Changes in secondary glutamate release underlie the developmental regulation of excitotoxic neuronal cell death

Vulnerability to excitotoxicity increases during development in vivo and in vitro. To determine whether the mere presence of mature N-methyl-d-aspartate (NMDA) receptors coincides with the emergence of excitotoxicity or whether post-receptor signaling processes may also contribute, we examined the temporal relationship of NMDA receptor expression, function and toxicity using cortical cell cultures. Surface expression of all NMDA receptor subunits increased with time in culture. This correlated with NMDA receptor function, assessed both biochemically and electrophysiologically, but not with the appearance of excitotoxicity. Specifically, cells at day in vitro (DIV) 10 were less susceptible to NMDA receptor-induced neurotoxicity than those cultured for 14 days, even though receptor expression/function was identical. In addition, cell-attached single channel recordings revealed that NMDA receptor conductance, open probability, and frequency of channel openings were not significantly different between the two days. Intriguingly, depolarization-induced release of glutamate from cultures grown for 10 days was significantly lower than that released from cultures grown for 14 days. Further, exogenous addition of glutamate receptor agonists immediately after removal of NMDA rendered cultures at DIV 10 susceptible to excitotoxicity, while toxicity was significantly reduced by addition of an NMDA receptor antagonist immediately after exposure to NMDA at DIV 14. These data are the first to demonstrate that the subsequent, secondary release of glutamate plays an equal, if not more important, role than NMDA receptor development per se, in mediating the enhanced vulnerability of neurons to excitotoxicity that occurs with age.