Excitatory amino acid‐induced alterations of cytoplasmic free Ca2+ individual cerebellar granule neurons: Role in neurotoxicity

Abstract

The effects of glutamate on intracellular free Ca²⁺ [Ca²⁺]_i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca²⁺]_i, was measured with fura‐2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dosedependent increases in [Ca²⁺]_XSi transient for glutamate concentrations in a range of : 0.01–0.5 μM and sustained for higher levels of glutamate. The ED₅₀ for the [Ca²⁺]_i response to glutamate was. 6 μM. The LQ₅₀ for glutamate‐induced neurotoxicity was similar, i.e., 10 μM The effect of glutamate on [Ca²⁺]_i was greatly dmiinished when external Ca²⁺ was removed and blocked by Mg²⁺+ N‐methyl‐D‐aspartate (NMDA)‐type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca²⁺ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10–20 min at 25°C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca²⁺]_i. Pretreatment cells with the ganglioside GM1 reduced this late increase in [Ca²⁺]_i, as well as the neurotoxic effects of glutamate. This indicates that glutamate‐induced neurotoxicity results from a composite of diverse temporal alterations in Ca²⁺ homeostasis and that blunting any of these components reduces excitotoxicity.