Dual mechanisms of Ca(2+) increases elicited by N-methyl-D-aspartate in immature and mature cultured cortical neurons.

Cortical primary cultures were loaded with the fluorescent indicator fluo-3 for assessment of intracellular-free Ca(2+) ions with the aid of a confocal laser-scanning microscope. The addition of N-methyl-D-aspartic acid (NMDA) markedly increased the number of fluorescent cells in a manner sensitive to prevention by both an NMDA channel blocker and MgCl(2). In the absence of added MgCl(2), NMDA induced a sustained increase in the number of fluorescent cells with a transient increase by KCl in cells cultured for 3 days in vitro (DIV). Both nifedipine and dantrolene were more potent in preventing the increase by NMDA in cortical preparations cultured for 9 DIV than those for 3 DIV. These results suggest that activation of NMDA receptors may lead to a sustained increase in intracellular-free Ca(2+) concentrations in immature cultured neurons, in a manner less dependent on the influx through L-type voltage-dependent channels as well as the release from intracellular stores than in mature neurons.