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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 [(3)H]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 [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) 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 [(3)H]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'.

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.

J. Neurochem. (2011) 117, 516–527. Abstract 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 co-localize 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.

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.

GABA(B) receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABA(B) 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 [(3)H]glycine through GLYT2 transporters. CGP35348 inhibited [(3)H]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 GABA(B) agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABA(B) antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100-300 microM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABA(B1) (GABA(B1-/-) mice) or the GABA(B2) (GABA(B2-/-) mice) subunit, the evoked exocytosis of [(3)H]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 GABA(B) receptor antagonists.

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.

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

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 [(3)H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR-mAChR agonists acetylcholine (ACh) and carbachol provoked [(3)H]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 micromol/L, together with 3, 10, or 100 micromol/L (-)nicotine provoked synergistic effect on [(3)H]DA overflow. The [(3)H]DA overflow elicited by 100 micromol/L (-)nicotine plus 30 micromol/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 [(3)H]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 alpha4beta2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, alpha4beta2 nAChRs exert a permissive role on the releasing function of reportedly M(5) mAChRs co-existing on the same dopaminergic nerve endings.

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.

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+]or= 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 inhibitors DL-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 by DL-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked by DL-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia.

Background and purpose: Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level. Experimental approach: Rat hippocampus and human neocortex synaptosomes were labelled with [3H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [3H]noradrenaline release was produced by removal of external Mg2+ or by simultaneous application of NMDA and AMPA in Mg2+-containing solutions. Key results: The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [3H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg2+ removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg2+ removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents. Conclusions and implications: Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5. British Journal of Pharmacology (2007) 151, 1087–1094; doi:10.1038/sj.bjp.0707280

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.

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 [(3)H]d-aspartate form gliosomes in a concentration-dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [(3)H]d-aspartate was independent of modifications of cytosolic Ca(2+) , but it was blocked by dl-threo-beta-benzyloxyaspartate (dl-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca(2+)-independent and dl-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 Ca(2+). 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 Ca(2+). These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

Background and purpose: Two metabolites of tryptophan, 5-hydroxyindole and kynurenic acid (kynurenate) affect the function of α7 nicotinic acetylcholine receptors (nAChRs), as measured by electrophysiological and Ca2+ fluorescence techniques. To better understand the modulations by 5-hydroxyindole and kynurenate of the function of nAChR subtypes, we compared the effects of 5-hydroxyindole and kynurenate on the release of various transmitters evoked by nAChR activation. Experimental approach: The function of α7nAChRs located on glutamatergic terminals was investigated by monitoring the release of [3H]D-aspartate or of endogenous glutamate from neocortical synaptosomes. We also comparatively considered non-α7 release-enhancing nAChRs localized on hippocampal noradrenergic or cholinergic terminals, as well as on striatal dopaminergic terminals. Key results: Epibatidine or nicotine, inactive on their own on basal release, enhanced [3H]D- aspartate and glutamate efflux in presence of 5-hydroxyindole. The release evoked by nicotine plus 5-hydroxyindole was abolished by methyllycaconitine or α-bungarotoxin. Presynaptic nAChRs mediating the release of [3H]noradrenaline ([3H]NA), [3H]dopamine ([3H]DA), or [3H]ACh were inhibited by 5-OHi. The α7nAChR-mediated release of [3H]D-aspartate was reduced by kynurenate at concentrations unable to affect the non-α7 receptor-mediated release of tritiated NA, DA or ACh. Conclusions and Implications: (i) 5-hydroxyindole permits selective activation of α7nAChRs mediating glutamate release; (ii) kynurenate down-regulates the permissive role of 5-hydroxyindole on α7nAChR activation; (iii) the non-α7nAChRs mediating release of NA, DA or ACh can be inhibited by 5-hydroxyindole, but not by kynurenate. These findings suggest up the possibility of developing novel drugs able to modulate selectively the cholinergic-glutamatergic transmission. British Journal of Pharmacology (2006) 149, 724–732. doi:10.1038/sj.bjp.0706914

The release of cholecystokinin-like immunoreactivity (CCK-LI) from the frontal cortex of freely moving rats has been studied using a transcerebral microdialysis technique coupled to a radioimmunoassay procedure. Basal levels of CCK-LI in the dialysate were above detection limits (2.4 +/- 0.7 pg/20 min; n = 8). High-K+ media evoked CCK-LI overflow in a concentration-dependent manner. The threshold concentration was 50 mM KCl. The peak overflow evoked by 100 mM K+ amounted to 42.7 +/- 2.8 pg/20 min (n = 6); it was totally Ca2+ dependent but insensitive to 1 microM tetrodotoxin. Infusion of 4-aminopyridine (1 mM; 20 min) evoked an overflow of CCK-LI (32 +/- 2.3 pg/20 min; n = 4), which was totally Ca2+ dependent and tetrodotoxin sensitive. Depolarization with 100 micrograms/ml of veratrine (20 min) provoked a CCK-LI overflow (62.2 +/- 10 pg/20 min; n = 6), which was also blocked by tetrodotoxin or by the absence of Ca2+ ions. The CCK-LI material collected under basal conditions or during veratrine infusion consisted essentially of CCK octapeptide sulfate. The veratrine-induced CCK-LI overflow did not change significantly when the infusion time was prolonged to 100 min. A second 20-min stimulus with 100 micrograms/ml of veratrine applied 200 min after a first 20-min stimulus evoked a barely significant CCK-LI overflow. These data suggest that one single 20-min stimulus with 100 micrograms/ml of veratrine may be sufficient to deplete the CCK-LI releasable stores and that > 200 min are required to replenish the depleted CCK-containing vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

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, 1 microM 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 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM 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 1 microM 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.

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 alpha7 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 Ca(2+)-dependent release of pre-accumulated D-[(3)H]aspartate ([(3)H]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 approximately 30 times more potent; the potentiation of the BzATP-evoked release was blocked by methyllycaconitine or alpha-bungarotoxin. Adding inactive concentrations of (-)-nicotine, epibatidine or choline together with inactive concentrations of BzATP resulted in significant elevation of the [(3)H]D-ASP release mediated by alpha7 nAChRs. To conclude, P2X(7) receptors and alpha7 nAChRs seem to co-exist and interact on rat neocortex glutamatergic terminals; in particular, P2X(7) receptors exert a permissive role on the activation of alpha7 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.

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.

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-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 following in vivo acute 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.

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.

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) 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 [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. 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.

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.

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 omega-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.

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.

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 [(3)H]d-ASP and [(3)H]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.

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 [ 3 H ]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 [ 3 H ]DA, the NMDA-evoked, but not the AMPA-evoked, release of [ 3 H ]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.

L-Glutamate, NMDA, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg(2+)-containing medium, the maximal effects (reached at approximately 100 microM) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 microM (AMPA), 39 microM (glutamate), 41 microM (KA), and 70 microM (NMDA). The metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 microM) was abolished by 10 microM dizocilpine (MK-801) plus 30 microM 1-aminophenyl-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA+AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca(2+) dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8-epi-PGF2α following infusion of N-methyl-d-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE2 and 8-epi-PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) 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 alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.

Glycine and GABA are likely co-transmitters in the spinal cord. Their possible interactions in presynaptic terminals have, however, not been investigated. We studied the effects of glycine on GABA release using superfused mouse spinal cord synaptosomes. Glycine concentration dependently elicited [3H]GABA release which was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was Na+ dependent and sensitive to the glycine uptake blocker glycyldodecylamide. The glycine effect was external Ca2+ independent, but was reduced when intraterminal Ca2+ was chelated with 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid or depleted with thapsigargin, or when vesicular storage was impaired with bafilomycin. Glycine-induced [3H]GABA release was prevented, in part, by blocking GABA transport. The glycine effect was halved by sarcosine, a GLYT1 substrate/inhibitor, or by amoxapine, a GLYT2 blocker, and abolished by a mixture of the two. The sensitivity to sarcosine, used as a transporter inhibitor or substrate, persisted in synaptosomes prelabelled with [3H]GABA in the presence of β-alanine, excluding major gliasome involvement. To conclude, in mice spinal cord, transporters for glycine (both GLYT1 and GLYT2) and for GABA coexist on the same axon terminals. Activation of the glycine transporters elicits GABA release, partly by internal Ca2+-dependent exocytosis and partly by transporter reversal.

Rat cerebellar synaptosomes labeled with [ 3 H ] d -aspartate ( [ 3 H ] d -ASP) were exposed in superfusion to muscimol. The GABAA receptor agonist did not affect [ 3 H ] d -ASP basal release or the overflow provoked by 15 mM K+; muscimol potentiated the 35 mM K+-evoked overflow of [ 3 H ] d -ASP or endogenous glutamate. Membrane potential measured by Rhodamine 6G fluorescence was −65 mV under resting conditions and −32 mV in the presence of 35 mM K+. The membrane potential was not significantly affected by muscimol. The muscimol effect on the K+(35 mM)-evoked [ 3 H ] d -ASP overflow was not inhibited by omitting external Ca2+ or by entrapping BAPTA to chelate cytosolic Ca2+. Muscimol lost its ability to release glutamate following superfusion with d -aspartate to deplete cytosolic glutamate by heteroexchange suggesting that GABAA receptor activation elicits release of cytosolic glutamate. The non-transportable glutamate carrier blockers dihydrokainate or dl -TBOA did not reduce the muscimol potentiation. This was abolished by the anion channel blockers niflumic acid and NPPB. To conclude, when cerebellar parallel fiber terminals are sufficiently depolarized, activation of α6 GABAA receptors on these terminals mediates glutamate release in addition to that evoked by depolarization. This extra-release does not occur by exocytosis or transporter reversal but involves the opening of anion channels present on parallel fiber terminals.

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

In the hippocampus of freely-moving rats, basal extracellular levels of cGMP are inhibited by L-NARG or ODQ whereas they are increased by NO donors or phosphodiesterase inhibitors. Activation of NMDA receptors also augments cGMP dialysate levels in a MK-801 and L-NARG sensitive manner, an effect dramatically diminished during ageing. Experiments with AMPA, AMPA receptor antagonists and cyclothiazide revealed complex relationships with GABAergic circuits that potently control the NO/cGMP pathway. Furthermore, the activity of this neurochemical cascade is also modulated by hippocampal nicotinic receptors via enhancement of endogenous glutamate release and stimulation of NMDA receptors. From a behavioural point of view, increased hippocampal excitation leads to the appearance of epileptic-like manifestations that, however, seem unrelated to the increase of NO/cGMP formation.

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5-hydroxytryptamine (5-HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery. The cyclic GMP elevation produced by 100 microM NMDA was blocked by 100 microM of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by 10 microM of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha] quinoxaline-1-one (ODQ). The NMDA effect was prevented by 5-HT or by the 5-HT(2) agonist (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane ((+/-)-DOI; EC(50)=22 nM). The (+/-)-DOI inhibition was insensitive to the 5-HT(2A) receptor antagonist MDL 100907 or the 5-HT(2B) antagonist rauwolscine; it was largely prevented by 1 microM of the non-selective 5-HT(2C) antagonists mesulergine (5-HT(2A,B,C)), ketanserin (5-HT(2A,C)) or SB 200646A (5-HT(2B,C)); it was completely abolished by 0.1 microM of the selective 5-HT(2C) receptor antagonist SB 242084. The NMDA-induced cyclic GMP elevation also was potently inhibited by the selective 5-HT(2C) agonist RO 60-0175 and by the antidepressant trazodone, both added at 1 microM, in an SB 242084-sensitive manner. Finally, the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 1 microM) inhibited the NMDA-evoked cyclic GMP response, an effect blocked by the selective 5-HT(1A) receptor antagonist WAY 100635. In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5-HT(2C) or 5-HT(1A) receptors.

The 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.

The release of [3H]-dopamine ([3H]-DA) from human neocortex nerve terminals was studied in synaptosomes prepared from brain specimens removed in neurosurgery and exposed during superfusion to different releasing stimuli. Treatment with 15 mM KCl, 100 μM 4-aminopyridine, 1 μM ionomycin or 30 mM caffeine elicited almost identical overflows of tritium. Removal of external Ca2+ ions abolished the overflow evoked by K+ or ionomycin and largely prevented that caused by 4-aminopyridine; the overflow evoked by caffeine was completely independent of external Ca2+. Exposure of synaptosomes to 25 μM of the broad spectrum calcium channel blocker CdCl2 strongly inhibited the 4-aminopyridine-induced tritium overflow while that evoked by ionomycin remained unaffected. The Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′ tetraacetic acid (BAPTA), reduced significantly the K+- and the caffeine-induced tritium overflow. The effect of caffeine was attenuated by exposure to the ryanodine receptor blocker dantrolene or when the membrane-impermeant inositol trisphosphate receptor antagonist, heparin, was entrapped into synaptosomes; the combined treatment with dantrolene and heparin abolished the release elicited by caffeine. Tetanus toxin, entrapped into human neocortex synaptosomes to avoid prolonged incubation, inhibited in a concentration-dependent manner the K+- or the 4-aminopyridine-evoked tritium overflow; in contrast, the release stimulated by ionomycin and by caffeine were both totally insensitive to the same concentrations of tetanus toxin. Western blot analysis showed about 50% reduction of the content of the vesicular protein, synaptobrevin, in synaptosomes poisoned with tetanus toxin. In conclusion, the release of dopamine from human neocortex nerve terminals can be triggered by Ca2+ ions originating from various sources. It seems that stimuli not leading to activation of voltage-sensitive Ca2+ channels elicit Ca2+-dependent, probably exocytotic, release that is insensitive to tetanus toxin. British Journal of Pharmacology (2000) 129, 1780–1786; doi:10.1038/sj.bjp.0703251

Superfused synaptosomes have been utilized in studies of neurotransmitter release during 25 years. This review summarizes the aspects of neurotransmission that have been and could be successfully investigated with this technique. The major aim of the article is to draw attention on the versatility of superfused synaptosomes and to suggest how the system could be exploited in clarifying several aspects of synaptic neurochemistry including neurotransmitter transport, receptor localization, receptor-receptor interactions, functional aspects of multi-sited receptor complexes, receptor heterogeneity and mechanisms of neurotransmitter exocytosis-endocytosis.

The releases of [3H]5-hydroxytryptamine ([3H]5-HT) and of endogenous glutamic acid and their modulation through presynaptic h5-HT1B autoreceptors and h5-HT1D heteroreceptors have been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery. The inhibition by 5-HT of the K+ (15 mM)-evoked overflow of [3H]5-HT was antagonized by the 5-HT1B/5-HT1D receptor ligand GR 127935, which was ineffective on its own; this drug was previously found to behave as a full agonist at the h5-HT1D heteroreceptor regulating glutamate release. The recently proposed selective h5-HT1B receptor ligand SB-224289 also prevented the effect of 5-HT at the autoreceptor, being inactive on its own; in contrast, SB-224289, at 1 μM, was unable to interact with the h5-HT1D heteroreceptor. The inhibitory effect of 5-HT on the K+-evoked overflow of glutamate was antagonized by the h5-HT1D receptor ligand BRL-15572; added in the absence of 5-HT the compound was without effect. BRL-15572 (1 μM) was unable to modify the effect of 5-HT at the autoreceptor regulating [3H]5-HT release. The selective 5-HT1A receptor antagonist (+)-WAY 100135, previously found to be an agonist at the h5-HT1D heteroreceptor regulating glutamate release, could not interact with the h5-HT1B autoreceptor when added at 1 μM. It is concluded that native h5-HT1B and h5-HT1D receptors exhibit a hitherto unexpected pharmacological diversity. British Journal of Pharmacology (1999) 126, 607–612; doi:10.1038/sj.bjp.0702336

1. The effects of NK-3 receptor agonists on the release of substance P-immunoreactivity (SP-LI) have been investigated using superfused rat spinal cord synaptosomes. 2. The Ca2+-dependent overflow of SP-LI evoked by 35 mM KCl was concentration-dependently enhanced by senktide (EC50 = 52 nM; maximal effect = 70%) or [MePhe7]NKB (EC50 = 5.5 nM; maximal effect 125%), both selective agonists at receptors of the NK-3 type. 3. The potentiation of the SP-LI overflow elicited by 100 nM senktide or [MePhe7]NKB was prevented by the NK-3 receptor antagonist (+)-SR142801. The antagonist halved, at 10 nM, and almost abolished, at 100 nM, the effect of both agonists. The effect of senktide or [MePhe7]NKB was insensitive to antagonists at NK-1 or NK-2 receptors. 4. Capsaicin (0.1-1 microM) stimulated SP-LI release in a concentration-dependent manner from spinal cord synaptosomes. The SP-LI overflow elicited by 1 microM capsaicin was completely dependent on external Ca2+. Senktide could not affect the capsaicin-evoked release of SP-LI. 5. Senktide failed to potentiate the K+-evoked overflow of SP-LI from synaptosomes previously exposed for 15 min in superfusion to capsaicin. 6. The results show that release-enhancing NK-3 receptors are located on axon terminals of capsaicin-sensitive primary afferent neurones in the spinal cord. Antagonists at NK-3 receptors might help controlling pain transmission.

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+ 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 microM) and by Zn2+ 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.

A number of experiments have shown that neurotrophins are involved in the development and plasticity of the visual cortex (Bonhoeffer, T., Curr. Op. Neurobiol., 6, 119, 1996). A possible mechanism underlying these effects is the neurotrophin modulation of synaptic transmission. We investigated whether nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) can modulate the release of neurotransmitter in the rat visual cortex at the peak of the critical period for plasticity (P23). The release of glutamate, acetylcholine and gamma-aminobutyric acid (GABA) from visual cortical synaptosomes was analysed in continuous perfusion conditions. We found that NGF enhances the depolarization-evoked release of glutamate (approximately 90%) and acetylcholine (approximately 35%) but not that of GABA. By contrast, BDNF enhances the depolarization-evoked release of all three neurotransmitters investigated (approximately 30%). BDNF and NGF were ineffective on basal release of neurotransmitters. The effect of NGF was not blocked by cholinergic antagonists atropine and mecamylamine. NGF and BDNF potentiation of transmitter release was strongly but not completely blocked by K252a, a tyrosine kinase inhibitor. The role of TrkA and p75NTR receptors was investigated in NGF-induced potentiation of glutamate release. Block of NGF binding to p75NTR using specific blocking antibodies (REX-IgG) slightly but significantly reduced the effect of NGF. Activation of TrkA in isolation by RTA-IgG, an antibody that specifically activates TrkA, was less effective than activation of both receptors by NGF. These results show that neurotrophin action on neurotransmitter release was mostly mediated by Trk receptors with p75NTR having a little but significant positive role. Antigen blot analysis showed the presence of TrkA, TrkB and p75NTR receptors in the visual cortex.

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA(A) receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA(A) receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3alpha,5alpha-P), at nanomolar concentrations, potentiated the GABA-induced [3H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10 microM. The enhancement by GABA of the K+-evoked [3H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3alpha,5alpha-P; in addition, at 1-10 microM, the steroid increased [3H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3alpha,5alpha-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10 microM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [3H]-D-aspartate release elicited by 3 microM 3alpha,5alpha-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3alpha,5alpha-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA(A) receptors under study. Low micromolar 3alpha,5alpha-P can directly activate the cerebellar receptor, whereas the hippocampal GABA(A) receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA(A) receptor having distinct pharmacology, neuronal localization and function.

The release of glutamic acid and its modulation by 5-hydroxytryptamine (5-HT) in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply sited tumours. The Ca2+-dependent K+ (15 mM)-evoked overflow of glutamate was inhibited by 5-HT in a concentration-dependent manner (EC50=2.9 nM; maximal effect ≃50%). The inhibition caused by 5-HT was antagonized by the 5-HT1/5-HT2 receptor antagonist methiothepin. The 5-HT1B/5-HT1D receptor agonist sumatriptan mimicked 5-HT (EC50=6.4 nM; maximal effect ≃50%); the effect of sumatriptan was also methiothepin-sensitive. Selective 5-HT1A receptor antagonists could not prevent the inhibition of glutamate release by 5-HT. The 5-HT1B/5-HT1D receptor ligand GR 127935 and the 5-HT2C/5-HT1B/5-HT1D receptor ligand metergoline were unable to prevent the 5-HT effect; instead they inhibited glutamate release, their effects being abolished by methiothepin. Some 5-HT1A receptor antagonists also displayed intrinsic agonist activity. The effect of sumatriptan was prevented by ketanserin, a drug known to display much higher affinity for recombinant h 5-HT1D than for h 5-HT1B receptors. We propose that neocortical glutamatergic nerve terminals in human brain cortex possess release-inhibiting presynaptic heteroreceptors that appear to belong to the h 5-HT1D subtype. British Journal of Pharmacology (1998) 123, 45–50; doi:10.1038/sj.bjp.0701581

1 Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[3H]-arginine to L-[3H]-citrulline. 2 Elevation of extracellular K+ to 20, 35 or 60 mM concentration-dependently augmented L-[3H]-citrulline production. The response to 35 mM KCl was abolished by NG-nitro-L-arginine (100 μM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3 However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2–3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4 The 35 mM K+ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K+-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω-conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX. 5 It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca+ entering cells mainly through channels of the P/Q-type. Other Ca2+ channels (L- and N-type) appear to contribute when P/Q-channels are blocked. British Journal of Pharmacology (1997) 122, 930–934; doi:10.1038/sj.bjp.0701457

Intrahippocampal perfusion of bicuculline (50 microM) in Mg2+-free medium caused elevation of extracellular cGMP and epileptic-like behaviour. Both effects were partially prevented by blocking NMDA receptors with MK-801 or Mg2+ ions. Similarly, the GABA(B) receptor antagonists CGP52432 (0.1-30 microM) and CGP35348 (0.3-1 mM) evoked increases of extracellular cGMP. CGP52432 also elicited behavioural responses ranging from wet dog shakes to convulsions. MK-801 or Mg2+ ions reduced the effects of CGP52432. Local application of muscimol (100-300 microM) or (-)baclofen (300 microM) caused inhibition of extracellular cGMP. Administration of the AMPA/kainate receptor antagonist NBQX (100 microM) caused cGMP elevation which was almost abolished by co-perfusion of muscimol and (-)baclofen. In the presence of physiological Mg2+, perfusion of AMPA (30 microM) failed to affect cGMP levels, although rats displayed wet dog shakes episodes. When AMPA was co-perfused with low concentrations of bicuculline or CGP52432, cGMP elevations were observed in 60% of the rats. Addition of both antagonists to AMPA resulted in 85% of rats displaying a cGMP response. To conclude: (a) extracellular hippocampal cGMP is controlled by inhibitory GABA(A) and GABA(B) receptors tonically activated through GABAergic interneurons receiving AMPA/kainate-mediated glutamatergic inputs; (b) the GABAergic receptors are not endogenously saturated and can be further stimulated by exogenous agonists; (c) blockade of the GABA-mediated inhibition causes increase of cGMP and epileptic-like behaviour, due largely to endogenous activation of NMDA receptors; (d) reproducible cGMP responses to AMPA can be observed when the inhibitory GABAergic inputs to the NO/guanylyl cyclase system are blocked, confirming the previously proposed existence of AMPA/kainate receptors able to increase the nucleotide synthesis.