Hippocampal neurons exhibit both persistent Ca²⁺ 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 Ca²⁺ imaging techniques to determine if glutamate-induced prolonged elevations in [Ca²⁺]_i 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 μM 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⁺]_i to resting levels. Longer, excitotoxic glutamate exposures (≥5 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 neuronal Ca²⁺ physiology, including a persistent Ca²⁺ influx and damage to Ca²⁺ regulation mechanisms.