ROLE OF GLUTAMATE RECEPTORS AND VOLTAGE-DEPENDENT CALCIUM AND SODIUM-CHANNELS IN THE EXTRACELLULAR GLUTAMATE ASPARTATE ACCUMULATION AND SUBSEQUENT NEURONAL INJURY-INDUCED BY OXYGEN/GLUCOSE DEPRIVATION IN CULTURED HIPPOCAMPAL-NEURONS/

Ischemia is believed to induce neuronal damage by causing a sustained increase in the level of extracellular excitatory amino acids. In our study, we have examined the relationship between oxygen/glucose depriv ation-induced changes in extracellular glutamate/aspartate level and s ubsequent neuronal injury by pharmacological manipulation of glutamate receptors and calcium and sodium channels. Cultured hippocampal neuro ns were exposed to combined deprivation of oxygen/glucose for 40 to 50 min. These cultures developed acute neuronal swelling and widespread neuronal degeneration over the next 20 hr. The extracellular levels of glutamate and aspartate at the end of the oxygen/glucose deprivation period were measured by high-performance liquid chromatography, and ne uronal injury was assessed by lactate dehydrogenase efflux assay after subsequent aerobic incubation of the cells in normal medium for 20 hr . Both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor anta gonists attenuated the extracellular level of glutamate/aspartate and the neuronal injury. L-type, N-type and P-type calcium channel blocker s each significantly attenuated the neuronal injury, although the incr ease in the extracellular glutamate/aspartate was not significantly in hibited by any subtype-specific calcium channel blocker alone. A combi nation of calcium channel blockers of the three subtypes showed the mo st prominent neuroprotective effect and inhibited glutamate release. T he sodium channel blocker tetrodotoxin also attenuated both glutamate efflux and neuronal injury. These observations suggest that the overac tivation of glutamate receptors, calcium channels and sodium channels leads to excitotoxic neuronal injury through enhancing glutamate efflu x into the extracellular space under the condition of oxygen/glucose d eprivation.