring mossy fiber synapses or associational/commisural (A/C) synapses results in the activation of these presynaptic ionotropic KARs. These results, along with previous studies, indicate that KARs, through the endogenous release of glutamate, mediate excitatory postsynaptic potentials (EPSPs), alter presynaptic excitability, and modulate transmitter release.     

Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism.

In the spinal cord dorsal horn, excitatory sensory fibers terminate adjacent to interneuron terminals. Here, we show that kainate (KA) receptor activation triggered action potential-independent release of GABA and glycine from dorsal horn interneurons. This release was transient, because KA receptors desensitized, and it required Na+ entry and Ca2+ channel activation. KA modulated evoked inhibitory transmission in a dose-dependent, biphasic manner, with suppression being more prominent. In recordings from isolated neuron pairs, this suppression required GABA(B) receptor activation, suggesting that KA-triggered GABA release activated presynaptic GABA(B) autoreceptors. Finally, glutamate released from sensory fibers caused a KA and GABA(B) receptor-dependent suppression of inhibitory transmission in spinal slices. Thus, we show how presynaptic KA receptors are linked to changes in GABA/glycine release and highlight a novel role for these receptors in regulating sensory transmission.     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

Activation of presynaptic ionotropic glutamate receptors stimulates GABA release from hippocampal and cortical nerve terminals in rats

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

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.     

Characteristics of GABA release modified by glutamate receptors in mouse hippocampal slices

The major part of hippocampal innervation is glutamatergic, regulated by inhibitory GABA-releasing interneurons. The modulation of [(3)H]GABA release by ionotropic and metabotropic glutamate receptors and by nitric oxide was here characterized in superfused mouse hippocampal slices. The ionotropic glutamate receptor agonists kainate, N-methyl-D-aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate potentiated the basal GABA release. These effects were blocked by their respective antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX), dizocilpine and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX), indicating receptor-mediated mechanisms. The NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), sodiumnitroprusside and hydroxylamine enhanced the basal GABA release. Particularly the sodiumnitroprusside-evoked release was attenuated by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) and the inhibitor of soluble guanylyl cyclase 1H-(1,2,4)oxadiazolo(4,3a)quinoxalin-1-one (ODQ), indicating the involvement of the NO/cGMP pathway. This inference is corroborated by the enhancing effect of zaprinast, a phosphodiesterase inhibitor, which is known to increase cGMP levels. The K(+)-stimulated hippocampal GABA release was reduced by the groups I and III agonists of metabotropic glutamate receptors (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4), which effects were abolished by their respective antagonists (RS)-1-aminoindan-1,5-dicarboxylate (AIDA) and (RS)-2-cyclopropyl-4-phosphonophenylglycine (CPPG), again indicating modification by receptor-mediated mechanisms.     

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

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.     

Reactive oxygen species induced by presynaptic glutamate receptor activation is involved in [H]GABA release from rat brain cortical nerve terminals

We investigated the production of reactive oxygen species (ROS) as a response to presynaptic glutamate receptor activation, and the role of ROS in neurotransmitter (GABA) release. Experiments were performed with rat brain cortical synaptosomes using glutamate, NMDA and kainate as agonists of glutamate receptors. ROS production was evaluated with the fluorogenic compound dichlorodihydrofluorescein diacetate (H₂DCF-DA), and GABA release was studied using synaptosomes loaded with [³H]GABA. All agonists were found to stimulate ROS production, and specific antagonists of NMDA and kainate/AMPA receptors, dizocilpine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-done (CNQX), significantly inhibited the ROS increase. Spontaneous as well as agonist-evoked ROS production was effectively attenuated by diphenyleneiodonium (DPI), a commonly used potent inhibitor of NADPH oxidase activity, that suggests a high contribution of NADPH-oxidase to this process. The replacement of glucose with pyruvate or the simultaneous presence of both substrates in the medium led to the decrease in spontaneous and NMDA-evoked ROS production, but to the increase in ROS production induced by kainate. Scavenging of agonist-evoked ROS production by a potent antioxidant N-acetylcysteine was tightly correlated with the inhibition of agonist-evoked GABA release. Together, these findings show that the activation of presynaptic glutamate receptors induces an increase in ROS production, and there is a tight correlation between ROS production and GABA secretion. The pivotal role of kainate/AMPA receptors in ROS production is under discussion.     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Characterization of divalent cation-induced [3H]Acetylcholine release from EGTA-treated rat hippocampal synaptosomes

Calcium-naive synaptosomes were used to assess the effects of divalent cations on [³H]acetylcholine release from rat hippocampal homogenates. Following equilibration with calcium-free buffer (containing 10M EGTA), calcium reversibly increased [³H]acetylcholine efflux (up to five-fold) while causing no measurable efflux of lactate dehydrogenase. When substituted for calcium, strongtium and barium behaved similarly although barium exhibited three-fold greater efficacy. In the presence of elevated potassium, 4-aminopyridine or tetraethylammonium, the secretagogue efficacy of calcium (but not barium) was markedly increased. The release-promoting effects of both cations were inhibited by lanthanum, magnesium, cadmium, and -conotoxin but were insensitive to nifedipine and cobalt (both 10 M). In addition, stimulation of muscarnic cholinergic autoreceptors substantially inhibited both calcium and barium-evoked [³H]acetylcholine release. Taken together, these results indicate that cation-evoked transmitter release from calcium-naive synaptosomes is subject to normal neuroregulatory mechanisms and therefore should be useful for investigating presynaptic modulation of neuronal exocytosis.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.