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Removal from receptors of neurotransmitters just released into synapses is one of the major steps in neurotransmission. Transporters situated on the plasma membrane of nerve endings and glial cells perform the process of neurotransmitter (re)uptake. Because the density of transporters in the membranes can fluctuate, transporters can determine the transmitter concentrations at receptors, thus modulating indirectly the excitability of neighboring neurons. Evidence is accumulating that neurotransmitter transporters can exhibit multiple functions. Being bidirectional, neurotransmitter transporters can mediate transmitter release by working in reverse, most often under pathological conditions that cause ionic gradient dysregulations. Some transporters reverse to release transmitters, like dopamine or serotonin, when activated by ‘indirectly acting’ substrates, like the amphetamines. Some transporters exhibit as one major function the ability to capture transmitters into nerve terminals that perform insufficient synthesis. Transporter activation can generate conductances that regulate directly neuronal excitability. Synaptic and non-synaptic transporters play different roles. Cytosolic Na + elevations accompanying transport can interact with plasmalemmal or/and mitochondrial Na + /Ca 2+ exchangers thus generating calcium signals. Finally, neurotransmitter transporters can behave as receptors mediating releasing stimuli able to cause transmitter efflux through multiple mechanisms. Neurotransmitter transporters are therefore likely to play hitherto unknown roles in multiple therapeutic treatments. [ABSTRACT FROM AUTHOR]

Abstract: The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [3H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K+-evoked overflow of [3H]glycine in a concentration-dependent manner (EC50 about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC50 about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [3H]glycine overflow with extraordinary potency (EC50 about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [3H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’. [Copyright &y& Elsevier]

Abstract: GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [3H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [3H]glycine release were characterized. GABA stimulated [3H]glycine release in a concentration-dependent manner (EC50 =8.26μM). The GABA-evoked release was insensitive to GABAA and GABAB receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca2+entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca2+-independent release involved mitochondrial Ca2+, as it was prevented by the Na+/Ca2+exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca2+ did not trigger exocytotic release because the [3H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [3H]glycine so that less [3H]glycine can become cytosolic when GABA taken up exchanges with [3H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [3H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca2+-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition. [Copyright &y& Elsevier]

J. Neurochem. (2010) 114, 647–653. Glycine transporter 1 (GLYT1) and GLYT2 are the glycine transporters in CNS. While GLYT2 is largely expressed in glycinergic neurons, GLYT1 has long been considered to be exclusively present in glial cells. There is increasing evidence that significant amounts of the ‘glial’ transporter also exist on neurons, particularly on pre-synaptic nerve endings of glutamatergic neurons. The functions of ‘neuronal GLYT1’ may be manifold and are discussed in this review. Of major interest are the interactions between neuronal GLYT1 and glutamatergic receptors of the NMDA type the activity of which is modulated not only by astrocytic GLYT1 but also by neuronal GLYT1. Pathophysiological roles and therapeutic implications of neuronal GLYT1 are emerging from recent studies with genetically modified mice, particularly with animals lacking forebrain neuron-specific GLYT1 transporters. These mutant mice exhibit promnesic phenotypes reflecting enhancement of NMDA receptor function, as it occurs following administration of GLYT1 inhibitors. Inactivation of neuronal GLYT1 in the forebrain may represent an effective therapeutic intervention for the treatment of schizophrenia. [ABSTRACT FROM AUTHOR]

It is now well established that nitric oxide is involved in a variety of physiopathological processes in the central nervous system, which mainly result from the interaction of this gaseous molecule with the heme group of soluble guanylyl cyclase and the elevation of intracellular cGMP in target neurons. During the last decade, several studies have monitored extracellular cGMP, by means of intracerebral microdialysis, to investigate in vivo the functioning and modulation of this neurochemical pathway under different experimental conditions and in various brain regions. In this review, we summarise some of the most relevant results obtained in this research field. [Copyright &y& Elsevier]

Transporters able to recapture released neurotransmitters into neurons can no longer be considered as cell-specific neuronal markers. In fact, colocalization on one nerve terminal of transporters able to selectively recapture the released endogenously synthesized transmitter (homotransporters) and of transporters that can selectively take up transmitters/modulators originating from neighboring structures (heterotransporters) has been demonstrated to occur on several families of nerve terminals. Activation of heterotransporters often increases the release of the transmitter stored in the terminals on which the heterotransporters are localized. The release caused by heterotransporter activation takes place through multiple mechanisms including exocytosis, either dependent on external Ca2+ or on Ca2+ mobilized from intraterminal stores, and homotransporter reversal. Homocarrier-mediated release elicited by heterocarrier activation represents a clear case of transporter–transporter interaction. Although the functional significance of transporter coexpression on one nerve terminal remains to be established, it may in some instances reflect cotransmission. In other cases, heterotransporters may mediate modulation of basal transmitter release in addition to the modulation of the evoked release brought about by presynaptic heteroreceptors. Heterotransporters are also increasingly reported to exist on neuronal soma/dendrites. With the exception of EAAT4, the glutamate transporter/chloride channel situated on GABAergic Purkinje cells in the cerebellum, the functions of somatodendritic heterocarriers is not understood. [Copyright &y& Elsevier]

Small molecules present during brain tissue homogenization are known to be entrapped within sub-sequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx-LC), pertussis toxin (PTx), anti-syntaxin, or anti-SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx-LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (-)-baclofen inhibited amino acid overflow more potently in standard than in PTx-containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti-syntaxin or anti-SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane-impermeant agents of different size in studies of presynaptic mechanisms. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2011) 119, 50-63. Abstract Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [3H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca2+-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 μM), but not of 4-AP (1 mM), involved also external Ca2+-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca2+-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca2+ channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca2+-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na+/Ca2+ exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca2+ originated through mitochondrial Na+/Ca2+ exchangers. Ca2+-induced Ca2+ release (CICR) mediated by inositoltrisphosphate receptors (InsP3Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP3R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract: GABAB receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABAB receptor antagonist CGP35348 and, in part, the compound CGP52432, are now found to inhibit on their own the K+-evoked exocytosis of glycine when added at low micromolar concentrations to superfused mouse glycinergic nerve endings prelabelled with [3H]glycine through GLYT2 transporters. CGP35348 inhibited [3H]glycine release both in spinal cord and in hippocampus, but was also able to prevent the inhibitory effect of (−)-baclofen; CGP52432 exhibited intrinsic activity only in the hippocampus; in spinal cord, it behaved exclusively as a silent orthosteric antagonist by blocking the release inhibition brought about by (−)-baclofen. The intrinsic activity of CGP35348 in spinal cord was not prevented by CGP52432, indicating that CGP35348 is not a partial GABAB agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABAB antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100–300μM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABAB1 (GABAB1−/− mice) or the GABAB2 (GABAB2−/− mice) subunit, the evoked exocytosis of [3H]glycine was no longer inhibited by (−)-baclofen, whereas the intrinsic activity of CGP35348 and CGP52432 was not decreased. Activation of unknown sites on glycinergic terminals is likely to be involved. These unexpected effects should not be ignored when interpreting results obtained with the above GABAB receptor antagonists. [Copyright &y& Elsevier]

Abstract: Glycine release and modulation of glycine exocytosis by presynaptic receptors have been rarely studied. We here investigate on the existence and the pharmacological profile of GABAB receptors regulating glycine release in mouse spinal cord and hippocampus. Synaptosomes were preincubated with [3H]glycine in presence of the glycine transporter 1 inhibitor NFPS, in order to study release from glycinergic terminals selectively labelled through the glycine transporter 2, and depolarized in superfusion with concentrations of KCl (12 and 15mM) previously found to elicit exocytosis of glycine. Exposure of synaptosomes to (−)-baclofen caused inhibition of [3H]glycine overflow: EC50 =0.62±0.08μM, in the spinal cord and EC50 =0.58±0.07μM, in the hippocampus. The effect of 3μM (−)-baclofen in the spinal cord was prevented by the GABAB receptor antagonists CGP 52432 (IC50 =22.6±2.15μM) and CGP 35348 (IC50 =5.99±1.21μM), whereas phaclofen was ineffective. In the hippocampus, the CGP antagonists were more potent than in the spinal cord, their IC50 values amounting to 0.81±0.08μM and to 0.94±0.09μM, respectively; phaclofen (100–300μM) was a weak antagonist. It is concluded that: (i) glycinergic nerve terminals in spinal cord and hippocampus possess GABAB receptors mediating inhibition of the evoked glycine exocytosis; (ii) the pharmacological profiles of the receptors are qualitatively similar in the two regions; (iii) low concentrations of GABAB antagonists appear able to preferentially inhibit the receptors located on hippocampal glycinergic nerve endings. [Copyright &y& Elsevier]

It is known that nicotine can activate several subtypes of release-regulating presynaptic nicotinic receptors (nAChRs) including those situated on central noradrenergic, dopaminergic, cholinergic and glutamatergic axon terminals. The objective of this study was to investigate the effects of chronic administration of (–)nicotine on the function of the above autoreceptors and heteroreceptors using rat superfused synaptosomes. In hippocampal synaptosomes prelabelled with[3H]noradrenaline (NA) the nicotine-evoked overflow of[3H]NA was higher in rats treated with nicotine for 10 days (via osmotic mini-pumps) than in vehicle-treated rats. In striatal synaptosomes, prelabelled with[3H]dopamine (DA), chronic nicotine did not modify the releasing effect of nicotine. No significant change was observed in experiments with synaptosomes from nucleus accumbens prelabelled with[3H]DA. Exposure of hippocampal synaptosomes prelabelled with[3H]choline to nicotine elicited release of[3H]acetylcholine; this effect was almost abolished in synaptosomes from animals administered nicotine for 10 days, suggesting down-regulation of nicotinic autoreceptors. In hippocampal synaptosomes prelabelled with[3H]d-aspartate, the releasing effect of epibatidine following chronic nicotine treatment did not differ from that in controls. The K+-evoked exocytotic release of the neurotransmitters tested was not modified by long-term nicotine administration. The results show that chronic nicotine differentially affects the function of release-regulating nAChR subtypes. [ABSTRACT FROM AUTHOR]

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg2+ (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg2+-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg2+, while dizocilpine, cis-4-[phosphomethyl]- piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg2+ strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4¢-fluorophenyl)- 3-(4¢-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [3H]glycine was taken up during superfusion, while lowering the external concentration of Na+, the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors colocalize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 343-352. Abstract We have investigated the spontaneous and the depolarisation-induced release of [3H] d-aspartate ([3H] d-ASP), a non-metabolisable analogue of glutamate, in spinal cord slices, synaptosomes and gliosomes from mice with experimental autoimmune encephalomyelitis (EAE) at 13, 21 and 55 days post-immunisation (d.p.i.), representing onset, peak and chronic phases of the pathology. At 13 and 21 d.p.i., the KCl-evoked, calcium-dependent overflow of [3H] d-ASP in spinal cord slices was significantly lower (30-40%), whereas at 55 d.p.i. it was significantly higher (30%), than that elicited in matched controls. When the release was measured from spinal cord synaptosomes and gliosomes in superfusion, a different picture emerged. The spontaneous and the KCl(15 mM)-induced release of [3H] d-ASP were significantly increased both in synaptosomes (17% and 45%, respectively) and gliosomes (26% and 25%, respectively) at 21, but not at 13, d.p.i. At 55 d.p.i., the KCl-induced [3H] d-ASP release was significantly increased (40%) only in synaptosomes. Finally, uptake of [3H] d-ASP was markedly (50-60%) increased in spinal cord synaptosomes, but not in gliosomes, obtained from EAE mice at 21 d.p.i., whereas no differences could be detected at 13 d.p.i. Our data indicate that glutamatergic neurotransmission is altered in the spinal cord of EAE mice. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 1074–1084. Energy deprivation during ischemia causes dysregulations of ions, particularly sodium, potassium and calcium. Under these conditions, release of neurotransmitters is often enhanced and can occur by multiple mechanisms. The aim of this work was to characterize the modes of exit of glycine and GABA from nerve endings exposed to stimuli known to reproduce some of the ionic changes typical of ischemic conditions. Their approach was chosen instead of application of ischemic conditions because the release evoked during ischemia is mechanistically too heterogeneous. Mouse hippocampus and spinal cord synaptosomes, pre-labeled with [3H]glycine or [3H]GABA, were exposed in superfusion to 50 mM KCl or to 10 μM veratridine. The evoked overflows differed greatly between the two transmitters and between the two regions examined. Significant portions of the K+- and the veratridine-evoked overflows occurred by classical exocytosis. Carrier-mediated release of GABA, but not of glycine, was evoked by high K+; GABA and, less so, glycine were released through transporter reversal by veratridine. External calcium-dependent overflows were only in part sensitive to ω-conotoxins; significant portions occurred following reversal of the plasmalemmal Na+/Ca2+ exchanger. Finally, a relevant contribution to the overall transmitter overflows came from cytosolic calcium originating through the mitochondrial Na+/Ca2+ exchanger. To conclude, ionic dysregulations typical of ischemia cause neurotransmitter release by heterogeneous mechanisms that differ depending on the transmitters and the CNS regions examined. [ABSTRACT FROM AUTHOR]

Background: Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release. Methodology/Findings: Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability. Conclusions/Significance: Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs. [ABSTRACT FROM AUTHOR]

The effects of the recombinant chemokine human RANTES (hRANTES) on the release of glutamate from human neocortex glutamatergic nerve endings were investigated. hRANTES facilitated the spontaneous release of d [³H]D-aspartate ([³H]D-ASP) by binding Pertussis toxin-sensitive G-protein-coupled receptors (GPCRs), whose activation caused Ca2+ mobilization from inositol trisphosphate-sensitive stores and cytosolic tyrosine kinase-mediated phosphorylations. Facilitation of release switched to inhibition when the effects of hRANTES on the 12mMK+-evoked [³H]D-ASP exocytosis were studied. Inhibition of exocytosis relied on activation of Pertussis toxin-sensitive GPCRs negatively coupled to adenylyl cyclase. Both hRANTES effects were prevented by met-RANTES, an antagonist at the chemokine receptors (CCRs) of the CCR1, CCR3, and CCR5 subtypes. Interestingly,humanneocortex glutamatergic nerve endings seem to possess all three receptor subtypes. Blockade of CCR1 and CCR5 by antibodies against the extracellular domain of CCRs prevented both the hRANTES effect on [ 3H]D-ASP release, whereas blockade of CCR3 prevented inhibition, but not facilitation, of release. The effects ofRANTESon the spontaneous and the evoked release of [³H]D-ASP were also observed in experiments with mouse cortical synaptosomes, which may therefore represent an appropriate animal model to study RANTES-induced effects on neurotransmission. It is concluded that glutamate transmission can be modulated in opposite directions by RANTES acting at distinct CCR receptor subtypes coupled to different transduction pathways, consistent with the multiple and sometimes contrasting effects of the chemokine. [ABSTRACT FROM AUTHOR]

Reduced activity of the mitochondrial respiratory chain and in particular of complex I is implicated not only in the etiology of Parkinson's disease but also in other forms of parkinsonism in which striatal neurodegeneration occurs, such as progressive supranuclear palsy. The pesticide rotenone inhibits mitochondrial complex I and reproduces features of these basal ganglia neurological disorders in animal models. We have characterized the electrophysiological effects of rotenone in the striatum as well as potential neuroprotective strategies to counteract the detrimental effects of this neurotoxin. We found that rotenone causes a dose-dependent and irreversible loss of the corticostriatal field potential amplitude, which was related to the development of a membrane depolarization/inward current in striatal spiny neurons, coupled to an increased release of both excitatory amino acids and dopamine (DA). In particular, we have investigated whether glutamate, DA, and GABA systems might represent possible targets for neuroprotection against rotenone-induced striatal neuronal dysfunction. Interestingly, whereas modulation of glutamatergic transmission was not neuroprotective, blockade of D2-like but not D1-like DA receptors significantly reduced the rotenone-induced effects via a GABA-mediated mechanism. In addition, because antiepileptic drugs (AEDs) modulate multiple transmitter systems, we have analyzed the possible neuroprotective effects of some AEDs against rotenone. We found that carbamazepine, unlike other tested AEDs, exerts a potent neuroprotective action against rotenone-induced striatal neuronal dysfunction. This neuroprotection was observed at therapeutically relevant concentrations requiring endogenous GABA. Differential targeting of GABAergic transmission may represent a possible therapeutic strategy against basal ganglia neurodegenerative disorders involving mitochondrial complex I dysfunction. [ABSTRACT FROM AUTHOR]

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [3H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [3H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [3H]DA overflow. The [3H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [3H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M5 mAChRs co-existing on the same dopaminergic nerve endings. [ABSTRACT FROM AUTHOR]

Abstract: It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the ‘neuronal’ GLYT2 contributed more efficiently than the ‘glial’ GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord. [Copyright &y& Elsevier]

Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]d-aspartate ([3H]d-ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] ≤ 15 mmol/L provoked [3H]d-ASP and glutamate overflows almost totally dependent on external Ca2+. Consistent with release by exocytosis, the overflow of [3H]d-ASP evoked by 12 mmol/L K+ was sensitive to clostridial toxins. The overflows evoked by 35/50 mmol/L K+ remained external Ca2+-dependent by more than 50%. The Ca2+-independent components of the [3H]d-ASP overflows evoked by [K+] > 15 mmol/L were prevented by the glutamate transporter inhibitorsdl- threo-beta-benzyloxyaspartate (dl-TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+-independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+-evoked release of endogenous glutamate or [3H]D-ASP was inhibited bydl-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked bydl-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia. [ABSTRACT FROM AUTHOR]

Abstract: Previous evidences showed that, besides noradrenaline (NA) and 5-hydroxytryptamine (5-HT), glutamate transmission is involved in the mechanism of action of antidepressants (ADs), although the relations between aminergic and glutamatergic systems are poorly understood. The aims of this investigation were to evaluate changes in the function of glutamate AMPA and NMDA receptors produced by acute and chronic administration of the two ADs reboxetine and fluoxetine, selective inhibitors of NA and 5-HT uptake, respectively. Rats were treated acutely (intraperitoneal injection) or chronically (osmotic minipump infusion) with reboxetine or fluoxetine. Isolated hippocampal nerve endings (synaptosomes) prepared following acute/chronic treatments were labelled with [3H]NA or [3H]5-HT and [3H]amine release was monitored during exposure in superfusion to NMDA/glycine, AMPA or K+-depolarization. Acute and chronic reboxetine reduced the release of [3H]NA evoked by NMDA/glycine or by AMPA. The NMDA/glycine-evoked release of [3H]NA was also down-regulated by chronic fluoxetine. Only acute, but not chronic, fluoxetine inhibited the AMPA-evoked release of [3H]5-HT. The release of [3H]NA and [3H]5-HT elicited by K+-depolarization was almost abolished by acute reboxetine or fluoxetine, respectively, but recovered during chronic ADs administration. ADs reduced NMDA receptor-mediated releasing effects in noradrenergic terminals after acute and chronic administration, although by different mechanisms. Chronic treatments markedly reduced the expression level of NR1 subunit in synaptic membranes. The noradrenergic and serotonergic release systems seem to be partly functionally interconnected and interact with glutamatergic transmission to down-regulate its function. The results obtained support the view that glutamate plays a major role in AD activity. [Copyright &y& Elsevier]

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [3H]d-aspartate form gliosomes in a concentration-dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [3H]d-aspartate was independent of modifications of cytosolic Ca2+ , but it was blocked bydl-threo-β-benzyloxyaspartate (dl-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca2+-independent anddl-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca2+. Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca2+. These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3′,5′-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1μM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1μM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1μM). Dependency of the 1μM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1μM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production. [Copyright &y& Elsevier]

To understand the events underlying the clinical efficacy of deep brain stimulation (DBS) of the subthalamic nucleus (STN), electrophysiological recordings and microdialysis evaluations were carried out in the substantia nigra pars reticulata (SNr), one of the two basal ganglia (BG) nuclei targeted by STN output, in patients with Parkinson's disease (PD). Clinically effective STN-DBS caused a significant increase of the SNr firing rate. The poststimulus histogram (PSTH) showed an excitation peak at 1.92–3.85 ms after the STN stimulus. The spontaneous discharge of SNr neurons was driven at the frequency of the stimulation (130 Hz), as shown in the autocorrelograms (AutoCrl). The fast Fourier transform (FFT) analysis showed a peak at 130 Hz, and a less pronounced second one at 260 Hz. Accordingly, in the distribution of the interspike intervals (ISIs), the mode was earlier, and skewness more asymmetric. Biochemically, the increased excitatory driving from the STN was reflected by a clear-cut increase in cyclic guanosine 3',5'-monophosphate (cGMP) levels in the SNr. These results indicate that the beneficial effect of DBS in PD patients is paralleled with a stimulus-synchronized activation of the STN target, SNr. Our findings suggest that, during STN-DBS, a critical change towards a high-frequency oscillatory discharge occurs. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine triphosphate (ATP) has been reported to enhance the release of glutamate by acting at P2X presynaptic receptors. Acetylcholine (ACh) can elicit glutamate release through presynaptic nicotinic cholinergic receptors (nAChRs) of the α7 subtype situated on glutamatergic axon terminals, provided that the terminal membrane is weakly depolarized. Considering that ATP and ACh are co-transmitters, we here investigate on the possibility that P2X and nAChRs co-exist and interact on the same glutamatergic nerve endings using purified rat neocortex synaptosomes in superfusion. ATP evoked Ca2+-dependent release of preaccumulated d-[3H]aspartate ([3H]d-ASP) as well as of endogenous glutamate; (−)-nicotine, inactive on its own, potentiated the ATP-evoked release. The ATP analogue benzoylbenzoylATP (BzATP) behaved like ATP, but was ∼30 times more potent; the potentiation of the BzATP-evoked release was blocked by methyllycaconitine or α-bungarotoxin. Adding inactive concentrations of (−)-nicotine, epibatidine or choline together with inactive concentrations of BzATP resulted in significant elevation of the [3H]d-ASP release mediated by α7 nAChRs. To conclude, P2X7 receptors and α7 nAChRs seem to co-exist and interact on rat neocortex glutamatergic terminals; in particular, P2X7 receptors exert a permissive role on the activation of α7 nAChRs, suggesting that ATP may not only evoke glutamate release on its own, but may also regulate the release of the amino acid elicited by ACh. [Copyright &y& Elsevier]

Abstract: Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2–GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2–PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2–NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated. [Copyright &y& Elsevier]

Abstract: The existence of metabotropic glutamate receptors (mGluRs) on hippocampal noradrenergic nerve terminals and their interaction with coexisting nicotinic acetylcholine receptors (nAChRs) were investigated in superfused rat synaptosomes using [3H]-noradrenaline ([3H]-NA) release as a readout. The selective agonist of group I mGluRs, (S)-3,5-dihydroxyphenylglycine (DHPG), inactive on its own, acquired ability to release [3H]-NA when added together with (−)-nicotine. The effect of DHPG was prevented by 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of mGluR5, but not by 7-(hydroxyimino)cyclopropane[b]chromen-1-carboxylate ethyl ester (CPCCOEt), selective antagonist of mGluR1. The [3H]-NA release evoked by (−)-nicotine plus DHPG was totally abrogated by the nAChR antagonist mecamylamine. Veratrine mimicked the permissive role of (−)-nicotine on the activation of mGluR5 mediating [3H]-NA release. The mGluR5-mediated component of the [3H]-NA release provoked by DHPG plus (−)-nicotine was blocked by xestospongin C, a selective antagonist of inositoltrisphosphate (IP3) receptors. It can be concluded that (i) release-enhancing mGluRs of subtype 5 exist on hippocampal noradrenergic axon terminals; (ii) activation of mGluR5 to mediate IP3-dependent NA release requires activation of depolarizing nAChRs coexisting on the same terminals. [Copyright &y& Elsevier]

Abstract: Amyotrophic lateral sclerosis is a progressive and fatal neurodegenerative disease, involving both upper and lower motor neurons, the cause of which is obscure, although glutamate (GLU)-induced excitotoxicity has been suggested to play a major role. We studied the release of [3H]d-aspartate ([3H]d-ASP) and endogenous glutamate evoked by glycine (GLY) or GABA from spinal cord synaptosomes in mice expressing a mutant form of human SOD1 with a Gly93Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, in mice expressing the non-mutated form of human SOD1 [SOD1(+)], and in non-transgenic littermates [SOD1(−)/G93A(−)]. In parallel experiments, we also studied the release of [3H]GABA evoked by GLY and that of [3H]GLY evoked by GABA. Mutant mice were killed at advanced phase of pathology or during the pre-symptomatic period. In SOD1(−)/G93A(−) or SOD1(+) mice GLY evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]GLY, release. The GLY-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]GLY, were more pronounced in SOD1-G93A(+) than in SOD1(+) or SOD1(−)/G93A(−) mice. Furthermore, the excessive potentiation of [3H]d-ASP by GLY or GABA was already present in asymptomatic 30–40 day-old SOD1-G93A(+) mice. The releases of endogenous glutamate and GABA also were enhanced by GLY and the GLY-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in control animals. Potentiation of the spontaneous amino acid release is likely to be mediated by activation of a GLY or a GABA transporter, since the effect of GLY was counteracted by the GLY transporter blocker glycyldodecylamide but not by the GLY receptor antagonists strychnine and 5,7-dichlorokynurenate while the effect of GABA was diminished by the GABA transporter blocker SKF89976-A but not by the GABA receptor antagonists SR9531 and CGP52432. It is concluded that the glutamate release machinery seems excessively functional in SOD1-G93A(+) animals. [Copyright &y& Elsevier]

Using intracerebral microdialysis, we reported previously that acutein vivoactivation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) andF2-isoprostane 15-F2t-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites– produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,– was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage followingin vivoacute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F2t-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity. [ABSTRACT FROM AUTHOR]

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mM KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr 286-phosphorylated αCaM kinase II (α-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals. [ABSTRACT FROM AUTHOR]

The effects of pH changes on the basal and evoked release of[3H]noradrenaline ([3H]NA) and[3H]5-hydrohytryptamine ([3H]5-HT) from hippocampal synaptosomes and of[3H]dopamine ([3H]DA) and[3H]acetylcholine ([3H]ACh) from striatal and cortical synaptosomes were investigated in rat brain.Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four[3H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of[3H]NA,[3H]5-HT and[3H]DA, but not that of[3H]ACh. In contrast, lowering pH to 6.4 decreased the K+-evoked overflows of[3H]NA,[3H]5-HT,[3H]DA and[3H]ACh.AMPA released transmitters in a Ca2+-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca2+ or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of[3H]NA,[3H]5-HT,[3H]DA and[3H]ACh.Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of[3H]NA,[3H]DA and[3H]5-HT, whereas the proton-insensitive AMPA-evoked release of[3H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4.To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H+ ions may prevent desensitization of some AMPA receptor subtypes.British Journal of Pharmacology (2005) 144, 293-299. doi:10.1038/sj.bjp.0705960 Published online 27 September 2004 [ABSTRACT FROM AUTHOR]

Abstract: Glycine concentration-dependently elicited [3H]d-aspartate ([3H]d-ASP) release from superfused mouse spinal cord synaptosomes. Glycine effect was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was prevented by the glycine transporter blocker glycyldodecylamide. Glycine also evoked release of endogenous glutamate, which was sensitive to glycyldodecylamide and abolished in low-Na+ medium. Experiments with purified synaptosomes and gliasomes show that the glycine-evoked [3H]d-ASP release largely originates from glutamatergic nerve terminals. The glycine-evoked [3H]d-ASP release was halved by NFPS, a selective blocker of GLYT1 transporters, or by Org 25543, a selective GLYT2 blocker, and almost abolished by a mixture of the two, suggesting that activation of GLYT1 and GLYT2 present on glutamatergic terminals triggers the release of [3H]d-ASP. Accordingly, confocal microscopy experiments show localization of GLYT1 and GLYT2 in purified synaptosomes immuno-stained for the vesicular glutamate transporter vGLUT1. The glycine effect was independent of extra- and intraterminal Ca2+ ions. It was partly inhibited by the glutamate transporter blocker dl-TBOA and largely prevented by the anion channel blockers niflumic acid and NPPB. To conclude, transporters for glycine (GLYT1 or/and GLYT2) and for glutamate coexist on the same spinal cord glutamatergic terminals. Activation of glycine heterotransporters elicits glutamate release partly by homotransporter reversal and largely through anion channels. [Copyright &y& Elsevier]

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 μM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A2A receptor antagonist SCH 58261 (1 μM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A2A receptors that enhance vesicular-like glutamate release during ischemia; A2A receptor antagonists would deserve consideration for their neuroprotective potential. [Copyright &y& Elsevier]

It was previously reported that the K+-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or d-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or d-serine on the K+-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K+-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca2+-free medium and with voltage-sensitive Ca2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30–50%) dependent on external Ca2+ and Ca2+ channel activation. In contrast, the alkalinization-evoked release of [3H]noradrenaline was highly sensitive to external Ca2+ removal and to blockade of Ca2+ channels with ω-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis. [Copyright &y& Elsevier]

The coexistence of nicotinic cholinergic receptors (nAChRs) and of N-methyl-d-aspartate (NMDA) receptors on the same noradrenergic axon terminals and the nAChR/NMDA receptor cross-talk were investigated by monitoring the release of noradrenaline (NA) evoked in superfused rat hippocampal synaptosomes by (-)-nicotine and NMDA alone or in combination. In medium containing a physiological concentration (1.2 mM) of Mg2+, the release of [3H]NA was very slightly increased by NMDA plus glycine, whereas it was significantly enhanced by (-)-nicotine. The (-)-nicotine/NMDA combination elicited supraadditive release which was totally abolished by the nAChR blocker mecamylamine and partly prevented by selectively blocking NMDA receptors. Supraadditive [3H]NA release was also observed by exposing synaptosomes to veratrine, but not to ionomycin. The supraadditive release elicited by the (-)-nicotine/NMDA or the veratrine/NMDA combination was sensitive to the protein kinase A/C inhibitor staurosporine and the selective protein kinase A inhibitor H89, but insensitive to the protein kinase C inhibitor Ro 31-8220. It is concluded that (i) release-modulating nAChRs and NMDA receptors coexist on hippocampal noradrenergic axon terminals; and (ii) nicotine permits NMDA receptor activation in the presence of Mg2+, possibly because the nicotine-induced influx of Na+ depolarizes the nerve ending membrane sufficiently to remove the Mg2+ block. [Copyright &y& Elsevier]

The release of [3H]d-aspartate ([3H]d-ASP) or [3H]GABA evoked by glycine and that of [3H]d-ASP or [3H]glycine evoked by GABA from spinal cord synaptosomes were studied in SOD1-G93A(+) mice, a transgenic model of amyotrophic lateral sclerosis, SOD1(+) mice and SOD1(−)/G93A(−) animals. Mutant mice were killed at advanced phase of pathology or during the presymptomatic period. In SOD1(−)/G93A(−) or SOD1(+) mice glycine evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]glycine, release. The glycine-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]glycine, were more pronounced in SOD1-G93A(+) than in SOD1(+) mice. Furthermore, these potentiations were already present in asymptomatic 30- to 40-day-old mice. Basal [3H]d-ASP release was also higher in SOD1-G93A(+) than SOD1(+) or SOD1(−)/G93A(−) mice. The release of endogenous glutamate and GABA was also enhanced in asymptomatic animals; the glycine-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in SOD1(+) animals. The effects of glycine and GABA were insensitive to receptor blockers, but sensitive to transporter inhibitors, indicating coexistence of glutamate and glycine transporters and of glutamate and GABA transporters on glutamate-releasing terminals. The glutamate release machinery seems excessively functional in SOD1-G93A(+) animals. [Copyright &y& Elsevier]

It has been proposed that (−)-nicotine can activate release-stimulating presynaptic nicotinic acetylcholine receptors (nAChRs) on glutamatergic nerve terminals to release glutamate, which in turn stimulates the release of noradrenaline (NA) and dopamine (DA) via presynaptic ionotropic glutamate receptors on catecholaminergic terminals. The objective of this study was to compare the function of N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazide-4-propionic acid (AMPA) glutamate receptors in synaptosomes of rat hippocampus and striatum following acute and chronic (−)-nicotine administration. In hippocampal synaptosomes, prelabeled with [3H]NA, both the NMDA- and AMPA-evoked releases were higher in (−)-nicotine-treated (10 days) than in (−)-nicotine-treated (1 day) or vehicle-treated (1 or 10 days) rats. In striatal synaptosomes prelabeled with [3H]DA, the NMDA-evoked, but not the AMPA-evoked, release of [3H]DA was higher in (−)-nicotine-treated (10 days) than in nicotine-treated (1 day) or vehicle-treated (1 or 10 days) animals. Chronic (−)-nicotine did not affect catecholamine uptake, basal release and release evoked by high-K+ depolarization. Thus, chronic exposure to nicotine enhances the function of ionotropic glutamate receptors mediating noradrenaline release in the hippocampus and dopamine release in the striatum. [Copyright &y& Elsevier]

We have investigated the functional relationships between NMDA receptors and the NOS/sGC system in the rat pre-frontal cortex in vivo by microdialysis. cGMP basal levels were sensitive to NOS or sGC inhibitors (l-NARG or ODQ) or NO donors (SNAP) when enzymatic breakdown was blocked by the phosphodiesterase inhibitor IBMX, indicating that basal cGMP production derives, at least in part, from the NOS/sGC pathway activity and that the pre-frontal cortex possesses a very efficient degradation system for cGMP. The glutamate receptor agonist NMDA did not alter extracellular cGMP either in absence or presence of IBMX. cGMP was not augmented when NMDA was co-infused with the NOS substrate l-arginine, the glycine site agonist d-serine or the glutamate receptor agonist AMPA. Interestingly, the selective GABAA receptor antagonist bicuculline enhanced cGMP production, revealing that the cortical NOS/sGC system is tonically inhibited by endogenous GABA. However, in the presence of bicuculline, NMDA did not increase extracellular cGMP. In the presence of bicuculline, blockade of 5-HT1/2 receptors, known to inhibit the NMDA/NOS/sGC pathway, with the antagonist methiothepin did not unmask cGMP elevations by NMDA. Thus, it would seem that NMDA receptors do not regulate cortical NOS/sGC activity that, on the other hand, is modulated by endogenous GABA acting at GABAA receptors. [Copyright &y& Elsevier]

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A1 and A2A receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A1 and A2A receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [3H]d-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca2+ ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [3H]d-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor dl-TBOA prevented the ischemia-evoked efflux of [3H]d-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [3H]d-aspartate or glutamate was increased by the A1 receptor antagonist DPCPX. The A2A antagonist SCH 58261 decreased [3H]d-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC50: 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A1 or the A2A receptor antagonist was found on the ischemia-evoked efflux of [3H]d-aspartate in Ca2+-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A1 and stimulatory A2A receptors that down- or up-regulate the vesicular-like component of glutamate release. [Copyright &y& Elsevier]

Investigates the effect of the human immunodeficiency virus-1 protein Tat on neurotransmitter release from human and rat cortical nerve endings. Status of the protein in human nerve terminals; Failure of Tat to affect the release of several neurotransmitters; Factor that sustains the occurrence of neurotoxic events.

The release of [(3)H]D-aspartate ([(3)H]D-ASP) or [(3)H]GABA evoked by glycine from spinal cord synaptosomes was compared in mice expressing mutant human SOD1 with a Gly(93) Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, and in control mice. Mice expressing mutated SOD1 were killed at the advanced phase of the pathology, when they showed signs of ingestion disability, because of paralysis of the posterior limbs. In control mice glycine concentration-dependently evoked [(3)H]D-ASP and [(3)H]GABA release. Potentiation of the spontaneous release of both amino acids is likely to be mediated by activation of a glycine transporter, since the effects of glycine were counteracted by the glycine transporter blocker glycyldodecylamide but not by the glycine receptor antagonists strychnine and 5,7-dichlorokynurenate. The glycine-evoked release of [(3)H]D-ASP, but not that of [(3)H]GABA, was significantly more pronounced in SOD1-G93A(+) than in control animals. [ABSTRACT FROM AUTHOR]

Presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [3H]noradrenaline or [3H]serotonin release, striatal [3H]dopamine release and cortical [3H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [3H]noradrenaline, [3H]dopamine or [3H]serotonin, while it weakly affected the AMPA-mediated release of [3H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [3H]dopamine and [3H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [3H]noradrenaline, while the AMPA-mediated [3H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [3H]dopamine, [3H]acetylcholine and [3H]serotonin, but not that of [3H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [3H]noradrenaline, [3H]dopamine, [3H]acetylcholine or [3H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [3H]noradrenaline and [3H]acetylcholine, but it potently blocked the AMPA-induced release of [3H]serotonin and, less so, of [3H]dopamine. AMOA inhibited the AMPA-induced release of [3H]serotonin or [3H]acetylcholine, but not that of [3H]noradrenaline or [3H]dopamine. GAMS prevented the AMPA-mediated release of [3H]acetylcholine and, more weakly, that of [3H]dopamine, but it failed to inhibit the release of [3H]noradrenaline or [3H]serotonin elicited by AMPA. γ-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes. [Copyright &y& Elsevier]

1. The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands. 2. Adenosine (0.1 microM) produced a significant potentiation of the Ca2+-dependent K+ (15 mM)-evoked [3H]-D-aspartate overflow (20.4+/-3.5%), which was blocked by A2A blocker SCH58261 (0.1 microM). At higher concentrations (10 - 1000 microM) adenosine inhibited in a DPCPX-sensitive manner the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow. The inhibitory effect of adenosine at 1000 microM was significantly increased by SCH58261. This inhibition was antagonized by 1 microM DPCPX. Adenosine did not produce any effect on basal release. 3. The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+-dependent K+-evoked overflow of [3H]-D-aspartate (EC50 approximately 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 approximately 5 nM). 4. The A1 receptor agonist CCPA inhibited the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow (EC50 approximately 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX. 5. The K+ (15 mM)-evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 microM). The effect of CGS 21680 was abolished by SCH58261 (0.1 microM) and that of CCPA by DPCPX (0.1 microM). 6. It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex. [ABSTRACT FROM AUTHOR]

1 The effects of adenosine A[sub2A] and A[sub1] receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands. 2 Adenosine (0.1μM) produced a significant potentiation of the Ca[sup2+]-dependent K[sup+](15mM)-evoked [[sup3]H]-D-aspartate overflow (20.4±3.5%), which was blocked by A[sub2A] blocker SCH5826I (0.1 μM). At higher concentrations (10-1000 μM) adenosine inhibited in a DPCPX-sensitive manner the Ca[sup2+]-dependent K[sup+]-evoked [[sup3]H]-D-aspartate overflow. The inhibitory effect of adenosine at 1000 μM was significantly increased by SCH58261. This inhibition was antagonized by 1 μM DPCPX. Adenosine did not produce any effect on basal release. 3 The A[sub2A] receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca[sup2+]-dependent K[sup+]-evoked overflow of [[sup3]H]-D-aspartate (EC[sub50] ≅ 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A[sub2A] receptor antagonist SCH5826I (IC[sub50] ≅5 nM). 4 The A[sub1] receptor agonist CCPA inhibited the Ca[sup2+] -dependent K[sup+]-evoked [[sup3]H]-D-aspartate overflow (EC[sub50] ≅ 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A[sub1] receptor antagonist DPCPX. 5 The K[sup+](15 mM)-evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 μM). The effect of CGS 21680 was abolished by SCH58261 (0.1 μM) and that of CCPA by DPCPX (0.1 μM). 6 It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A[sub1] and excitatory A[sub2A] receptors present on glutamatergic terminals of the rat cerebral cortex. [ABSTRACT FROM AUTHOR]

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [³H]-u-aspartate ([³H]-D-ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [³H]-D-ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca[sup 2+]-dependent exocytotic release evoked by K[sup +] (12 mM) depolarization. Their rank order of potency were anatoxin-a > epibatidine > nicotine > ACh (+ atropine). The anatoxin-a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, α-bungarotoxin or methyllycaconitine. The basal release of [³H]ACh from human cortical synaptosomes was increased by (-)-nicotine (EC[sub 50] = 1.16±0.33 µM) or by ACh plus atropine (EC[sub 50] = 2.0 ± 0.04 µM). The effect of ACh plus atropine was insensitive to α-bungarotoxin, methyllycaconitine or α-conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro-β-erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess α7[sup *] nicotinic heteroreceptors mediating enhancement of glutamate release. Release-enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the α4β2 subunit combination. [ABSTRACT FROM AUTHOR]

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [3H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 mM or 1 mM) plus glycine (1 mM)-elicited [3H]NA, but not [3H]DA release. The NMDA (100 mM)-evoked release of [3H]NA and [3H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1±1 mM); in contrast, the HIV-1 envelope protein gp120 potently (30±100 pM) enhanced the NMDAevoked release of [3H]NA, but not that of [3H]DA. Gp120 also potentiated quinolinate-evoked [3 H]NA release. Ifenprodil (0.1±0.5 mM) or CP-101,606 (0.1±10 mM) inhibited the NMDA plus glycine-evoked release of both [3H]catecholamines. Zinc (0.1±1 mM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [3H]NA elicited by NMDA plus glycine, whereas the release of [3H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 mM) or H7 (10 mM) selectively prevented the effect of NMDA plus glycine on the release of [3H]NA. GF 109203X also blocked the release of [3H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit. [ABSTRACT FROM AUTHOR]

AbstractThe objective of this study was to investigate the effects of the HIV-1 envelope protein gp120 and its peptide fragments on the function of N-methyl-d-aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or d-serine ‘only’. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mm K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU-C-34-A, but not by RP-135 (a central portion of BRU-C-34-A). The EC50 values of gp120 were 0.02 nm (CCK-LI release) and 0.01 nm (SRIF-LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK- and SRIF-releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near-maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B. [ABSTRACT FROM AUTHOR]

In the cerebellum, infusion of NMDA (200 μM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABAA receptor agonist muscimol (0.01-100 μM) was able to counteract the NMDA effect with an EC50 of 0.65 μM ; the inhibitory effect of muscimol (10 μM) was prevented by bicuculline (50 μM). Diazepam (10 μM) significantly potentiated the muscimol (1 μM) inhibition ; furthermore, when coinfused with 0.1 μM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 μM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 μM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 μM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABAA receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the α subunit composition, we can deduce that the cerebellar GABAA receptor does not contain α6 or β4 subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of α5 subunits in the receptor composition and suggest the involvement of an α1 subunit. [ABSTRACT FROM AUTHOR]

In this study we have examined the effects of N-methyl-d-aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mm K[sup +] from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration-dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration-dependent manner the depolarization-evoked release of both CCK-LI and SRIF-LI. This effect of glycine was strychnine-insensitive and could be mimicked by d-serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/d-serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by d-serine was strongly inhibited by ifenprodil (0.3 μm) and by Zn[sup 2+] ions (50 nm), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK- and SRIF-releasing neurons possess non-conventional NMDA receptors whose channels can be operated by glycine or d-serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B. [ABSTRACT FROM AUTHOR]

Abstract Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 nm–10 μm) inhibited tritium release with a calculated EC50 of 17 nm and a maximal inhibition of ≈ 75% reached at 1 μm. In the presence of the D2/D3 receptor antagonist (–)-sulpiride (0.1 and 1 μm), the concentration–response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14–8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor-selective antagonist [7-N,N-dipropylamino-5,6,7,8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane] (S 14297) and the D4 receptor-selective antagonist 3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine (L-745,870) (0.01–1 μm in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype. [ABSTRACT FROM AUTHOR]