DEVELOPMENT OF INTRACELLULAR CALCIUM RESPONSES TO DEPOLARIZATION AND TO KAINATE AND N-METHYL-D-ASPARTATE IN CULTURED MOUSE HIPPOCAMPAL-NEURONS

We have investigated the initial appearance of voltage-gated Ca channels and kainate- and NMDA-type glutamate receptors in cultured embryonic mouse hippocampal neurons. The Ca-dependent fluorescence change of the dye fura-2 was used as a sensitive assay for the presence of functional channels and receptors. Expression of functional NMDA receptors was observed on some hippocampal neurons as early as E14. By the equivalent of E15-16, 40-50% of cells responded to K(o)-depolarization (50 mM), indicating the presence of functional voltage-gated Ca channels, approximately 20% of cells responded to kainate (50-mu-M), and just under 20% responded to NMDA (50-mu-M; in the presence of glycine and strychnine). By the equivalent of the end of the embryonic period 70-80% of cells responded to all 3 stimuli. As approximately 20% of cells in these cultures are glia, these data indicate that by the time of birth close to 100% of neurons express functioning kainate and NMDA receptors, and voltage-gated Ca channels. Increases in [Ca2+]i in embryonic neurons after application of NMDA were sensitive to APV and to external Mg, as are responses in mature neurons. The IC50 for block by external Mg of the [Ca2+]i increase induced by NMDA was 130-mu-M, and there was a slight positive correlation between the amplitude of the response to NMDA and sensitivity to external Mg.