Intracellular survival pathways against glutamate receptor agonist excitotoxicity in cultured neurons. Intracellular calcium responses

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 (< 10 sec) and the pattern of response heterogeneous in that cells responsive to glutamate increased their [Ca⁺⁺](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.