Release of [H]GABA evoked by glutamate agonists from hippocampal slices: effects of dithiothreitol and glutathione

The effects of dithiothreitol (DTT) and, reduced (GSH) and oxidized (GSSG), glutathione on the release of [³H]GABA evoked by glutamate and its agonists were studied in rat hippocampal slices. DTT had no effect on the basal release of [³H]GABA but it enhanced and prolonged the glutamate agonist-evoked release. This effect was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801), a noncompetitive NMDA antagonist, and blocked by Mg²⁺ ions. It was only slightly attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, and not affected by -(+)-2-amino-3-phosphonopropionate ( -AP3), a selective antagonist of the metabotropic glutamate receptor. The effect of DTT on the NMDA-evoked release of GABA was only slightly affected by extracellular Ca²⁺ but completely blocked by verapamil even in the absence of Ca²⁺. GSH and GSSG attenuated or abolished the effects of DTT on the agonist-induced release of [³H]GABA. The results imply that the enhanced and prolonged release of GABA evoked by the coexistence of DTT and excitatory amino acids and attenuated by endogenous GSH and GSSG is a consequence of sustained activation of the NMDA receptor-governed ionophores, which contain functional thiol groups. DTT, GSH and GSSG may regulate the redox state and accessibility of these groups. In addition to the influx of extracellular Ca²⁺, DTT mobilizes Ca²⁺ from intracellular pools distinct from those regulated by metabotropic glutamate receptors.     

Inhibition by sodium bromide of acetylcholine release and synaptic transmission in the superior cervical ganglion of the rat

In the superior cervical ganglion (SCG) of rats, the interaction of sodium bromide (NaBr) with various drugs which interfere with the GABA system, such as 3-(4-chlorophenyl)-4-aminobutyrate [( + )baclofen, Bac], ( + )bicuculline (Bic), picrotoxin (Pic) and chlorpromazine (CPZ), and the effects of NaBr on the K⁺-induced release of [³H]acetylcholine ([³H]ACh) were studied in vitro. The effects on the evoked potentials induced by preganglionic stimulation were analysed in situ. The in vitro experiments revealed that 1 mM NaBr inhibits both the basal and the K⁺-induced release of [³H]ACh in a Ca²⁺-dependent manner. This NaBr effect was additive with the similar effect of the GABA agonist Bac, but it could not be blocked with any of the drugs applied. In vivo, 1 mM NaBr depressed the amplitude of the evoked potentials in the SCG. It is concluded that, in the SCG of rats, NaBr interacts with the presynaptic and postsynaptic membranes. The inhibitory effects of NaBr on both the [³H]ACh release and the potentials evoked by preganglionic stimulation cannot be attributed to a direct interference with GABA receptor complexes; some other binding site/s on the presynaptic and postsynaptic membranes might be responsible for the bromide-induced reduction of the synaptic transmission in the SCG of rats.     

Homocysteate-Evoked Release of Acetylcholine from the Rabbit Retina

Abstract: The cholinergic amacrine cells of the rabbit retina can be labeled with [³H]choline and the activity of the cholinergic population monitored by following the release of [³H]acetylcholine. It has been proposed that l -homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [³H]acetylcholine. In magnesium-free medium, d -homocysteate was slightly more potent than the l -isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to l -homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked l -homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked d -homocysteate-evoked responses. Therefore, in the rabbit retina, l -homocysteate is an NMDA-preferring agonist, whereas d -homocysteate is a kainate-preferring agonist. In addition, we found that l -homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of l -homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of l -homocysteate at the bipolar-to-cholinergic amacrine cell synapse.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

The Effect of Colchicine and Cytochalasin B on the Release of Taurine from the Chick Retina

Abstract: The effect of colchicine (0.5 mM) and of cytochalasin B (10⁻⁴ M) on the release of [³⁵S]taurine from the isolated chick retina, upon stimulation by 68.5 mM-KCl, 10⁻⁵ M-veratridine and 10 mM-glutamate, was studied. Cytochalasin and colchicine effects on taurine release were compared with those on K⁺-stimulated release of [³H]dopamine and [³H]GABA. Colchicine caused a marked decrease of the [³⁵S]taurine release evoked by the three stimulatory agents; it also decreased [³H]dopamine release without affecting that of [³H]GABA. Cytochalasin B significantly decreased the efflux of [³⁵S]taurine stimulated by glutamate or veratridine without altering that evoked by 68.5 mM-KCl. Cytochalasin practically suppressed the [³H]dopamine-stimulated release and slightly decreased that of [³H]GABA. This drug produced a high increase in the spontaneous release of labeled GABA and taurine. These results suggest that the release of taurine and GABA from the chick retina probably occurs through different mechanisms. It is suggested that taurine release may be related to a process involving contractile proteins.     

Type A and B gaba receptors mediate inhibition of acetylcholine release from cholinergic nerve terminals in the superior cervical ganglion of rat

The effects of γ-amino-n-butyric acid (GABA), (+)bicuculline, isoguvacine and 3-(4-chlorophenyl)-4-aminobutyrate [(±)baclofen] on the K-induced release of [³H]acetylcholine (ACh) were studied in the superior cervical ganglia of the rat in vitro. GABA and isoguvacine inhibited [³H]ACh release and these inhibitions were reversible by (+)bicuculline. Furthermore, the release of [³H]ACh was also inhibited by (±)baclofen. In receptor-binding studies, binding of [³H]GABA to membrane preparations from the superior cervical ganglia was inhibited by both (±)baclofen and (+)bicuculline. It is concluded that the inhibitory effect of GABA on the release of ACh can be mediated by GABA_A(bicuculline-sensitive) and by GABA_B (baclofen-activated) receptors. Our findings are compatible with the existence of a non-synaptic GABAergic inhibitory system involving GABA_A and GABA_B receptors on cholinergic nerve terminals in the superior cervical ganglion of rat.     

THE FORMATION OF GLUTAMATE, ASPARTATE AND GABA IN THE RAT RETINA; GLUCOSE AND GLUTAMINE AS PRECURSORS

Abstract— The distribution of the neuroactive amino acids taurine, GABA, glycine, glutamate and aspartate, together with glutamine, have been studied in the rat retina. Peak levels of taurine were found in photoreceptor cells and of GABA and glycine in a retinal fraction enriched in amacrine cells and, synaptic terminals. In vitro , GABA formation from [³H]glutamine and [¹⁴C]glucose was also most prominent in this fraction; at 500 μ m [³H]glutamine was the better precursor.
Observations on metabolism in the photoreceptor cell layer of the tissue suggest an active turnover of glutamate, aspartate and GABA, and show that glutamine may serve as an alternative substrate to glucose here, perhaps via the GABA bypath.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

Effect of oxidative stress on excitatory amino acid release by cerebral cortical synaptosomes

Previous studies in our laboratory have suggested that an oxidation reaction is responsible for the actions of free radicals to decrease synaptic potentials. Recently we observed that free radicals both decreased depolarization-induced vesicular release and enhanced basal, nonvesicular release of the excitatory amino acid, [³H]L-glutamate. In order to evaluate the contribution of oxidative reactions to this latter effect, we evaluated the actions of the oxidizing agent chloramine-T on synaptosomal release of excitatory amino acids, using [³H]D-aspartate as the exogenous label. Basal and depolarization evoked [³H]D-aspartate release were calcium-independent and nonvesicular. Chloramine-T pretreatment significantly increased basal release, while having no effect on high K⁺-evoked release. These data suggest that an oxidative process can mimic the free radical increase of basal release, as well as the decrease in synaptic potentials. On the other hand, the calcium-independent-evoked release may involve a different mechanism. Our results demonstrate that under basal, nondepolarizing conditions, oxidative stress exerts an adverse effect on the presynaptic nerve terminal, resulting in an increased release of potentially damaging excitatory amino acid neurotransmitters.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

Multiple neurotransmitter systems influence the release of adenosine derivatives from the rabbit retina

Rabbit retinac preloaded with [³H]adenosine were superfused in vitro and the effect of neurotransmitter agonists and antagonists on the release of [³H]purines was studied. Glutamic acid, aspartic acid, kainic acid (KA), quisqualic acid (QUIS) and acid (NMDA) all stimulated the efflux of [³H] labelled and endogenous purines. Their effect was reduced in a Ca²⁺-free medium except when using a high concentration (100 μM) of KA, QUIS and NMDA. The effect of aspartic acid and of NMDA were blocked by 2-amino-7-phosphono-heptanoic acid (APH) and 2-amino-5-phosphono-valeric acid (APV). Carbachol also increased the release of adenosine-derived radioactivity and this effect was reduced by the removal of Ca²⁺ and by pretreatment with atropine. τ-Aminobutyric acid (GABA) and muscimol, induced a small increase in the release which was Ca²⁺-dependent and was blocked by bicuculline and picrotoxin. Dopamine elicited an increase in the release which was partially reduced in a Ca²⁺-free medium and was blocked by haloperidol. Glycine and 5-hydroxytryptamine (5-HT) also induced small but significant increases. The neurotransmitter antagonists had an effect of their own. Superfusion with APH and APV depressed the outflow of radioactivity whereas bicuculline, picrotoxin, strychnine and haloperidol enhanced it. The K⁺-evoked release of [³H]purines was reduced by haloperidol and by 5-HT. The observations indicate that stimulation of several important neurotransmitter receptors in the retina elicits the release of adenosine derivatives. The results with the antagonists also suggest that purines are continuously released as a result of a tonic activation of the respective membrane receptors.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [³H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC-3) increased the spontaneous efflux of [³H]choline. Electrical stimulation at 4/s increased the efflux of [³H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [³H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [³H]choline evoked by stimulation probably originates from stores of [³H]ACh synthetized during incubation.     

Characteristics of nitric oxide-evoked [H]taurine release from cerebral cortical neurons

Pharmacological characteristics of [³H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [³H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [³H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [³H]taurine releases, whereas the removal of calcium showed no alterations in the [³H]taurine release evoked by NO generators. β-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [³H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [³H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.     

Inhibition of Na+,K+-ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Abstract: Ouabain, an Na⁺,K⁺-ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca²⁺-independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [³H]ACh. Release of [³H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the ω-conotoxins GVIA (selective N-type calcium channel blocker) and MVIIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [³H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [³H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, ω-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [³H]ACh. Thus, extracellular Ca²⁺ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na⁺,K⁺-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.     

Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes

Abstract: To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K⁺ -evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 m M K⁺. In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, five sequential K⁺ -depolarizations caused a decremental release of preformed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [¹⁴C]ACh was unaltered while new ACh was being synthesized from exogenous [³H]choline. Since the evoked release of [³H]ACh was maintained while that of [¹⁴C]ACh was decreasing, the [³H]ACh/[¹⁴C]ACh ratio in perfusate increased with each successive depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.     

Serotonin and histamine receptor-mediated inhibition of serotonin and noradrenaline release in rat brain cortex under nimodipine treatment

Rat brain cortex slices preincubated with [³H]serotonin or [³H]noradrenaline (25 100 nmol/l each) were superfused and the effects of serotonin and histamine on the electrically (0.3 or 3 Hz) evoked tritium overflow were studied.

In slices preincubated with [³H]serotonin the extent of inhibition of the electrically (3 Hz) evoked tritium overflow produced by histamine was increased when the concentration of [³H]serotonin used for incubation was decreased. The evoked overflow tended to be lower in slices from 2-year-old rats than in slices from 6-month-old animals whereas the inhibitory effect of histamine on the evoked overflow did not differ. Treatment of rats with nimodipine for at least 6 weeks did not significantly affect the evoked overflow in slices from 6-month and 2-year-old rats nor did it significantly alter the serotonin- and histamine-mediated inhibition of the evoked overflow in slices from young adult rats. The extent of histamine-mediated inhibition of the electrically evoked tritium overflow from slices (of young adult rats) preincubated with [³H]noradrenaline did not change when the concentration of [³H]noradrenaline used for incubation was decreased; the degree of inhibition markedly increased when the frequency of stimulation was lowered from 3 to 0.3 Hz. The inhibitory effect of histamine on the electrically (0.3 Hz) evoked overflow was mimicked by the H₃ receptor agonist R-(−)--methylhistamine and antagonized by the H₃ receptor antagonist thioperamide. The electrically evoked overflow and its inhibition by histamine were not affected by nimodipine, irrespective of whether the Ca²⁺ antagonist was administered in vivo (for at least 6 weeks) or added to the superfusion medium in vitro.

It is concluded that (1) the extent of the H₃ receptor-mediated effect in rat brain cortex slices can be markedly increased by lowering the concentration of the tracer in slices preincubated with [³H]serotonin and by lowering the stimulation frequency in slices preincubated with [³H]noradrenaline; (2) the H₃ receptor-mediated inhibition of serotonin release is not changed during aging and (3) nimodipine does not significantly influence serotonin release and noradrenaline release and their serotonin and/or histamine receptor-mediated modulation.     

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [³H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 n M ) significantly increased [³H]glutamate uptake, and this effect persisted in a Ca²⁺-free medium. On the other hand, melatonin significantly increased [³H]glutamate release in retinas excised at 2400 h, but this effect was Ca²⁺ sensitive. Melatonin significantly increased ⁴⁵Ca²⁺ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [³H]glutamate uptake, [³H]glutamate release, or ⁴⁵Ca²⁺ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'- O -dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [³H]glutamate uptake, [³H]glutamate release, and ⁴⁵Ca²⁺ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.     

N-Methyl-d-Aspartate Releases γ-Aminobutyric Acid from Rat Striatum In Vivo: A Microdialysis Study Using a Novel Preloading Method

Abstract: In vivo microdialysis was used in conjunction with a novel dual-label preloading method to monitor changes in extracellular levels of γ-aminobutyric acid (GABA) and glutamate due to N -methyl- d -aspartate (NMDA) infusion in the striatum of conscious, unrestrained rats. [¹⁴C]GABA and [³H]glutamate were applied in the dialysis stream for a preloading period of 30 min, after which dialysis perfusion was continued for up to 6 h and dialysate samples were collected for analysis by liquid scintillation spectrometry. NMDA (300 μ M in the dialysate) caused significant rises in both ¹⁴C and ³H content measured in the dialysates, the majority of which remained associated with the preloaded GABA and glutamate, respectively. The NMDA-evoked release of both GABA and glutamate was blocked by the specific NMDA receptor antagonist 3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP), indicating that the response was receptor mediated. The NMDA-stimulated release of glutamate was also totally abolished by concomitant application of the adenosine agonist 2-chloroadenosine or by prior frontal decortication. However, these two treatments caused little change in NMDA-evoked GABA release. These results show that NMDA causes release of GABA from the striatum in vivo by an NMDA receptor-mediated mechanism and that the majority of this release is not secondary to glutamate release from terminals of the corticostriate pathway. In addition, they confirm the results of previous studies investigating the effect of NMDA on endogenous glutamate release.