Intracellular Survival Pathways against Glutamate Receptor Agonist Excitotoxicity in Cultured Neurons: Intracellular Calcium Responsesa

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Abstract
Cultured rat cerebellar granule cells are resistant to the excitotoxic effects of N‐methyl‐d‐aspartate (NMDA) and non‐NMDA receptor agonists under three conditions: 1) prior to day seven in vitro when cultured in depolarizing concentrations of potassium [25 mM]; 2) at any time in vitro when cultured in non‐depolarizing concentrations of potassium [5 mM]; and 3) when neurons, cultured in depolarizing concentrations of potassium [25 mM] for eight days in vitro, are pretreated with a subtoxic concentration of NMDA. The focus of this paper is to determine: a) whether the resistance to excitotoxicity by NMDA and non‐NMDA receptor agonists is due to a decreased intracellular calcium [Ca++]i response to glutamate receptor agonists in cultured rat cerebellar granule cells; or b) whether [Ca++]i levels induced by the agonists are similar to those observed under excitotoxic conditions.Granule cells, matured in non‐depolarizing growth medium, treated with glutamate resulted in an increase in [Ca++]i followed by a plateau that remained above baseline in virtually all neurons that responded to glutamate. The response was rapid in onset (++]i to different extents; some cells did not respond to glutamate. Kainate also produced significant elevations in [Ca++]i.The [Ca++]i response to glutamate in neurons matured in depolarizing (25 mM K+) growth medium for three days was rapid, transient and heterogeneous, which reached a plateau that was elevated above baseline levels; removing the glutamate markedly reduced the [Ca++]i concentration. Activation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate receptors by kainic acid produced similar changes in [Ca++]i responses. At a time when cultured cerebellar granule cells become susceptible to the excitotoxic effects of glutamate acting at NMDA receptors (day in vitro (DIV) 8) in depolarizing growth medium, glutamate elicited [Ca++]i responses similar to those observed at a culture time when the neurons are not susceptible to the excitotoxic effects of glutamate (DIV 3). Pretreatment of the cultured neurons with a subtoxic concentration of NMDA, which protects all neurons against the excitotoxic effects of glutamate, did not alter the maximal [Ca++]i elicited by an excitotoxic concentration of glutamate.