Multifaces of neuropeptide Y in the brain--neuroprotection, neurogenesis and neuroinflammation.

Neuropeptide Y (NPY) has been implicated in the modulation of important features of neuronal physiology, including calcium homeostasis, neurotransmitter release and excitability. Moreover, NPY has been involved as an important modulator of hippocampal and thalamic circuits, receiving particular attention as an endogenous antiepileptic peptide and as a potential master regulator of feeding behavior. NPY not only inhibits excessive glutamate release (decreasing circuitry hyperexcitability) but also protects neurons from excitotoxic cell death. Furthermore, NPY has been involved in the modulation of the dynamics of dentate gyrus and subventricular zone neural stem cell niches. In both regions, NPY is part of the chemical resource of the neurogenic niche and acts through NPY Y1 receptors to promote neuronal differentiation. Interestingly, NPY is also considered a neuroimmune messenger. In this review, we highlight recent evidences concerning paracrine/autocrine actions of NPY involved in neuroprotection, neurogenesis and neuroinflammation. In summary, the three faces of NPY, discussed in the present review, may contribute to better understand the dynamics and cell fate decision in the brain parenchyma and in restricted areas of neurogenic niches, in health and disease.

Functional identification of cell phenotypes differentiating from mice retinal neurospheres using single cell calcium imaging.

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca(2+) transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca(2+) fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity: a role for microglia.

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Susceptibility of hippocampal neurons to Abeta peptide toxicity is associated with perturbation of Ca2+ homeostasis.

Neuritic dystrophy, loss of synapses and neuronal death in the cerebral cortex and hippocampus are hallmarks of Alzheimer's disease. The aim of the present study was to investigate the differential susceptibility of cortical and hippocampal neurons to amyloid-beta (Abeta)-induced toxicity. For that, we have used primary neuronal cultures prepared from rat brain cortex and hippocampus which were treated with the synthetic peptides Abeta25-35 or Abeta1-40. Abeta-induced apoptotic cell death was analyzed by determining caspase-3-like activity. Neuritic dystrophy was evaluated by cobalt staining and MAP2 immunoreactivity. Perturbation of Ca(2+) homeostasis caused by exposure to Abeta was evaluated by determining basal cytosolic calcium levels in the whole neuronal population and by single cell calcium imaging under basal and KCl-depolarization conditions. Finally, levels of GluR2 subunit of glutamate AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate) receptors were quantified by western blotting. Our results demonstrated that hippocampal neurons in culture are more susceptible than cortical neurons to Abeta-induced apoptosis and also that this mechanism involves the perturbation of Ca(2+) homeostasis. Accordingly, the exposure of hippocampal neurons to Abeta peptides decreases the protein levels of the GluR2 subunit of glutamate AMPA receptors that may be associated with a significant rise of cytosolic Ca(2+) concentration, leading to dendritic dystrophy and activation of apoptotic neuronal death.

Neuropeptide Y can rescue neurons from cell death following the application of an excitotoxic insult with kainate in rat organotypic hippocampal slice cultures.

In the present work we investigated the neuroprotective role of neuropeptide Y (NPY) after an excitotoxic insult in rat organotypic hippocampal slice cultures. Exposure of 2 week-old rat hippocampal slice cultures to 12muM kainate (KA) for 24h induced neuronal death in dentate gyrus (DG) granular cell layer, CA1 and CA3 pyramidal cell layers, as quantified by cellular propidium iodide (PI) uptake. The activation of Y(1) or Y(2) receptors 30min after starting the exposure to the excitotoxic insult with kainate resulted in neuroprotection by reducing the PI uptake in DG, CA1 and CA3 cell layers. The use of Y(1) or Y(2) receptors antagonists, BIBP3226 (1muM) or BIIE0246 (1muM), resulted in the loss of the neuroprotection induced by the activation of Y(1) or Y(2) receptors, respectively, in all hippocampal subfields. Taken together these results suggest that activation of NPY Y(1) or Y(2) receptors activates neuroprotective pathways that are able to rescue neurons from excitotoxic cell death.

Inflammation and neurogenesis in temporal lobe epilepsy.

The aim of the present review is to discuss the evidence supporting the hypothesis that inflammation and neurogenesis play an important role in temporal lobe epilepsy (TLE) and to examine whether possible strategies that involve the pharmacological manipulation of inflammation/neurogenesis can lead to the development of novel approaches for the treatment of epilepsy. Since it is not yet clear whether the neuron-glia response obtained in this pathology is a secondary effect of an aggressive inflammation or if it is somehow related to the cause of the epileptic condition, with the present review we guide the readers through the complex and ambiguous crosstalk between neuroimmunology and epilepsy.

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

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

Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus.

In the present work, we investigated the role of pre- and post-synaptic neuropeptide Y1 (NPY1) and Y2 receptors on the calcium responses and on glutamate release in the rat hippocampus. In cultured hippocampal neurones, we observed that only NPY1 receptors are involved in the modulation of intracellular free calcium concentration ([Ca(2+)](i)). In 88% of the neurones analysed, the increase in the [Ca(2+)](i), in response to depolarization with 50 mM KCl, was inhibited by 1 microM [Leu31,Pro34]NPY, whereas 300 nM NPY13-36 was without effect. However, studies with hippocampal synaptosomes showed that both NPY1 and Y2 receptors can modulate the [Ca(2+)](i) and glutamate release. The pharmacological characterization of the NPY-induced inhibition of glutamate release indicated that Y2 receptors play a predominant role, both in the modulation of Ca(2+)-dependent and -independent glutamate release. However, we could distinguish between Y1 and Y2 receptors by using [Leu31,Pro34]NPY and NPY13-36. Active pre-synaptic Y1 receptors are present in the dentate gyrus (DG) as well as in the CA3 subregion, but its activity was not revealed by using the endogenous agonist, NPY. Concerning the Y2 receptors, they are present in the three subregions (CA1, CA3 and DG) and were activated by either NPY13-36 or NPY. The present data support a predominant role for NPY2 receptors in mediating NPY-induced inhibition of glutamate release in the hippocampus, but the physiological relevance of the presently described DG and CA3 pre-synaptic NPY1 receptors remains to be clarified.

Role of kainate receptor activation and desensitization on the [Ca(2+)](i) changes in cultured rat hippocampal neurons.

We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca(2+)](i) caused by kainate showed cell-to-cell variability. The [Ca(2+)](i) increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca(2+)](i) changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca(2+)](i) and toxicity.

Inhibition of glutamate release by BIA 2-093 and BIA 2-024, two novel derivatives of carbamazepine, due to blockade of sodium but not calcium channels.

We investigated the mechanism(s) of action of two new putative antiepileptic drugs (AEDs), (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), by comparing their effects on the release of endogenous glutamate in hippocampal synaptosomes, with those of carbamazepine (CBZ) and oxcarbazepine (OXC). The AEDs inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) or veratridine in a concentration-dependent manner, being CBZ more potent than the other AEDs. Using conditions of stimulation (30 mM KCl), where Na(+) channels are inactivated, the AEDs did not inhibit either the Ca(2+)-dependent or -independent release of glutamate. The results indicate that BIA 2-093 and BIA 2-024 have sodium channel-blocking properties, but CBZ and OXC are more potent than the new AEDs. Moreover, the present data also indicate that Ca(2+) channels coupled to the exocytotic release of glutamate and the activity of the glutamate transporter were not affected by the AEDs.

Presynaptic N-methyl-D-aspartate receptor activation inhibits neurotransmitter release through nitric oxide formation in rat hippocampal nerve terminals.

In brain synapses, nitric oxide synthase activation is coupled to N-methyl-D-aspartate-mediated calcium entry at postsynaptic densities through regulatory protein complexes, however a presynaptic equivalent to this signaling mechanism has not yet been identified. Novel evidence indicates that N-methyl-D-aspartate glutamate receptors may play a presynaptic role in synaptic plasticity. Thus, we investigated whether ionotropic glutamate receptor activation in isolated nerve terminals regulates neurotransmitter release, through nitric oxide formation. N-Methyl-D-aspartate dose-dependently inhibited the release of glutamate evoked by 4-aminopyridine (IC(50)=155 microM), and this effect was reversed by the N-methyl-D-aspartate receptor antagonist D-(-)-2-amino-5-phosphopentanoic acid and by the nitric oxide synthase inhibitor, L-nitroarginine, in synaptosomes isolated from whole hippocampus, CA3 and CA1 areas, but not from the dentate gyrus. In contrast, the 4-aminopyridine-evoked release of glutamate was reduced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate by a nitric oxide-independent mechanism, since it was not blocked by L-nitroarginine, and N-methyl-D-aspartate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, significantly increased cGMP formation. Presynaptic N-methyl-D-aspartate receptors are probably involved since removing extracellular nitric oxide with the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide did not block the depression of glutamate release by N-methyl-D-aspartate. The mechanism underlying this depression involves the inhibition of synaptic vesicle exocytosis since N-methyl-D-aspartate/nitric oxide inhibited the release of [3H]glutamate and [14C]GABA evoked by hypertonic sucrose. The results also suggest that presynaptic N-methyl-D-aspartate receptors may function as auto- and heteroreceptors.

Neurotoxic/neuroprotective profile of carbamazepine, oxcarbazepine and two new putative antiepileptic drugs, BIA 2-093 and BIA 2-024.

We investigated and compared the toxicity profile, as well as possible neuroprotective effects, of some antiepileptic drugs in cultured rat hippocampal neurons. We used two novel carbamazepine derivatives, (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b, f]azepine-5-carboxamide (BIA 2-093) and 10, 11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), and compared their effects with the established compounds carbamazepine and oxcarbazepine. The assessment of neuronal injury was made by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay, as well as by analysing morphology and nuclear chromatin condensation (propidium iodide staining), after hippocampal neurons were exposed to the drugs for 24 h. The putative antiepileptic drugs, BIA 2-093 or BIA 2-024 (at 300 microM), only slightly decreased MTT reduction, whereas carbamazepine or oxcarbazepine were much more toxic at lower concentrations. Treatment with the antiepileptic drugs caused nuclear chromatin condensation in some neurons, which is characteristic of apoptosis, and increased the activity of caspase-3-like enzymes, mainly in neurons treated with carbamazepine and oxcarbazepine. The toxic effect caused by carbamazepine was not mediated by N-methyl-D-aspartate (NMDA) or by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors. Moreover, the antiepileptic drugs failed to protect hippocampal neurons from the toxicity caused by kainate, veratridine, or ischaemia-like conditions.

Role of desensitization of AMPA receptors on the neuronal viability and on the [Ca2+]i changes in cultured rat hippocampal neurons.

We investigated the role of desensitization of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors on the neurotoxicity and on the [Ca2+]i changes induced by kainate or by AMPA in cultured rat hippocampal neurons. The neuronal viability was evaluated either by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, or by analysis of cell morphology. Short-term exposure of the neurons to kainate or AMPA (30 min) was not toxic, but the exposure for 24 h to the excitotoxic drugs caused a concentration-dependent neurotoxic effect which was prevented by LY 303070, a noncompetitive AMPA receptor antagonist. In the presence of cyclothiazide (CTZ), kainate or AMPA was toxic (30 min exposure), or the toxic effect was significantly enhanced (24 h exposure), but in this case LY 303070 did not completely protect the cells against kainate-induced toxicity. The alterations in the [Ca2+]i caused by kainate or AMPA showed a great cell-to-cell variability. LY 303070 completely or partially inhibited the responses stimulated by kainate. CTZ differentially affected the responses evoked by kainate or AMPA. In the majority of hippocampal neurons, CTZ did not potentiate, or only slightly potentiated, the kainate-stimulated responses but in 11% of neurons there was a great potentiation. In AMPA-stimulated neurons, the responses were slightly or greatly potentiated in the majority of neurons, but not in all of them. The results show that AMPA and kainate may be toxic, depending on the time of exposure and on the blockade of the desensitization of the AMPA receptors. Overall, our results clearly show that there exist different populations of hippocampal neurons with different sensitivities to kainate, AMPA, CTZ and LY 303070. Moreover, the effects of CTZ on both [Ca2+]i alterations and neurotoxicity are not fully correlated.

Pertussis toxin prevents presynaptic inhibition by kainate receptors of rat hippocampal [(3)H]GABA release.

Kainate receptors are ionotropic receptors, also reported to couple to G(i)/G(o) proteins, increasing neuronal excitability through disinhibition of neuronal circuits. We directly tested in hippocampal synaptosomes if kainate receptor-mediated inhibition of GABA release involved a metabotropic action. The kainate analogue, domoate (3 microM), inhibited by 24% [(3)H]GABA-evoked release, an effect reduced by 76% in synaptosomes pre-treated with pertussis toxin. Protein kinase C inhibition attenuated by 82% domoate-induced inhibition of GABA release whereas protein kinase C activation did not change kainate receptor binding. Thus, domoate inhibition of GABA release recruits G(i)/G(o) proteins and a protein kinase C pathway.

Carbamazepine inhibits L-type Ca2+ channels in cultured rat hippocampal neurons stimulated with glutamate receptor agonists.

In order to better understand the mechanism(s) of action of carbamazepine (CBZ), we studied its effects on the increase in [Ca2+]i and [Na+]i stimulated by glutamate ionotropic receptor agonists, in cultured rat hippocampal neurons, as followed by indo- or SBFI fluorescence, respectively. CBZ inhibited the increase in [Ca2+]i stimulated either by glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), or N-methyl-D-aspartate (NMDA), in a concentration-dependent manner. In order to discriminate the effects of CBZ on the activation of glutamate receptors from possible effects on Ca2+ channels, we determined the inhibitory effects of Ca2+ channel blockers on [Ca2+]i changes in the absence or in the presence of CBZ. The presence of 1 microM nitrendipine, 0.5 microM omega-conotoxin GVIA (omega-CgTx GVIA), or of both blockers, inhibited the kainate-stimulated increase in [Ca2+]i by 51.6, 32.9 or 68.7%, respectively. In the presence of both 100 microM CBZ and nitrendipine, the inhibition was similar (54.1%) to that obtained with nitrendipine alone, but in the presence of both CBZ and omega-CgTx GVIA, the inhibition was greater (54%) than that caused by omega-CgTx GVIA alone. However, CBZ did not inhibit the increase in [Na+]i stimulated by the glutamate receptor agonists, but inhibited the increase in [Na+]i due to veratridine. Tetrodotoxin, or MK-801, did not inhibit the influx of Na+ stimulated by kainate, indicating that Na+ influx occurs mainly through the glutamate ionotropic non-NMDA receptors. Moreover, LY 303070, a specific AMPA receptor antagonist, inhibited the [Na+]i response to kainate or AMPA by about 70 or 80%, respectively, suggesting that AMPA receptors are mainly involved. Taken together, the results suggest that CBZ inhibits L-type Ca2+ channels and Na+ channels, but does not inhibit activation of glutamate ionotropic receptors.

Kainate receptors coupled to G(i)/G(o) proteins in the rat hippocampus.

Kainate receptors are a subtype of ionotropic glutamate receptors, permeable to cations and thus expected to have an excitatory depolarizing action on neurons. However, kainate receptor activation inhibits gamma-aminobutyric acid release in the hippocampus through activation of protein kinase C in a pertussis toxin-dependent manner, suggesting a coupling of kainate receptors to G proteins. Thus, we directly investigated the G protein coupling of kainate receptors in the rat hippocampus by using a selective kainate receptor agonist, [(3)H](2S,4R)-4-methylglutamate ([(3)H]MGA). [(3)H]MGA bound to a single site to hippocampal membranes with a K(D) value of 32 nM and a B(max) value of 1024 fmol/mg protein. This binding likely represents kainate receptors because it was displaced by domoate (K(i) = 4 nM), kainate (K(i) = 11 nM), and 6-cyano-7-nitroquinoxaline-2,3-dione (K(i) = 1.4 microM), but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (K(i) > 10 microM), (RS)-alpha-methyl-4-phosphonophenylglycine (K(i) > 10 microM), or (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (K(i) > 10 microM). Guanylylimidodiphosphate (30 microM), which uncouples all G protein-coupled receptors, shifted to the right the saturation curve of [(3)H]MGA (K(D) = 133 nM). This effect was mimicked by pretreatment of hippocampal membranes with modifiers of G(i)/G(o) proteins [30 microM N-ethylmaleimide (K(D) = 98 nM) or 25 microgram/ml pertussis toxin (K(D) = 95 nM)] but not by a modifier of G(s) proteins [50 microgram/ml cholera toxin (K(D) = 32 nM)]. Treatment of solubilized hippocampal membranes with pertussis toxin (25 microgram/ml) decreased [(3)H]MGA affinity (K(D) = 105-113 nM), which was recovered by reconstitution of these pretreated solubilized hippocampal membranes with G(i)/G(o) proteins (K(D) = 41-76 nM). These results indicate that hippocampal kainate receptors are coupled to G(i)/G(o) proteins.

Kainate receptors in hippocampal CA3 subregion: evidence for a role in regulating neurotransmitter release.

The hippocampal CA3 subregion of the rat is characteristically enriched in kainate receptors. At the synaptic level, the subcellular localization of these receptors is still a matter of debate. The CA3 pyramidal cells are particularly sensitive to excitotoxicity induced by kainate, which is in agreement with the high levels of kainate receptors in the stratum lucidum of the hippocampal CA3 subregion. Immunocytochemical studies, using antibodies against kainate receptor subunits, clearly demonstrated the presence of postsynaptic kainate receptors. However, it was not possible at the time to identify the activity of postsynaptic kainate receptors as mediators of the synaptic transmission. There are also reports showing the labeling of unmyelinated axons and nerve terminals with antibodies against kainate receptor subunits. The evidence for the presence of presynaptic kainate receptors in the hippocampus is further substantiated by the demonstration that stimulation of kainate receptors in synaptosomes isolated from the rat hippocampal CA3 subregion increases the intracellular free Ca2+ concentration ([Ca2+]i) coupled to the release of glutamate. These results support the model proposed by Coyle (1983), in which the excitotoxicity induced by kainate involves the activation of presynaptic kainate receptors, causing the release of glutamate. According to this model, the neurotoxic effect of kainate in the rat hippocampal CA3 subregion involves a direct effect on presynaptic kainate receptors and an indirect effect on postsynaptic glutamate receptors due to the enhanced release of glutamate.

Increase of the intracellular Ca2+ concentration mediated by transport of glutamate into rat hippocampal synaptosomes: characterization of the activated voltage sensitive Ca2+ channels.

The changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate and D-aspartate into rat hippocampal synaptosomes was studied. Glutamate increased the [Ca2+]i in a dose-dependent manner with an EC50 of 1.87 microM and a maximal increase of 31.5 +/- 0.9 nM. We also observed that stimulation of the synaptosomes with 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 100 microM kainate, or 100 microM D-aspartate increased the synaptosomal [Ca2+]i. The effect of either of these non-NMDA receptor agonists and of D-aspartate was additive, suggesting the activation of two different components (the ionotropic non-NMDA receptors or the glutamate transporters). Stimulation of synaptosomes with 100 microM glutamate increased the [Ca2+]i and prevented the effect of either non-NMDA receptor agonists and the effect of D-aspartate. We also observed that incubation of the synaptosomes with D-aspartate induced the Ca(2+)-independent release of glutamate, possibly through the reversal of the glutamate carrier. The aim of incubating the synaptosomes with D-aspartate was to avoid undesirable secondary activation of glutamate receptors. After incubating the synaptosomes with 100 microM D-aspartate (10 min at 37 degrees C), the subsequent stimulation with D-aspartate increased the [Ca2+]i due to glutamate transport. This increase in [Ca2+]i induced by 100 microM D-aspartate was insensitive to 1 microM nitrendipine, but was inhibited by about 50% by the presence of both 500 nM omega-CgTx GVIA and 100 nM omega-Aga IVA or by 500 nM omega-CgTx MVIIC. We clearly identified two different processes by which glutamate increased the [Ca2+]i in rat hippocampal synaptosomes: activation of non-NMDA receptors and activation of the glutamate transporters. We also characterized the voltage sensitive Ca2+ channels (VSCC) activated as a consequence of the glutamate transport, and determined that class B (N-type) and class A (P or Q-type) Ca2+ channels were responsible for about 50% of the signal.

Modulation of glutamate release from rat hippocampal synaptosomes by nitric oxide.

We used hippocampal synaptosomes to study the effect of NO originating from NO donors and from the activation of the NO synthase on the Ca2+-dependent release of glutamate due to 4-aminopyridine (4-AP) depolarization. We distinguished between the effects of NO on the exocytotic and on the carrier-mediated release of glutamate, which we found to be related to an increase in cGMP content and to a reduction of the ATP/ADP ratio, respectively. The NO donor hydroxylamine, at concentrations < or = 0.3 mM, inhibited the Ca2+-dependent glutamate release evoked by 4-AP, and addition of the NO donor, NOC-7, had a similar effect, which was reversed by the NO scavenger, carboxy-PTIO. Increasing the activity of NO synthase by addition of L-arginine also led to a decrease in the Ca2+-dependent release of glutamate induced by 4-AP, and this effect was reversed by inhibiting NO synthase with NG-nitro-L-arginine. This depression of the exocytotic release of glutamate was accompanied by an increase in cGMP levels due to the stimulation of soluble guanylyl cyclase by NO, produced either by the NO donors (hydroxylamine <0.3 mM) or by the endogenous NO synthase, but no significant decrease in ATP/ADP ratio was observed. However, at concentrations > or = 0.3 mM, hydroxylamine drastically increased the basal release and completely inhibited the Ca2+-dependent release of glutamate (IC50 = 168 microM). At these higher levels of NO, cGMP levels dropped to about 40% of the maximal values obtained at lower concentrations, and the ATP/ADP ratio decreased to about 50% (at 0.3 mM hydroxylamine). The large increase in the basal release could be partially inhibited by L-trans-2,4-PDC, previously loaded into the synaptosomes, suggesting that the nonexocytotic basal release occurred by reversal of the glutamate carrier. Therefore, the increase in cGMP induced by NO stimulation of the guanylyl cyclase decreases the exocytotic release of glutamate, but higher NO levels reduce the ATP/ADP ratio by inhibiting mitochondrial function, which therefore causes the massive release of cytosolic glutamate through the glutamate carrier.

Inhibition of N-,P/Q- and other types of Ca2+ channels in rat hippocampal nerve terminals by the adenosine A1 receptor.

The effects of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca2+ concentration ([Ca2+]i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca2+]i stimulated with 4-aminopyridine was neutralized by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca2+ channel blockers, omega-conotoxin GVIA, omega-conotoxin MVIIC or omega-conotoxin GVIA plus omega-conotoxin MVIIC, were greater than those caused by the Ca2+ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and omega-conotoxin GVIA or CPA and omega-conotoxin MVIIC were present was also greater than the inhibition by the Ca2+ channel blockers alone. The presence of both omega-conotoxin GVIA and omega-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [3H]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A1 receptor activation partly inhibits P/Q- and other non-identified types of Ca2+ channels.