Characterization of the excitoprotective actions of N-methyl-D-aspartate in cultured cerebellar granule neurons

Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N-methyl-D-aspartate (NMDA) has been shown previously to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations of glutamate. In the present study, we have further characterized the excitoprotective actions of NMDA in these neurons. NMDA- induced excitoprotection was concentration dependent (EC₅₀ ~30 μM) and time dependent, with maximal protection observed following 16 h of preexposure to NMDA. NMDA-induced excitoprotection did not require continuous exposure to NMDA, as a 4-h preincubation was sufficient to induce full excitoprotection when measured 8 h later. Maximal protection was manifest as a 'right shift' in the concentration-response relationship for glutamate toxicity of approximately three orders of magnitude (EC₅₀ ~30 μM in untreated neurons compared with ≥50 mM in NMDA-treated neurons). After removal of NMDA, complete reversal of the excitoprotection state was observed by 48 h (t( 1/2 ) ≃24 h). The ability of NMDA to induce excitoprotection was observed in neurons maintained for up to 14 days in vitro (DIV) [postnatal day (PND) 22], but was absent at 21 and 32 DIV (PND 29-40), despite little to no difference in the toxicity of glutamate at any DIV examined. Preexposure of cerebellar granule neurons to a maximally excitoprotection concentration of NMDA (50 μM) failed to alter the density of NMDA receptors measured by the specific binding of [³H] MK-801. Moreover, the immediate elevation in intracellular free calcium concentration ([Ca²⁺](i)) induced by glutamate exposure and measured by microfluorimetry and the Ca²⁺-sensitive indicator fura-2 was similar in NMDA-pretreated and untreated neurons. As reported previously, NMDA-induced excitoprotection in cerebellar granule neurons was, however, reversed by coincubation with the protein synthesis inhibitor cycloheximide. Taken together, these data suggest that NMDA receptor-mediated excitoprotection in cerebellar granule neurons is mediated via both a transcriptionally directed and a developmentally regulated postreceptor mechanism(s).