Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 lM) increased cell proliferation, whereas higher concentrations (100 lM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27 KIP1 , allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS 2/2 mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 µM AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 µM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampus.Key words: NPY • AMPA • kainate • neuroprotection • neurodegeneration N europeptide Y (NPY) is a 36-amino-acid peptide abundantly distributed in the brain and associated with a number of physiological and pathological conditions (1). This peptide has been shown to modulate anxiety, pain, memory, eating behavior, and many other functions in the central, as well as in the peripheral, nervous systems (1, 2). Another significant role of NPY that has become evident during the past decade is regulation of seizure activity (3).At least five NPY receptor subtypes have been identified based on different pharmacological profiles: Y1, Y2, Y4, Y5, and y6 (4). A putative Y3 receptor has also been identified (5 In the hippocampus, Y1 and Y2 receptors are highly expressed (11). Receptors of the Y1 subtype are preferentially located in granule cells of the dentate molecular layer, whereas Y2 receptors are expressed at high concentrations in the mossy fiber and Schaffer collateral fields (12). Moreover, Y5 receptor binding sites can also be found in the strata oriens and radiatum of the CA3 and CA1 areas (13,14). Presently, there is evidence for the involvement of these three NPY receptors subtypes in epilepsy. The activation of Y2 receptors suppresses seizure activity in hippocampal slices in vitro (15) and in vivo models (3). In human temporal lobe epilepsy, significant alterations in Y1 and Y2 receptor binding were observed (16). NPY Y5 receptors also seem to be involved in the suppression of seizure activity (17,18). In different animal models of epilepsy, NPY is highly expressed by granule cells and mossy fibers of the hippocampus (19,20), whereas in normal conditions only GABAergic interneurons express this peptide. Furthermore, knockout mice lacking the NPY gene develop spontaneous seizures and are more susceptible to pentylenetetrazol and kainic acid-induced motor seizures, which are inhibited by intracerebral infusion of NPY (21). Thus, these evidences suggest that N...

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca(2+) channels (VGCCs) in NPY receptors-induced inhibition of Ca(2+) influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 microM NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu(31),Pro(34)]NPY, NPY13-36, NPY (19-23)-(Gly(1),Ser(3),Gln(4),Thr(6),Ala(31),Aib(32),Gln(34))-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca(2+)](i) due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or omega-conotoxin GVIA (omega-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by omega-agatoxin IVA (omega-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca(2+)](i) changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs.

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.

Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNELpositive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro‐Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline‐treated rats. Immunohistochemistry and western blot analysis for the calpain‐derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus.

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.

Carbamazepine (CBZ) has been extensively used in the treatment of epilepsy, as well as in the treatment of neuropathic pain and affective disorders. However, the mechanisms of action of this drug are not completely elucidated and are still a matter of debate. Since CBZ is not very effective in some epileptic patients and may cause several adverse effects, several antiepileptic drugs have been developed by structural variation of CBZ, such as oxcarbazepine (OXC), which is used in the treatment of epilepsy since 1990. (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), which were recently developed by BIAL, are new putative antiepileptic drugs, with some improved properties. In this review, we will focus on the mechanisms of action of CBZ and its derivatives, OXC, BIA 2-093 and BIA 2-024. The available data indicate that the anticonvulsant efficacy of these AEDs is mainly due to the inhibition of sodium channel activity.