Cultured Rat Cerebellar Neurons Research Articles

Structure and Neuroprotector Properties of a Complex Compound of Lithium with Comenic Acid.

The structure, antioxidant and neuroprotective properties of lithium comenate (lithium 5-hydroxy-4-oxo-4H-pyran-2-carboxylate) were studied. Lithium comenate was obtained by reacting comenic acid (H2Com) with lithium hydroxide in an aqueous solution. The structure of lithium comenate was confirmed via thermal analysis, mass spectrometry, IR, NMR and UV spectroscopy. The crystal structure was studied in detail via X-ray diffraction. The compound crystallized in a non-centrosymmetric space group of symmetry of the orthorhombic system Pna21 in the form of a hydrate, with three water molecules entering the first coordination sphere of the cation Li+ and one molecule forming a second environment through non-valent contacts. The gross formula of the complex compound was established [Li(HCom)(H2O)3]·H2O. It has been established that lithium comenate has a pronounced neuroprotective activity under the excitotoxic effect of glutamate, increasing the survival rate of cultured rat cerebellar neurons more than two-fold. It has also been found that the pre-stress use of lithium comenate at doses of 1 and 2 mg/kg has an antioxidant effect, which is manifested in a decrease in oxidative damage to the brain tissues of mice subjected to immobilization stress. Based on the data available in the literature, we believe that the high neuroprotective and antioxidant efficacy of lithium comenate is a consequence of the mutual potentiation of the pharmacological effects of lithium and comenic acid.

Study of the Mechanism of the Neuron Sensitization to the Repeated Glutamate Challenge

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.

Collapse of Neuronal Energy Balance As a Basis of L-Homocysteine Neurotoxicity

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca2+ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca2+ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca2+ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca2+ overload, while only a small portion of neurons were able to compensate the intracellular Ca2+ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φmit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φmit so that an uncoupling agent FCCP did not cause any additional changes in φmit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φmit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.

Нейропротекторный эффект комената кальция при окислительном стрессе и глутаматной цитотоксичности на модели культивируемых нейронов мозжечка и спинальных ганглиев

Изучено влияние нового фармакологического соединения кальциевой соли коменовой кислоты (CaK) на генерацию активных форм кислорода в модельной системе ЦФЛ (цитрат фосфатный буфер с добавлением люминола), на токсичность глутамата в диссоциированных культурах нейронов мозжечка крыс, а также его влияние на ростовые процессы культивируемых нейронов спинальных ганглиев эмбрионов кур в условиях окислительного стресса в сравнении с коменовой кислотой (КК). Установлено, что CaK повышает устойчивость культивируемых нейронов мозжечка к глутаматной цитотоксичности, проявляет выраженный нейротрофический эффект — защищает нейроны спинальных ганглиев эмбрионов кур от окислительного воздействия перекиси водорода, значительно снижает содержание свободных радикалов в модельной системе ЦФЛ (на 64,6 %, p < 0,001). Антиокислительные свойства CaK практически не отличаются от КК, в тоже время максимальный нейропротекторный эффект CaK проявляется в более низких (1 и 10 мкМ), чем КК (1000 мкМ) концентрациях, а нейротрофическое действие, в отличие от КК, в более широком диапазоне концентраций: CaK — 0,01 и 0,001 мМ, КК — 0,01 мМ.

The neuroprotective efficacy of carnosine-containing nanoliposomes and S-trolox-carnosine under oxidative stress conditions in vitro and in vivo

We investigated the protective effect of carnosine (carnosine-containing nanoliposomes and newly synthesized S-trolox-carnosine) in experiments in vivo on a model of acute hypobaric hypoxia in rodents and in vitro on neuronal cultures under oxidative stress conditions. We demonstrated the ability of new carnosine compounds to increase resistance of animals to acute hypobaric hypoxia, protecting the brain from oxidative damage. This effect is accompanied by preservation of acquired skills in the Morris water maze test, which is likely related to an increased efficiency of the brain antioxidant system. When oxidative stress was induced by hydrogen peroxide, spermine, acrolein, or cadmium in a suspension of cerebellar granule cells of the SAMP1 mouse line, a primary culture of rat cerebellar neurons, and a culture of neuronally differentiated PC-12 cells, carnosine (carnosine-containing nanoliposomes and S-trolox-carnosine) was able to inhibit generation of reactive oxygen species and reduce death of cells, enhancing the cell viability. The study results demonstrate high antihypoxic and antioxidant activities of the new carnosine compounds and open up prospects for the development and use of carnosine-based drugs in neurology.

Neuroprotective effect of potassium comenate against glutamate toxicity on the model of cultured rat cerebellar neurons.

The study demonstrated neuroprotective action of novel chemical agent, potassium salt of comenic acid, against the glutamate-induced cytotoxicity on the model of cultured cerebral neurons. Potassium comenate (0.001-1.0 mM) significantly decreased the rate of glutamateinduced neuronal death. The highest viability of the cultured neurons during postglutamate time was observed when potassium comenate was applied in a concentration of 0.1 mM.

Gabapentin is neuroprotective through glutamate receptor-independent mechanisms in staurosporine-induced apoptosis of cultured rat cerebellar neurons

Abstract The anticonvulsants that are currently available modulate the activity of neuronal receptors and ion channels, which are equally involved in apoptotic pathways. We investigated the hypothesis that gabapentin (GP), an anticonvulsant without effect on glutamate receptors acting as GABA analog, has neuroprotective properties. For comparison, we chose topiramate (TPM), which has been reported to be neuroprotective via AMPA receptors blockade. For this purpose, we used rat cerebellar granule neuron (CGN) cultures and we triggered apoptosis independent of glutamate receptors with staurosporine, a broad-spectrum protein kinase inhibitor. GP at therapeutic range concentration significantly increased cell viability in CGN cultures maintained in physiological KCl concentration and reversed apoptosis induced by staurosporine. Blockade of NMDA or AMPA receptors by MK801 or NBQX, respectively, did not alter GP neuroprotection, which was reversed instead by GABA. In contrast, protective effect of TPM on STS-treated CGN cultures was annihilated by NBQX, and not altered by MK801 or GABA. Treatments with neuroprotective concentrations of GP or TPM did not modify the expression of neuronal cell adhesion molecule or synaptophysin or the morphological aspect of neuronal endings. In summary, we report that GP is neuroprotective through glutamate-receptor independent mechanisms and without alteration of neuronal plasticity markers, which makes it a possible candidate for clinical neuroprotection trials.

Effects of Selective Inhibitors of Neuronal and Inducible NO-Synthase on ATP Content and Survival of Cultured Rat Cerebellar Neurons during Hyperstimulation of Glutamate Receptors

We studied the effects of selective inhibitors of neuronal and inducible NO-synthase (7-nitroindazole and aminoguanidine) and non-selective NO-synthase inhibitor L-NAME on ATP content and survival of cultured rat cerebellar neurons during hyperstimulation of glutamate receptors with toxic doses of glutamate. Application of 100 μM glutamate reduced ATP content in the primary culture of 7-8- and 14-15-day-old cerebellar granule cells by 66 and 49%, respectively, in comparison with the control. Inhibition of nitric oxide synthesis with 7-nitroindazole during glutamate exposure in the culture of 7-8-day-old neurons and with 7-nitroindazole and aminoguanidine in the culture of 14-15-day-old neurons ensured better protection of cells from ATP level decrease than non-specific inhibition with L-NAME. In addition, inhibition of neuronal and inducible NO-synthase during glutamate exposure decreased death of "young" neurons, whereas death of "old" neurons remained high under these conditions.

Polychlorinated Biphenyls PCB 52, PCB 180, and PCB 138 Impair the Glutamate−Nitric Oxide−cGMP Pathway in Cerebellar Neurons in Culture by Different Mechanisms

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in the food chain and are present in human blood and milk. Children born to mothers exposed to PCBs show cognitive deficits, which are reproduced in rats perinatally exposed to PCBs. It has been proposed that PCB-induced cognitive impairment is due to impairment of the glutamate-nitric oxide (NO)-cGMP pathway. The aim of the present work was to assess whether chronic exposure to the nondioxin-like PCB52, PCB138, or PCB180 alters the function of this pathway in primary cultures of rat cerebellar neurons and to assess whether different PCBs have similar or different mechanisms of action. PCB180 and PCB138 impair the function of the glutamate-NO-cGMP pathway at nanomolar concentrations, and PCB52 impairs the function of the glutamate-NO-cGMP pathway at micromolar concentrations. The mechanisms by which different PCBs impair the function of the glutamate-NO-cGMP pathway are different. Each PCB affects the pathway at more than one step but with different potency and, for some steps, in opposite ways. Exposure to the PCBs alters the basal concentrations of intracellular calcium, NO, and cGMP. The three PCBs increase NO; however, PCB52 and PCB138 increase basal cGMP, while PCB180 decreases it. PCB52 and PCB138 decrease the activation of soluble guanylate cyclase by NO, and PCB180 increases it. Long-term exposure to PCB52, PCB180, or PCB138 reduces the activation of NO synthase and the whole glutamate-NO-cGMP pathway in response to activation of N-methyl-d-aspartate receptors. The EC(50) was 300 nM for PCB52 and 2 nM for PCB138 or PCB180. These results show that chronic exposure to nondioxin like PCBs impairs the function of the glutamate-NO-cGMP pathway in cerebellar neurons by different mechanisms and with different potencies. Impaired function of this pathway would contribute to the cognitive alterations induced by perinatal exposure to PCBs in humans.

Role of protein kinase C in Ca2+ homeostasis disorders in cultured rat neurons during hyperstimulation of glutamate receptors

The primary culture of rat cerebellar neurons was used to study protein kinase C activity, intracellular variations in calcium concentration ([Ca(2+)]i), changes in the mitochondrial potential, and neuronal death during hyperstimulation of glutamate receptors and after 24-h incubation with phorbol ester. Prolonged exposure of neurons to glutamate (100 microM, 45 min) was followed by the development of delayed calcium dysregulation. Protein kinase C activity depended on the time of cell incubation with glutamate. Protein kinase C activity increased in response to application of glutamate for 15 min. However, protein kinase C activity decreased after 45-min exposure to glutamate and development of delayed calcium dysregulation. Protein kinase C activity was nearly undetected after 24-h preincubation of neurons with phorbol ester. Under these conditions, delayed calcium dysregulation developed more slowly and was observed in a smaller number of neurons. Neuronal death decreased to 2+/-1%. Our results suggest that protein kinase C plays an important role in death of neurons, which exhibit delayed calcium dysregulation during glutamate treatment.

Potent Neurotoxic Action of the Shellfish Biotoxin Yessotoxin on Cultured Cerebellar Neurons

Yessotoxin (YTX) and its analogues are disulphated polyether compounds of increasing occurrence in seafood. The biological effects of these algal toxins on mammals and the risk associated to their ingestion have not been clearly established. We have used primary cultures of rat cerebellar neurons to investigate whether YTX affected survival and functioning of central nervous system neurons. Exposure to YTX (> or =25 nM) caused first (approximately 8 h) weakening, granulation, and fragmentation of neuronal network, and later (approximately 48 h) complete disintegration of neurites and extensive neuronal death, with a significant decrease in the amount of filamentous actin. The concentration of YTX that reduced by 50% the maximum neuronal survival (EC50(48)) was approximately 20 nM. Lower toxin concentrations (approximately 15 nM) also caused visible signs of toxicity affecting neuronal network primarily. Removal of YTX after 5 h exposure delayed the onset of neurotoxicity but did not prevent neuronal degeneration and death. YTX induced a two-fold increase in cytosolic calcium that was prevented by the voltage-sensitive calcium channel antagonists nifedipine and verapamil. These antagonists were, however, completely ineffective in reducing neurotoxicity. Voltage-sensitive sodium channel antagonists saxitoxin and nefopam, and the NMDA receptor antagonist MK-801 also failed to prevent YTX neurotoxicity. Neuronal death by YTX involved typical hallmarks of apoptosis and required the synthesis of new proteins. Our data suggest neuronal tissue to be a vulnerable biological target for YTX. The potent neurotoxicity of YTX we report raises reasonable concern about the potential risk that exposure to YTX may represent for neuronal survival in vivo.

The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons

We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a low-density, sphingolipid- and cholesterol-enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immuno-separation procedure. Under conditions designed to preserve lipid-mediated membrane organization, several proteins were found in the prion protein-enriched membrane domains (i.e. the non-receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The prion protein-rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid-enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid-mediated non-covalent interactions.

Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation

Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, α-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca 2+ concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca 2+ concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5–p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity.

Pathogenic autoantibodies in the lambert-eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission in which antibodies are directed against voltage-gated calcium channels (VGCCs). We studied the action of LEMS immunoglobulin G (IgG) on cloned human VGCCs stably transfected into human embryonic kidney cells (HEK293). All LEMS IgGs tested bound to the surface of the P/Q-type VGCC cell line and caused a significant reduction in whole-cell calcium currents in these cells. By contrast, only 2 out of 6 IgGs bound extracellularly to the N-type VGCC cell line, and none of the LEMS IgGs tested was able to reduce whole-cell calcium currents in these cells. We used this apparent specificity of LEMS IgG for the P/Q-type VGCC to investigate the action of these IgGs on model systems where a number of different VGCC populations exist in equilibrium. LEMS IgG caused a significant downregulation in the omega-agatoxin IVA-sensitive P/Q-type VGCCs of cultured rat cerebellar neurons, but this was accompanied by a concomitant rise in the "resistant" R-type VGCCs. By using the passive transfer model of LEMS, similar results were observed at the mouse neuromuscular junction, where a significant reduction in P/Q-type VGCCs was paralleled by an increase in L- and R-type VGCCs. These results demonstrate an unexpected plasticity in the expression of VGCCs in mammalian neurons and may represent a mechanism by which the pathogenic effects of LEMS IgG are reduced.

RNA synthesis-dependent potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated toxicity by antihistamine terfenadine in cultured rat cerebellar neurons

We have studied the effects of terfenadine on neurotoxicity and elevation of free cytoplasmic Ca 2+ levels upon stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured cerebellar neurons. Pre-exposure to terfenadine (5 μM, 5 h) significantly increased neuronal death following specific stimulation of receptors by 100 μM AMPA or by subtoxic concentrations of domoate (8 μM), stimuli that are non-toxic when applied to terfenadine-untreated sister cultures. Terfenadine potentiation was prevented by the transcription inhibitor actinomycin D and was significantly ameliorated by histamine (1 mM). In terfenadine-treated neurons, AMPA increased [Ca 2+] i by approximately five fold, while AMPA induced no significant increase in [Ca 2+] i in the absence of terfenadine. Terfenadine reduced neuronal steady-state concentrations of [Ca 2+] i by approximately 75%. Our results suggest a role for histamine H1 receptors and intracellular calcium in the modulation of the excitotoxic response via AMPA receptors.

Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.

Non-Linear Effects of Cycloheximide in Glutamate-Treated Cultured Rat Cerebellar Neurons

Multiple cell types and organisms across a wide array of phyla and a variety of toxins demonstrate non-linear dose responses to low-level chemical exposures with high doses inhibiting cellular function and low doses stimulating function. We tested whether such non-linear responses to low and ultra-low dose N-methyl- d-aspartate (NMDA), 1-methyl-4-phenylpyridinium (MPP +) or cycloheximide moderated toxic glutamate exposure in cultured cerebellar granule cells. Neurons were incubated over 72 h with successive NMDA, MPP + iodide or cycloheximide additions producing specified low (10 −5, 10 −7, 10 −9, 10 −11, and 10 −13 M) and ultra-low (10 −27, 10 −29, 10 −63, and 10 −65 M) concentrations. Subsequently these neuronal cells were exposed to a 50% excitotoxic concentration of glutamate for 24 h. Neuronal viability was significantly reduced in neurons treated with micromolar (10 −5 M) cycloheximide whereas viability was enhanced in neurons treated with an ultra-low dose exposure of 10 −27 M cycloheximide. Neither NMDA nor MPP + elicited harmful or protective responses. This is the first report demonstrating non-linear dose–response effects of cycloheximide in low and ultra-low concentration ranges.

Carnitine prevents NMDA receptor-mediated activation of MAP-kinase and phosphorylation of microtubule-associated protein 2 in cerebellar neurons in culture

Activation of N-methyl- d-aspartate (NMDA) receptors leads to increased phosphorylation of the microtubule-associated protein MAP-2 by a mechanism that involves activation of nitric oxide synthase and nitric oxide-induced activation of mitogen-activated protein kinase (MAP-kinase). We have assessed the effects of carnitine on this signal transduction pathway in primary cultures of rat cerebellar neurons. We show that carnitine inhibits NMDA-induced phosphorylation of MAP-2 and that this is due to decreased activation of MAP-kinase. This effect is not due to inhibition by carnitine of NMDA-induced activation of nitric oxide synthase or to quenching of the nitric oxide formed, which are not affected by carnitine. Carnitine also inhibits the increase in phosphorylation of MAP-2 induced by the nitric oxide-generating agent S-nitroso- N-acetylpenicillamine, but not nitric oxide-induced activation of soluble guanylate cyclase. These results indicate that carnitine interferes with NMDA-induced, nitric oxide mediated activation of MAP-kinase at a step subsequent to nitric oxide formation.

Arsenic Induces Apoptosis in Rat Cerebellar Neurons via Activation of JNK3 and p38 MAP Kinases

Primary cultures of rat cerebellar neurons were used to study mechanisms of arsenic neurotoxicity. Exposure to 5, 10, or 15 μM sodium arsenite reduced cerebellar neuron viability and induced nuclear fragmentation and condensation as well as DNA degradation to oligonucleosome fragments. Exposure to 1 or 5 mM dimethylarsinic acid caused similar changes. Therefore, both inorganic arsenite and organic dimethylarsinic acid induce apoptosis in cerebellar neurons, with the inorganic form being more toxic. Cotreatment with cycloheximide or actinomycin D, inhibitors of protein or RNA synthesis, respectively, or with the caspase inhibitor zVAD, completely blocked arsenite-induced cerebellar neuron apoptosis. This implies that arsenite-induced cerebellar neuron apoptosis requires new gene expression and caspase activation. Interestingly, sodium arsenite selectively activated p38 and JNK3, but not JNK1 or JNK2 in cerebellar neurons. Blocking the p38 or JNK signaling pathways using the inhibitors SB203580 or CEP-1347 protected cerebellar neurons against arsenite-induced apoptosis. These data suggest that arsenite neurotoxicity may be due to apoptosis caused by activation of p38 and JNK3 MAP kinases.

RS-100642-198, a novel sodium channel blocker, provides differential neuroprotection against hypoxia/hypoglycemia, veratridine or glutamate-mediated neurotoxicity in primary cultures of rat cerebellar neurons.

The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), in in vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 micro M. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS-100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium [Ca(2+)]i) mobilization revealed that glutamate caused immediate and sustained increases in [Ca(2+)]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca(2+)]i mobilization produced by veratridine or KCl depolarization. QX-314 produced similar effects on glutamate-, veratridine- or KCl-induced [Ca(2+)]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca(2+)]i, and attenuating the sustained increase in [Ca(2+)]i mobilization. By using different in vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na(+) channels.