Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death

Excessive activation of neuronal glutamate receptors has been implicated in the pathophysiology of stroke, epilepsy, and traumatic brain injury. Previously, it has been demonstrated that excitotoxic glutamate exposure results in the induction of an extended neuronal depolarization (END), as well as protracted elevations in free intracellular calcium ([Ca²⁺]_i). Both END and the prolonged [Ca²⁺]_i elevations were shown to correlate with subsequent neuronal death. In the current study, we used whole-cell current-clamp electrophysiology and fura-ff Ca²⁺ imaging to determine the electrophysiological basis of END. We found that removal of extracellular Ca²⁺ but not Na⁺ in the post-glutamate period resulted in complete reversal of END, allowing neurons to rapidly repolarize to their initial resting membrane potential (RMP). In addition, removal of extracellular Ca²⁺ was sufficient to eliminate the protracted [Ca²⁺]_i elevations induced by excitotoxic glutamate exposure. To investigate the mechanism through which extracellular Ca²⁺ was effecting these changes, pharmacolgical antagonists of well-characterized routes of Ca²⁺ entry were tested for their effects on END. Antagonists of glutamate receptors and voltage-gated Ca²⁺ channels (VGCCs) had no significant effect on the membrane potential of neurons in END. Likewise, inhibitors of the Na⁺/Ca²⁺ exchange (NCX) were ineffective. In contrast, addition of 500 μM ZnCl₂ or 100 μM GdCl₃ to control extracellular medium (containing normal levels of exctracellular Ca²⁺) in the post-glutamate period resulted in rapid and complete reversal of END. Addition of 1 mM CdCl₂ to control medium had only modest effects on END. These data provide the first direct evidence that END induced by excitotoxic glutamate exposure is caused by an influx of extracellular Ca²⁺ and demonstrate that the previously irreversible condition of END can be reversed by removing extracellular Ca²⁺. In addition, understanding the electrophysiological basis of this novel Ca²⁺ -induced extended depolarization may provide an insight into the pathophysiology of stroke, traumatic brain injury, and other forms of neuronal injury.