In Vitro Ischemia Promotes Glutamate-Mediated Free Radical Generation and Intracellular Calcium Accumulation in Hippocampal Pyramidal Neurons

Ischemia-induced cell damage studies have revealed a complex mechanism that is thought to involve glutamate excitotoxicity, intracellular calcium increase, and free radical production. We provide direct evidence that free radical generation occurs in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic–hypoglycemic insult. The production of free radicals is temporally correlated with intracellular calcium elevation, as measured by injection of fluo-3 in individual pyramidal cells, using patch electrodes. Free radical production (measured as changes in the fluorescence emission of dihydrorhodamine 123) peaked during reoxygenation and paralleled rising intracellular calcium. Electrophysiological whole-cell recordings revealed membrane potential depolarization and decreased input resistance during the ischemic insult. Glutamate receptor blockade resulted in decreased free radical production and markedly diminished intracellular calcium accumulation, and prevented neuronal depolarization and input resistance decrease during the ischemic episode. These results provide evidence for a direct involvement of glutamate in oxidative damage resulting from ischemic episodes.