An alternative splicing variant in Clcn7-/- mice prevents osteopetrosis but not neural and retinal degeneration.

The ubiquitously expressed chloride channel 7 (CLCN7) is present within the ruffled border of osteoclasts. Mutations in the CLCN7 gene in humans (homologous to murine Clcn7) are responsible for several types of osteopetrosis in humans, and deficiencies in CLCN7 can present with retinal degeneration and a neuronal storage disease. A previously reported Clcn7(-/-) mouse showed diffuse osteopetrosis accompanied by severe retinal and neuronal degeneration. In contrast, the authors produced a novel Clcn7(-/-) mutant where mice did not develop osteopetrosis but still developed lethal neural and retinal degeneration. In these mice, there was a rapid progressive loss of the outer nuclear layer and photoreceptor layers of the retina. Laminar degeneration and necrosis of neurons in layers IV and V of the cerebral cortex and in the CA2/CA3 regions of the hippocampus were associated with intraneuronal accumulations of autofluorescent granules (periodic acid-Schiff positive). The extensive reactive gliosis was always associated with the accumulation of intraneuronal cytoplasmic material. The authors found, through quantitative real time polymerase chain reaction analyses, that an alternate Clcn7 transcript (previously identified only in bone marrow) showed minimal expression in the brain and eye but moderate expression in bone, which correlates with rescue of the osteopetrotic phenotype in the face of continued retinal and neuronal degeneration. Findings in this knockout mouse model prove that osteopetrotic compression of the brain is not responsible for neuronal and retinal degeneration in CLCN7-deficient mice; rather, they suggest that neurotoxicity is most likely due to lysosomal dysfunction as a result of the functional lack of this chloride channel in the central nervous system and eye.

Structural modifications to an N-methyl-D-aspartate receptor antagonist result in large differences in trapping block.

Differences in the degree of trapping of initial block by N-methyl-D-aspartate (NMDA) receptor antagonists may affect their safety and, hence, suitability for clinical trials. In this comparative study, 23 compounds structurally related to the low-affinity, use-dependent NMDA receptor antagonist (S)-alpha-phenyl-2-pyridineethanamine dihydrochloride (AR-R15896AR) were examined to determine the degree of trapping block they exhibit. Compounds were tested at concentrations that produced a comparable initial 80% block of NMDA-mediated whole-cell current in rat cortical cultures. A wide range of values of trapping block was found, indicating that trapping is not an all-or-none event. Fifteen of the compounds trapped significantly more than the 54 +/- 3% of initial block trapped by AR-R15896AR. The off-rates of these compounds were slower than that of AR-R15896AR. Only 2 of the 23 compounds trapped significantly less than AR-R15896AR. AR-R15808, the piperidine analog of AR-R15896AR, appeared to trap only 8 +/- 3% of its initial block, although its fast off-rate confounded accurate quantification of trapping. AR-R26952, which, like AR-R15896AR, contains a pyridine in place of a phenyl group, trapped 40 +/- 5% of its initial block and exhibited kinetics comparable with AR-R15896AR. Structure-activity analysis suggested that the presence of two basic nitrogen atoms and decreased hydrophobicity led to decreased trapping. There was no correlation between trapping and lipophilicity as would be expected if closed-channel egress was due to escape through the lipid bilayer. However, there was a positive correlation between off-rate and degree of trapping. Models that can account for partial trapping are presented.

Characterization of cyclin D1 expression in a rat global model of cerebral ischemia.

During normal development of the central nervous system there is expression of cyclins that regulate the progression of cells through various stages of mitosis. Cyclins have also been implicated in neuronal degeneration and apoptosis in adult brain, especially cyclin D1 as it is permissive for the transition from growth phase to synthesis phase in mitotic cell division. There is controversy as to whether cyclin D1 expression increases in both in vitro and in vivo models of cerebral ischemia. In this study we use immunohistochemistry and Western blot analysis to characterize cyclin D1 expression in an in vivo rat global model of cerebral ischemia to address the hypothesis that cyclin D1 alterations are involved in ischemic neuronal death. Although there was no change in cyclin D1 expression in either the vulnerable CA1 or resistant CA3 regions of the hippocampus prior to neuronal cell death (<3 days reperfusion), concomitant with the death of CA1 neurons and the loss of cyclin D1 in these cells, there was an increase in non-neuronal cyclin D1 positive cells. Some of the non-neuronal cyclin D1 expressing cells were identified to be activated microglia. In contrast to the cytoplasmic expression of cyclin D1 in neurons, the cyclin D1 expression in the microglia and other non-neuronal cells in CA1 was both nuclear and cytosolic. This study suggests that cyclin D1 does not play a role in the death of vulnerable CA1 neurons in global ischemia.

Ion channels involved in stroke.

Ion channels are membrane proteins that flicker open and shut to regulate the flow of ions down their electrochemical gradient across the membrane and consequently regulate cellular excitability. Every living cell expresses ion channels, as they are critical life-sustaining proteins. Ion channels are generally either activated by voltage or by ligand interaction. For each group of ion channels the channels' molecular biology and biophysics will be introduced and the pharmacology of that group of channels will be reviewed. The in vitro and in vivo literature will be reviewed and, for ion channel groups in which clinical trials have been conducted, the efficacy and therapeutic potential of the neuroprotective compounds will be reviewed. A large part of this article will deal with glutamate receptors, focusing specifically on N-methyl-D-aspartate (NMDA) receptors. Although the outcome of clinical trials for NMDA receptor antagonists as therapeutics for acute stroke is disappointing, the culmination of these failed trials was preceded by a decade of efforts to develop these agents. Sodium and calcium channel antagonists will be reviewed and the newly emerging efforts to develop therapeutics targeting potassium channels will be discussed. The future development of stroke therapeutics targeting ion channels will be discussed in the context of the failures of the last decade in hopes that this decade will yield successful stroke therapeutics.

Animal models.

Animal models of cerebral ischaemia mimic at best less than 25% of all strokes. Compounds which prove efficacious in animal models should, therefore, only be expected to improve outcome in a quarter of all strokes. If trials for acute stroke are to succeed, stroke subgroups represented by the animal models should be targeted. For the other subgroups, e.g. lacunar stroke, appropriate animal models need to be developed. Moreover, thrombolysis should be included in animal models because it is likely to be used as a first line treatment for ischaemic stroke and any future therapeutics will need to be compatible with it.

Pharmacological and molecular characterization of glutamate receptors in the MIN6 pancreatic beta-cell line.

The MIN6 pancreatic beta-cell line responds to glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, but not N-methyl-D-aspartate (NMDA) or 1S,3R-trans-ACPD, with increases in [Ca2+]i. This correlates with MIN6 expression of AMPA receptor subunits (GluR1-4) but only weak expression of NMDA NR2 receptor subunits, as determined by reverse transcriptase polymerase chain reaction (RT-PCR). Pharmacological characterization of the MIN6 AMPA receptors showed that AMPA-triggered [Ca2+]i responses were blocked by GYKI 52466, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and pentobarbital. AMPA-triggered [Ca2+]i responses were also blocked in Na(+)-free medium and by the voltage-sensitive Ca2+ channel antagonist La3+. Unlike cortical neuronal cultures, which show a loss of membrane-associated protein kinase C (PKC) activity and die in response to excitatory amino acid exposure, glutamate was not toxic to MIN6 cells and it did not decrease PKC activity. These studies indicate that MIN6 cells possess Ca(2+)-impermeable AMPA receptors that secondarily allow Ca2+ influx following AMPA-induced depolarization and that, despite elevating [Ca2+]i, AMPA is not toxic to these cells. The effects of glutamate and glutamate receptor antagonists on pancreatic cells needs to be better understood if these compounds are to be used as therapeutic agents to treat stroke.

NS-7 (Nippon Shinyaku).

Nippon Shinyaku is developing NS-7, a sodium and calcium channel blocker, as a neuroprotectant for use following cerebral infarction [249336]. It entered phase I trials for the treatment of acute stage cerebrovascular disorders in 1997 [271963]. As of November 1999, Nippon Shinyaku was preparing to start phase II studies in Japan [347972]. In November 1999, Nippon Shinyaku granted Schering exclusive developing and marketing rights to NS-7. Schering will conduct clinical trials in Europe and the US [347972].

Biology of ischemic cerebral cell death.

With the approval of alteplase (tPA) therapy for stroke, it is likely that combination therapy with tPA to restore blood flow, and agents like glutamate receptor antagonists to halt or reverse the cascade of neuronal damage, will dominate the future of stroke care. The authors describe events and potential targets of therapeutic intervention that contribute to the excitotoxic cascade underlying cerebral ischemic cell death. The focal and global animal models of stroke are the basis for the identification of these events and therapeutic targets. The signalling pathways contributing to ischemic neuronal death are discussed based on their cellular localization. Cell surface signalling events include the activities of both voltage-gated K+, Na+, and Ca2+ channels and ligand-gated glutamate, gamma-aminobutyric acid and adenosine receptors and channels. Intracellular signalling events include alterations in cytosolic and subcellular Ca2+ dynamics, Ca2+ -dependent kinases and immediate early genes whereas intercellular mechanisms include free radical formation and the activation of the immune system. An understanding of the relative importance and temporal sequence of these processes may result in an effective stroke therapy targeting several points in the cascade. The overall goal is to reduce disability and enhance quality of life for stroke survivors.

Loss of cyclin D1 in necrotic and apoptotic models of cortical neuronal degeneration.

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle and it is characterized by increased expression of cyclins, such as cyclin D1, prior to death. Cyclin D1 increases to permit transition from growth phase (G0/G1) to synthesis phase (S) during normal development but there is controversy as to which of the cyclins are activated prior to apoptotic cell death. We looked at the expression of cyclin D1 in cortical neuronal cultures treated with either staurosporine to produce apoptotic death, or with glutamate, to produce a non-apoptotic death. Cyclin D1 immunoreactivity was observed in the cytoplasm and nucleus of virtually all neurons under control conditions. Following the addition of either staurosporine or glutamate, cyclin D1 immunoreactivity did not change within 4 h. The cyclin D1 immunoreactivity was lost by 6 h with the appearance of either staurosporine-induced fragmented nuclei or glutamate-induced pyknotic nuclei. These immunocytochemical observations were confirmed with immunoblot analysis. Therefore, cyclin D1 is not a reliable indicator of apoptosis in cortical neuronal cultures and should not be used as an indicator of apoptotic cell death.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT-1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1-P21) and mature (P50), show distinct patterns of GLT-1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl-cyclic adenosine monophosphate. The transcripts for GLT-1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1-P4 astrocyte cultures, declined in P10-P21, and was undetectable in P50 astrocytes. Uptake of 3H-glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db-cAMP (250-500 microM; 6 days), resulted in a pronounced stellation, up-regulation of GLT-1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db-cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10-fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

Differences in degree of trapping of low-affinity uncompetitive N-methyl-D-aspartic acid receptor antagonists with similar kinetics of block.

This study characterizes the trapping of block of N-methyl-D-aspartic acid (NMDA)-induced currents by three structurally distinct, use-dependent NMDA receptor antagonists with similar rapid on-off rates. The antagonism of whole-cell currents in cultured rat cortical neurons by AR-R15896AR, ketamine, and memantine was examined. All three compounds produced a steady-state block after a 30-s coapplication, which was fully relieved after 50 s of NMDA exposure. The amplitudes of block caused by 50 microM AR-R15896AR, 10 microM ketamine, or 10 microM memantine were not significantly different, being 82 +/- 1%, 80 +/- 2%, and 81 +/- 2%, respectively. All three NMDA receptor antagonists exhibited trapping of block that was not significantly increased by extending the agonist/antagonist coapplication beyond 30 s. Although the initial blocks were similar, after 120 s of washout without agonist present, there were significant differences in trapping of block between antagonists, as only 54 +/- 3% of the AR-R15896AR block, 86 +/- 1% of the ketamine block, and 71 +/- 4% of the memantine block remained trapped. The lack of complete trapping is consistent with closed-channel egress by these compounds. Higher antagonist concentrations produced larger initial blocks, but the degree of trapping block was not significantly different from that at lower antagonist concentrations. The results demonstrate that differences in the degree of trapping exist among use-dependent NMDA receptor antagonists even when on and off rates are similar. These differences are correlated with measures of therapeutic index.

Brain derived neurotrophic factor induction of N-methyl-D-aspartate receptor subunit NR2A expression in cultured rat cortical neurons.

N-methyl-D-aspartate (NMDA) receptor subunit expression changes during development and following injury in several brain regions. These changes may be mediated by neurotrophic factors, such as brain derived neurotrophic factor (BDNF). Exposure of cultured cortical neurons to BDNF (100 ng/ml) for 24 h produced a significant decrease in the NMDA-induced whole-cell currents sensitive to the NR2B subunit selective NMDA receptor antagonist, CP-101,606, suggesting a relative decrease in NR2B subunit expression. There was a significant increase in NR2A by Western blot analysis. Consistent with the electrophysiology and Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) amplification revealed that BDNF caused a significant increase in relative NR2A subunit expression, a significant decrease in relative NR2B subunit expression and no change in relative NR2C subunit expression. These results suggest that BDNF enhances NMDA receptor maturation, warranting further study of the mechanism of BDNF effects on NMDA receptor subunit expression and the role these effects play in development and neuronal injury.

Neuroprotective effects of a novel AMPA receptor antagonist, YM872.

Quinoxalinediones such as NBQX are neuroprotective in most models of cerebral ischemia but their poor solubility results in nephrotoxicity limiting their clinical utility. We have investigated the neuroprotective effects of a water soluble AMPA receptor antagonist, YM872, using two in vitro models. The viability of cortical cultures exposed to 400 microM AMPA for 15 min (16.4 +/- 2.6%; n = 10) was significantly (p < 0.05) increased (84.7 +/- 4.6%; n = 6) with YM872 (10 microM) in a concentration-dependent manner. Evoked post-synaptic response amplitudes in oxygen-glucose deprived hippocampal slices treated with 10 microM YM872 (3.5 +/- 0.3 mV; n = 27) were significantly different from untreated deprived slices (0.3 +/- 0.1 mV; n = 31, p < 0.05) and the CA1 neurons appeared viable using a confocal live/dead fluorescence assay with confocal microscopy. The neuroprotection seen with YM872 in vitro warrants further investigation in vivo.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells.

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

A fluorescence confocal assay to assess neuronal viability in brain slices.

Hippocampal slice models are used to study the mechanisms of ischemia-induced neurotoxicity and to assess the neuroprotective potential of novel therapeutic agents. A number of morphological and functional endpoints are available to assess neuronal viability. The slice model also allows the study of selectively vulnerable neuronal populations within the same preparation. The fluorescence procedure described here provides a method of assessing the viability of neurons in rat hippocampal slices exposed to hypoxic-hypoglycemic conditions. Control and/or treated slices that had been subjected to a 10 min oxygen-glucose deprivation insult are double stained with calcein-AM (4 microM), which stains live cells green, and ethidium homodimer (6 microM), which stains the nucleus of dead cells red. The stained slices are then imaged using confocal microscopy. Vulnerable neurons in the CA1 region of slices deprived of oxygen and glucose became increasingly permeant to ethidium homodimer over the 4 h reperfusion period. Exposure to low Ca2+ concentration (0.3 mM) or the N-, P- and Q-type Ca2+ channel antagonist MVIIC (100 nM), which have been shown to be neuroprotective in this model of ischemia using field evoked post-synaptic potential (EPSP) measures as an endpoint, were also shown to be protective using the fluorescence assay.

Refractive-Index Measurements of Undoped Yttrium Aluminum Garnet from 0.4 to 5.0 mum.

We report measurements of the refractive index of undoped yttrium aluminum garnet from 0.4 to 5.0 mum and the calculation of Sellmeier coefficients based on our data. The data differ considerably from previously published reports. The effect of the new data on the design of optical devices such as intracavity etalons for lasers is discussed.

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons.

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na+ through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Galpha[il]) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gbetagamma blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Galpha(il) with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices.

Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

Antagonism of N-methyl-D-aspartate-evoked currents in rat cortical cultures by ARL 15896AR.

The purpose of this study was to characterize the kinetics and voltage-dependence of the block of N-methyl-D-aspartate (NMDA)-induced currents in primary cultures of rat cortical neurons by the neuroprotective, low-affinity, NMDA antagonist ARL 15896AR, using whole-cell voltage-clamp techniques. ARL 15896AR caused rapid and reversible inhibition of NMDA (50 microM)-evoked currents from neurons held at -60 mV, with an IC50 of 9.8 microM. The EC50 for NMDA was not significantly affected by 10 microM ARL 15896AR (P > .05), consistent with a noncompetitive mechanism of block. ARL 15896AR antagonism was use-dependent, because application of the drug 60 sec before NMDA did not attenuate the initial NMDA-evoked current, although the block developed rapidly thereafter. Once bound, ARL 15896AR remained trapped upon removal of NMDA until subsequent NMDA re-exposure, whereupon currents recovered rapidly. The forward and reverse binding rate constants were estimated to be 2.406 x 10(4) M(-1) sec(-1) and 0.722 sec(-1), respectively. Antagonism was strongly voltage-dependent; the K(D) values at 0 and -60 mV were 60 and 11 microM, respectively. Additionally, there was a component of the block by ARL 15896AR that was voltage-insensitive. This component of the block did not act at the ligand binding site, because it was not influenced by NMDA concentration, or at the polyamine site, because it was not affected by spermine. However, there was an interaction of ARL 15896AR with the glycine regulatory site. In contrast to many uncompetitive NMDA antagonists, like MK-801, ARL 15896AR exhibited rapid kinetics. This property may result in a large margin of safety while maintaining the efficacy associated with use-dependent NMDA antagonists, making this compound an excellent candidate for clinical trials.

Identification of calcium channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation.

The presynaptic Ca2+-influx affecting glutamate release during neuropathological processes is mediated via voltage-sensitive calcium channels (VSCCs). There is controversy, however, over the fractional contribution of the specific channel types involved. We have addressed this by investigating the protective effects of various VSCC blockers on oxygen and glucose-deprived rat hippocampal slices. The viability of treated and non-treated slices was assayed electrophysiologically by measuring the evoked population spike (PS) amplitude in the stratum pyramidale of the CA1 region and by imaging slices loaded with fluorochrome dyes specific for dead (ethidium homodimer) and live (calcein) cells using confocal microscopy. PS amplitudes were significantly (P < 0.01) depressed from 4.4 +/- 0.2 mV (n = 38) to 0.2 +/- 0.1 mV (n = 40) after the deprivation insult. Responses from deprived slices treated with omega-conotoxin MVIIC (100 nM; 4.2 +/- 0.5 mV; n = 20) were not significantly different from control, non-deprived slice responses. In contrast, deprived slices treated with either L-type (0.1 or 1 microM nimodipine) or N-type (0.1 or 3 microM omega-conotoxin MVIIA) blockers showed no significant protection. The viability of CA1 neurons as revealed by the fluorescence live/dead confocal viability assay was consistent with the electrophysiological measurements. By comparison with previous studies using P- and Q-type blockers to attempt neuroprotection against the same deprivation insult, the rank order in which specific Ca2+-channel types contribute to neuronal death due to oxygen and glucose deprivation was determined to be Q > N >> P > L.