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. © 2001 Wiley‐Liss, Inc.