MITOCHONDRIA ACCUMULATE CA2+ FOLLOWING INTENSE GLUTAMATE STIMULATION OF CULTURED RAT FOREBRAIN NEURONS

1. In cultures of rat forebrain neurones, mitochondria buffer glutamat e-induced, NMDA receptor-mediated Ca2+ influx. Here, we have used the fluorescent calcium indicator, indo-1 AM to record [Ca2+](i) from sing le cells. We varied either the glutamate concentration or the duration of exposure to investigate the cellular mechanisms recruited to buffe r [Ca2+](i) within different stimulation protocols. 2. For a 15 s stim ulus, the recovery time doubled as the glutamate concentration was rai sed from 3 to 300 mu M. Changing the duration of exposure from 15 a to 5 min increased the recovery time tenfold even when the glutamate con centration was held at 3 mu M. 3. We used a selective inhibitor of the mitochondrial Na+-Ca2+ exchange, CGP-37175. When applied immediately after a 15 s, 100 mu M glutamate challenge, CGP-37157 consistently cau sed a rapid fall in [Ca2+](i) followed by a slow rise after the drug w as washed out. A similar pattern was seen with the 5 min, 3 mu M gluta mate stimulus. The effects of CGP-37157 are consistent with the releas e of substantial mitochondrial Ca2+ stores during recovery from an int ense glutamate stimulus. 4. These studies suggest that mitochondria be come progressively more important for buffering glutamate-induced Ca2 loads as the stimulus intensity increases. The recovery of [Ca2+](i) to baseline following glutamate removal is critically regulated by the release of Ca2+ from mitochondrial stores via mitochondrial Na+-Ca2exchange. The data highlight a previously under-appreciated role for [ Na+](i) in the regulation of [Ca2+](i) in central neurones.