Résumé
Reverse-transcription PCR assays were used to measure levels of NMDA receptor (NR) subunit mRNAs encoding splice variants of NR1 (NR1a, -exon 5; NR1b, +exon 5) and the major NR2 subunits (NR2A, NR2B, and NR2C) in dissociated cerebellar granule cell cultures. Cultures chronically exposed to 25 mM KCl or 100 mu M NMDA/15 mM KCl, which promote survival by stimulating Ca2+ influx through voltage-sensitive Ca2+ channels or NRs, were compared with 5 mM KCl culture conditions, which results in limited cell survival attributable to a lower level of NR stimulation by ambient glutamate. In situ granule-cell maturation is associated with downregulation of NR2B and increases both of NR2A and NR2C and in the ratio of NR1b/NR1a mRNAs. In culture, 25 mM KCl or NMDA rapidly induced NR2A and downregulated NR2B, followed by gradual induction of NR2C. In 5 mM KCl, a similar, rapid increase in NR2A was observed, but disappearance of NR2B occurred over a longer time course. By 9-12 d in vitro in 5 mM KCl, the relative proportions of all three NR2 mRNAs in surviving cells were not significantly different from cells cultured in 25 mM KCl. NR1a mRNA predominated at every stage of culture in 25 mM KCl or NMDA, however, whereas gradual induction of the mature-form NR1b was observed in 5 mM KCl. Although using high potassium- or NMDA-containing media enhanced granule cell survival, it did not reproduce the pattern of expression of NR mRNAs observed in situ, whereas this pattern was observed in granule cells surviving in 5 mM KCl.