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 (KCl) 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.