[A mechanism for modulation of N-methyl-D-aspartate receptor channels by free ferrous ions in cultured rat cortical neurons]

Rat cortical neurons cultured in DMEM/F-12 in the absence of fetal calf serum were harvested on days 3, 6 and 9 in vitro (DIV), followed by homogenization and subsequent SDS-PAGE for immunoblotting using an antibody against MAP-2 or GFAP. Expression of MAP-2 was not markedly changed during cultivation for 3 to 9 DIV, while GFAP was profoundly expressed after 6 DIV in a manner dependent on the duration of culturing. Cortical neurons cultured for 3 DIV were pre-incubated with fluo-3 AM and then subjected to fluorescence image analysis using a confocal microscope. The exposure to NMDA markedly increased the number of neurons with high fluorescence intensity in the absence of MgCl2, without prominently affecting that in the presence of MgCl2. The addition of FeCI2, but not hemoglobin and transferrin, markedly reduced the increase in a concentration-dependent manner in the presence of NMDA. This inhibition by FeCl2 was not affected by the addition of dithiothreitol or 2-mercaptoethanol. These results suggest that free ferrous ions may selectively prevent Ca2+ influx across NMDA receptor channels in a manner different from that done by the redox site on the channels in cultured rat cortical neurons.