Characterization of the Excitoprotective Actions of N‐Methyl‐d‐Aspartate in Cultured Cerebellar Granule Neurons

Abstract: 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 excitoprotective 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 excitoprotective 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).