INABILITY TO RESTORE RESTING INTRACELLULAR CALCIUM LEVELS AS AN EARLY INDICATOR OF DELAYED NEURONAL CELL-DEATH

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca2+](i)) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca2+](i) has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca2+](i) in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca2+](i) that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca2+](i), and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca2+](i) and cell death. Depolarization of neurons with potassium chloride (KCI) resulted in increases in [Ca2+](i), but these increases were buffered immediately upon removal of the KCL, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca2+](i) and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca2+](i) (IRRC) that is a statistically significant indicator of delayed neuronal cell death.