N-METHYL-D-ASPARTATE RECEPTORS ARE CRITICAL FOR MEDIATING THE EFFECTSOF GLUTAMATE ON INTRACELLULAR CALCIUM-CONCENTRATION AND IMMEDIATE-EARLY GENE-EXPRESSION IN CULTURED HIPPOCAMPAL-NEURONS

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, ca lcium imaging, specific RNA analysis and immunocytochemistry were carr ied out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2(+)](i)), and determinatio n of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fos B, c-jun,jun B, zif/268 (also te rmed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expressi on of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-m ethyl-D-aspartate subtype of glutamate receptor was necessary and suff icient for the transcriptional response. Non-N-methyl-D-aspartate rece ptors, while present in cultured hippocampal neurons, contributed rela tively little to the regulation of transcription. Calcium imaging show ed that glutamate-induced changes in [Ca2+](i) were almost entirely me diated by N-methyl-D-aspartate receptors, rather than by L-type Voltag e-sensitive calcium channels. Previous studies have shown that stimula tion with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can le ad to an increase in [Ca2+](i) and c-fos expression.(20,25) Here we de monstrate that in our hippocampal culture system glutamate controls [C a2+](i) and induces immediate early gene transcription primarily by ac tivating N-methyl-D-aspartate receptors.