Predictors of recurrent stroke in African Americans.

BACKGROUND: Stroke incidence and mortality are disproportionately higher among African Americans than among whites. OBJECTIVE: To describe the recurrent stroke characteristics and determine the predictability of known vascular risk factors for stroke recurrence in African Americans. METHODS: The authors followed 1,809 African Americans in the African-American Antiplatelet Stroke Prevention Study with recent noncardioembolic ischemic stroke for recurrent stroke, recurrent stroke subtype, and disability. RESULTS: Of the subjects, 10.6% experienced a recurrent stroke during follow-up. The mean interval between eligibility and recurrent stroke was 325 days (median 287 days, SD = 224 days). Stroke recurrence resulted in an average 1.5-point increase in the National Institute of Health Stroke Scale (p < 0.001) and a 3.5-point decrease in modified Barthel Index (p < 0.001). Of previously nondisabled subjects, 48% became disabled or died after stroke recurrence (p < 0.0001). Longitudinal analysis resulted in a hazard for recurrent stroke for each 10-mm Hg increase in systolic blood pressure of 1.103 (95% CI: 1.031 to 1.179, p = 0.004), pulse pressure 1.123 (95% CI: 1.041 to 1.213, p = 0.003), and mean arterial pressure 1.123 (95% CI: 1.001 to 1.260, p = 0.048). Multivariate analysis revealed increases in the recurrent stroke hazard for increases in baseline Glasgow Outcome Score (1.449, 95% CI: 1.071 to 1.961, p = 0.016) and increases in longitudinal pulse pressure (1.009, 95% CI: 1.001 to 1.017, p = 0.029). CONCLUSION: Recurrent stroke leads to disability and disability predicts recurrent stroke. Hypertension is the most predictive modifiable stroke risk factor.

Subcellular localization of calcium-permeable AMPA receptors in spinal motoneurons.

Activation of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been linked to potent effects on survival and dendritic outgrowth of spinal motoneurons. Ca(2+) permeability of AMPA receptors is controlled by the GluR2 subunit. Whole-cell electrophysiological studies have suggested that GluR2-containing and GluR2-lacking AMPA receptors may coexist in individual motoneurons. However, there has not been a direct demonstration of heterogeneity in AMPA receptor subunit composition in single motoneurons, nor of distinct subcellular distributions of GluR2-containing and GluR2-lacking receptors. In the present study, we have used confocal microscopy, immunocytochemistry and Ca(2+) imaging to characterize the subcellular localization of AMPA receptors in cultured rat spinal motoneurons. Immunoreactivity for GluR2 and GluR4 was concentrated in clusters, the vast majority of which were found in dendrites at synapses. Double-labelling for GluR2 and GluR4 revealed variability in relative expression of GluR2 and GluR4 between clusters within individual motoneurons; most AMPA receptor clusters were immunoreactive for both GluR2 and GluR4, but a significant minority of clusters were immunoreactive for GluR2 only or for GluR4 only. The majority of GluR2-immunonegative AMPA receptor clusters was present in dendrites, but the relative proportion of GluR2-immunonegative and GluR2-immunopositive clusters was similar in dendrites and soma. Imaging of [Ca(2+)](i) rises triggered by AMPA receptor activation confirmed Ca(2+) influx in motoneuron dendrites. These findings strongly support a model in which GluR2-containing and GluR2-lacking AMPA receptors coexist in motoneurons, clustered at synapses, and mixed in a relative proportion that varies considerably between cell membrane microdomains.

AMPA receptor current density, not desensitization, predicts selective motoneuron vulnerability.

Spinal motoneurons are more susceptible to AMPA receptor-mediated injury than are other spinal neurons, a property that has been implicated in their selective degeneration in amyotrophic lateral sclerosis (ALS). The aim of this study was to determine whether this difference in vulnerability between motoneurons and other spinal neurons can be attributed to a difference in AMPA receptor desensitization and/or to a difference in density of functional AMPA receptors. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. Single-cell RT-PCR quantification of the relative abundance of the flip and flop isoforms of the AMPA receptor subunits, which are known to affect receptor desensitization, did not reveal any difference between the two cell populations. Examination of AMPA receptor desensitization by patch-clamp electrophysiological measurements on nucleated and outside-out patches and in the whole-cell mode also yielded similar results for the two cell groups. However, AMPA receptor current density was two- to threefold higher in motoneurons than in dorsal horn neurons, suggesting a higher density of functional AMPA receptors in motoneuron membranes. Pharmacological reduction of AMPA receptor current density in motoneurons to the level found in dorsal horn neurons eliminated selective motoneuron vulnerability to AMPA receptor activation. These results suggest that the greater AMPA receptor current density of spinal motoneurons may be sufficient to account for their selective vulnerability to AMPA receptor agonists in vitro.

The role of ARNT2 in tumor angiogenesis and the neural response to hypoxia.

The Hypoxia-Inducible Factor-1 (HIF-1) activates the transcription of many genes required for cellular and organismal responses to oxygen deprivation. The HIF-1 complex is composed of the ubiquitously expressed basic helix-loop-helix/PAS (bHLH/PAS) proteins HIF-1alpha and Arylhydrocarbon Receptor Nuclear Translocator (ARNT). ARNT2 is a conserved ARNT homolog that is highly expressed in neurons, suggesting that ARNT2/HIF-1alpha heterodimers mediate transcriptional responses to oxygen deprivation in the nervous system. We show here that ARNT2 forms functional HIF complexes in vivo, and that ARNT2 restores hypoxia-induced gene expression to ARNT-deficient ES cells and hepatocytes. Formation of neural ARNT2/HIF-1alpha complexes in Arnt(-/-) ES cell-derived teratocarcinomas may explain why these tumors express VEGF, vascularize and grow efficiently, in contrast to ARNT-deficient hepatomas. Interestingly, all neural cell types studied accumulate both ARNT- and ARNT2-containing HIF complexes. We conclude that ARNT2 forms functional HIF complexes in neurons and plays an integral role in hypoxic responses in the CNS.

AMPA receptor calcium permeability, GluR2 expression, and selective motoneuron vulnerability.

AMPA receptor-mediated excitotoxicity is proposed to play a major pathogenic role in the selective motoneuron death of amyotrophic lateral sclerosis. Motoneurons have been shown in various models to be more susceptible to AMPA receptor-mediated injury than other spinal neurons. It has been hypothesized that this selective vulnerability of motoneurons is caused by the expression of highly Ca(2+)-permeable AMPA receptors and a complete or relative lack of the AMPA receptor subunit Glu receptor 2 (GluR2). The aim of this study was to quantify the relative Ca(2+) permeability of AMPA receptors and the fractional expression of GluR2 in motoneurons by combining whole-cell patch-clamp electrophysiology and single-cell RT-PCR and to compare these properties with those of dorsal horn neurons. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. As in previous studies, motoneurons were significantly more vulnerable to AMPA and kainate than dorsal horn neurons. However, all motoneurons expressed GluR2 mRNA ( approximately 40% of total AMPA receptor subunit mRNA), and their AMPA receptors had intermediate whole-cell relative Ca(2+) permeability (P(Ca(2+))/P(Cs(+)) approximately 0. 4). AMPA receptor P(Ca(2+))/P(Cs(+)) and the relative abundance of GluR2 varied more widely in dorsal horn neurons than in motoneurons, but the mean values did not differ significantly between the two cell populations. GluR2 was virtually completely edited at the Q/R site both in motoneurons and dorsal horn neurons. These results indicate that the selective vulnerability of motoneurons to AMPA receptor agonists is not determined solely by whole-cell relative Ca(2+) permeability of AMPA receptors.

Ca(2+) permeation of AMPA receptors in cerebellar neurons expressing glu receptor 2.

AMPA receptors in cultured cerebellar neurons were characterized by whole-cell electrophysiological studies and single cell PCR-based quantitation of subunit mRNA expression. Purkinje neurons consistently expressed high levels of Glu receptor 2 (GluR2) mRNA and AMPA receptors with low but nonzero Ca(2+) permeability. Other cerebellar neurons expressed AMPA receptors with a wide range of Ca(2+) permeability and of fractional GluR2. These properties correlated on a cell-by-cell basis. Their relationship was well fit by a model that assumed stochastic assembly of subunits and GluR2 dominance in controlling divalent cation permeation, suggesting that AMPA receptor properties in individual neurons may be determined primarily by relative levels of subunit transcription. A fraction of receptors, lacking GluR2, can contribute a highly Ca(2+)-permeable component to AMPA receptor responses, even in cells expressing GluR2.

Nitric oxide acutely inhibits neuronal energy production. The Committees on Neurobiology and Cell Physiology.

Disruption of mitochondrial respiration has been proposed as an action of nitric oxide (NO) responsible for its toxicity, but the effects of NO on the energetics of intact central neurons have not been reported. We examined the effects of NO on mitochondrial function and energy metabolism in cultured hippocampal neurons. The application of NO from NO donors or from dissolved gas produced a rapid, reversible depolarization of mitochondrial membrane potential, as detected by rhodamine-123 fluorescence. NO also produced a progressive concentration-dependent depletion of cellular ATP over 20 min exposures. The energy depletion produced by higher levels of NO (2 microM or more) was profound and irreversible and proceeded to subsequent neuronal death. In contrast to the effects of NO, mitochondrial protonophores produced complete depolarizations of mitochondrial membrane potential but depleted the neuronal ATP stores only partially. Inhibitors of mitochondrial oxidative phosphorylation (rotenone or 3-nitropropionic acid) or of glycolysis (iodoacetate plus pyruvate) also produced only partial ATP depletion, suggesting that either process alone could partially maintain ATP stores. Only by combining the inhibition of glycolytic energy production with the inhibition of mitochondria could the effects of NO in depleting energy and inducing delayed toxicity be duplicated. These results show that NO has rapid inhibitory actions on mitochondrial metabolism in living neurons. However, the severe ATP-depleting effects of high concentrations of NO are not fully explained by the direct effects on mitochondrial activity alone but must involve the inhibition of glycolysis as well. These inhibitory effects on energy production may contribute to the delayed toxicity of NO in vitro and in ischemic stroke.

Nitric oxide disrupts Ca2+ homeostasis in hippocampal neurons.

Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca2+ concentration ([Ca2+]i). In [Ca2+]i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S-nitrosocysteine produced a delayed rise in [Ca2+]i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca2+]i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca2+]i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca2+]i fluorimetry experiments, S-nitrosocysteine had no effect on the resting [Ca2+]i or on the recovery kinetics of [Ca2+]i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca2+]i homeostasis in hippocampal neurons by impairing Ca2+ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca2+]i may contribute to the delayed neurotoxicity of nitric oxide.

Disrupted [Ca2+]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons.

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 microM) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.

Mechanisms of hemolysate-induced [Ca2+]i elevation in cerebral smooth muscle cells.

The effects of hemolysate on free cytosolic [Ca2+] ([Ca2+]i) homeostasis were studied in freshly isolated rat basilar artery smooth muscle cells using fura 2 and dual excitation wavelength microfluorimetry. Hemolysate reversibly produced a transient [Ca2+]i peak followed by a slowly decaying plateau which was absent in Ca(2+)-free solution. This effect of hemolysate was attenuated by 1) the sarcoplasmic reticulum Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid, 2) the Ca2+ release-blocking agents ryanodine and dantrolene, 3) the cytochrome P-450 inhibitor econazole, and 4) the inorganic Ca2+ channel blocker lanthanum but was not significantly attenuated by 1) the receptor-regulated Ca2+ channel blocker SKF-96365 or 2) the voltage-dependent Ca2+ channel blocker nimodipine. Fractionation of hemolysate using membranes with specific pore sizes (0.5, 1, and 12-14 kDa) indicated that a component(s) > 0.5 but < 1 kDa could produce a similar [Ca2+]i peak and plateau while fractions > 1 and > 12-14 kDa produced a small and slow [Ca2+]i rise without a significant peak. ATP, which was found in hemolysate, produced a [Ca2+]i response similar to that of hemolysate. P2-purinoceptor antagonists significantly attenuated the effect of ATP, hemolysate, and the fractions < 1 and < 12-14 kDa. We conclude that hemolysate elevates [Ca2+]i by both releasing Ca2+ from internal stores and triggering Ca2+ entry, possibly from a voltage-independent Ca2+ influx pathway, an effect apparently identical to that of ATP.

Delayed antagonism of calpain reduces excitotoxicity in cultured neurons.

BACKGROUND AND PURPOSE: Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment. METHODS: Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval. RESULTS: The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170. This inhibitor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E-64, also blocked the proteolysis and toxicity produced by kainate in cerebellar neurons. Blocking nitric oxide synthase activity after 1 hour with the antagonist NG-nitro-L-arginine was also protective of cerebellar and hippocampal neurons, as was the combination of MDL-28170 and NG-nitro-L-arginine. CONCLUSIONS: The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutamate receptor stimulation, and blocking these postreceptor mechanisms can be effective in protecting neurons from excitotoxicity at delayed time points.

AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity.

Cerebellar Purkinje cells are selectively vulnerable to ischemia, although the reasons for this are unknown. In cultured embryonic rat cerebellar neurons, the steady state responses to the desensitizing agonist AMPA relative to responses to the nondesensitizing agonist kainate were greater in Purkinje cells compared to other cells, as measured by whole cell voltage clamp studies. Fluorimetric [Ca2+]i imaging experiments similarly found greater responses to AMPA relative to kainate in Purkinje cells than in other cerebellar neurons. In toxicity experiments measuring cell survival 24 hr following agonist exposure, AMPA and glutamate produced Ca(2+)-dependent toxicity which was selective for the Purkinje cell fraction of the neurons, whereas kainate produced nonselective toxicity, and NMDA selectively spared the mature Purkinje cells. Cyclothiazide, which inhibits AMPA receptor desensitization, enhanced steady state current responses to AMPA and increased the toxicity of AMPA. We conclude that the vulnerability of cerebellar neurons in culture to glutamate agonist-induced toxicity parallels the magnitude of the steady state currents produced, and that Purkinje cells may be selectively vulnerable because they express AMPA receptors which undergo less complete desensitization.

Increase in [Ca2+]i by CCh in adult rat sympathetic neurons are not dependent on intracellular Ca2+ pools.

We have examined the effects of the muscarinic agonists, carbachol (CCh) and oxotremorine (Oxo), on the intracellular free Ca2+ concentration ([Ca2+]i) in acutely dissociated sympathetic neurons from adult rats using fura 2-based microfluorometry. The drugs increased [Ca2+]i by 86 +/- 7 and 38 +/- 10 nM for CCh and Oxo, respectively (both 10 microM). Basal [Ca2+]i was 52 +/- 3 nM. Depletion of the caffeine-sensitive Ca2+ store or blockade of the Ca(2+)-adenosinetriphosphatase with thapsigargin did not alter the effect of either agonist on the rise in [Ca2+]i. On the other hand, the omission of Ca2+ from the perfusion solution or the use of TA-3090, a Ca2+ channel antagonist, blocked the effects of CCh and Oxo. In whole cell current-clamp recordings, the muscarinic agonists elicited a depolarization and action potential firing, which probably explained the rise in [Ca2+]i observed with microfluorimetric recording. In addition to their direct effects on the [Ca2+]i, muscarinic agonists also reduced the rise in [Ca2+]i induced by a nicotinic agonist. This inhibitory effect, observed in 68% of cells that responded to the nicotinic agonist, was blocked by atropine and pertussis toxin, whereas the muscarinic agonist-induced increase in [Ca2+]i was blocked by atropine but was pertussis toxin insensitive. These results suggest that at least two muscarinic receptors are present on sympathetic neurons and that they mediate opposite effect on the fluctuation of [Ca2+]i.

The Ca2+ influx induced by beta-amyloid peptide 25-35 in cultured hippocampal neurons results from network excitation.

Although a neurotoxic role has been postulated for the beta-amyloid protein (beta AP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (beta AP25-35), in particular, has been reported to be toxic in cultured neurons. We report that beta AP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, beta AP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The beta AP25-35-induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by beta AP1-40. Responses to beta AP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of beta AP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by beta AP25-35 in voltage-clamp studies. We conclude that beta AP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of beta AP25-35.

Glutamate receptor agonists stimulate diverse calcium responses in different types of cultured rat cortical glial cells.

We examined the effects of different types of glutamate receptor agonists on the intracellular calcium concentration, ([Ca2+]i), in cultured rat cortical glial cells. The cells in these cultures were characterized immunocytochemically using antibodies against glial fibrillary acidic protein, A2B5, and OX-42. The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3- dicarboxylic acid produced Ca2+ mobilization from intracellular stores in all classes of cells. Agonists at non-NMDA glutamate receptors also produced large increases in [Ca2+]i, primarily in cells of the O-2A lineage. Disruption of intracellular Ca2+ stores with thapsigargin showed that increases in [Ca2+]i produced by activating AMPA/kainate receptors were primarily due to Ca2+ influx rather than Ca(2+)-induced Ca2+ release. Agonists at NMDA receptors were ineffective. Electrophysiological studies revealed that cells of the O-2A lineage exhibited moderate inward currents in response to kainate in Na(+)-containing solutions, but only small inward currents and outward rectification in Na(+)-free solutions. However, in the presence of cyclothiazide, the kainate-induced currents were increased in size and a rightward shift of the reversal potential with increased [Ca2+]o could be demonstrated. Activation of cells by kainate, but not by depolarizing stimuli, stimulated the uptake of Co2+. Polymerase chain reaction studies showed that the glutamate receptor subunits GluR1-4 and GluR6 were all expressed in these cultures, but GluR5 was absent. The nature of the Ca2+ uptake pathway activated by non-NMDA receptor agonists in the O-2A lineage population is discussed. It is considered most likely that the O-2A lineage cells express both non-NMDA receptors that are relatively impermeable to divalent cations, as well as a smaller population that are Ca2+ permeable.

Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells.

Initial studies of glutamate receptors activated by kainate (KA) found them to be Ca2+ impermeable. Activation of these receptors was thought to produce Ca2+ influx into neurons only indirectly by Na(+)-dependent depolarization. However, Ca2+ entry via AMPA/KA receptors has now been demonstrated in several neuronal types, including cerebellar Purkinje cells. We have investigated whether such Ca2+ influx is sufficient to induce excitotoxicity in cultures of cerebellar neurons enriched for Purkinje cells. Agonists at non-NMDA receptors induced Ca2+ influx in the majority of these cells, as measured by whole-cell voltage clamp and by fura-2 [Ca2+]i microfluorimetry. To assess excitotoxicity, neurons were exposed to agonists for 20 min and cell survival was evaluated by a fluorescence assay 24 hr later. KA (100 microM) reduced neuronal survival relative to controls to 43 +/- 3% when applied in Na(+)-containing solution and to 45 +/- 3% in Na(+)-free solution. This toxicity was blocked completely by CNQX but only slightly by 100 microM Cd2+ and 50 microM D-(-)-2-amino-5-phosphonovaleric acid. Both Purkinje neurons and non-Purkinje cell types present in the cultures were similarly vulnerable to toxic KA exposure, but the population marked by KA-induced Co2+ uptake was selectively diminished by the excitotoxicity. Na(+)-independent excitotoxicity could also be induced by domoate, AMPA, or glutamate. Compared to KA, NMDA was relatively ineffective in inducing cell death. Most of the KA-induced excitotoxicity could be blocked by removal of extracellular Ca2+ during the KA exposure and for a 5 min period thereafter. Furthermore, antagonists of the Ca(2+)-activated enzymes nitric oxide synthase and calpain significantly reduced the KA-induced cell death. These results show that non-NMDA receptor activation can cause excitotoxicity in cerebellar Purkinje neurons by mechanisms not involving Na+ influx, but rather depending on direct Ca2+ permeation and activation of Ca(2+)-dependent enzymatic processes.

Characteristics of voltage sensitive calcium channels in dendrites of cultured rat cerebellar neurons.

We examined the effects of various pharmacological agents on the Ca2+ channels existing in the dendrites of cultured rat cerebellar neurons. The cultures consisted of Purkinje cells and non-Purkinje cells (deep cerebellar nuclear neurons and other non-granule neurons). Changes in intracellular free calcium concentration, [Ca2+]i, were monitored by digital imaging microfluorimetry using fura-2 as the indicator dye. In the Purkinje cell population increases in dendritic [Ca2+]i evoked by brief pulses of high K+ were very effectively blocked by (> 80%) by omega-Aga-IVA at low concentrations. Nimodipine and omega-conotoxins GVIA and MVIIC only blocked small components of the [Ca2+]i rise. In the non-Purkinje cells, omega-Aga-IVA was much less effective. omega-Conotoxin-GVIA blocked somewhat more and nimodipine blocked a similar percentage of the [Ca2+]i rise. omega-Conotoxin-MVIIC was quite ineffective in these cells. The results are discussed in terms of the types of voltage sensitive Ca2+ channels existing in the dendrites of these cells.

Calcium directly permeates kainate/alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors in cultured cerebellar Purkinje neurons.

In cultures of rat cerebellar neurons that were enriched in Purkinje cells, the non-N-methyl-D-aspartate glutamate receptor agonist kainate (KA) stimulated Ca2+ influx into all neurons in Na(+)-containing solutions. A large Ca2+ influx was also observed in most neurons when KA was applied in Na(+)-free solutions, even when the cells were voltage-clamped at negative potentials. KA also stimulated Co2+ uptake into both Purkinje and non-Purkinje neurons. The KA-induced Ca2+ influx was insensitive to pharmacological antagonists of voltage-sensitive Ca2+ channels and antagonists of N-methyl-D-aspartate receptors. Thus, different types of cerebellar neurons possess KA-gated ionophores that are permeable to Ca2+. This Ca2+ conductance may play an important role in glutamate-mediated physiological and pathological events in the cerebellum.