Hippocampal neurons exhibit both persistent C2+ influx and impairment of Ca2+ sequestration/extrusion mechanisms following excitotoxic glutamate exposure

Exposure of neurons to glutamate is an essential element of neuronal function, producing transient elevations in free intracellular-calcium ([Ca2+](i)) that are required for normal physiological processes. However, prolonged elevations in [Ca2+](i) have been observed following glutamate excitotoxicity and have been implicated in the pathophysiology of delayed neuronal cell death. In the current study, we utilized indo-1 and fura-2ff Ca-2+ imaging techniques to determine if glutamate-induced prolonged elevations in [Ca2+], were due to persistent influx of extracellular Ca2+ or from impairment of neuronal Ca2+ extrusion/sequestration mechanisms. By experimentally removing Ca2+ from the extracellular solution following glutamate exposure, influx of Ca2+ into the neurons was severely attenuated. We observed that brief glutamate exposures (<5 min, 50 <mu>M glutamate) resulted in a Ca2+ influx that continued after the removal of glutamate. The Ca2+ influx was reversible, and the cell was able to effectively restore [Ca2+], to resting levels. Longer, excitotoxic glutamate exposures (greater than or equal to5 min) generated a Ca2+ influx that continued for the duration of the recording period (>1 h). This persistent Ca2+ influx was not primarily mediated through traditionally recognized Ca2+ channels such as glutamate receptor-operated channels or voltage-gated Ca2+ channels. In addition to the persistent Ca2+ influx, longer glutamate exposures also produced a lasting disruption of Ca2+ extrusion/sequestration mechanisms, impairing the ability of the neuron to restore resting [Ca2+ ](i). These data suggest that glutamate-induced protracted [Ca2+](i) elevations result from at least two independent, simultaneously occurring alterations in nruronal Ca2+ physiology, including a persistent Ca2+ influx and damage to Ca2+ regulation mechanisms. (C) 2001 Elsevier Science B.V. All rights reserved.