Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones

1. In cultures of rat forebrain neurones, mitochondria buffer glutamate-induced, NMDA receptor-mediated Ca2+ influx. Here, we have used the fluorescent calcium indicator, indo-1 AM to record [Ca2+](i) from single cells. We varied either the glutamate concentration or the duration of exposure to investigate the cellular mechanisms recruited to buffer [Ca2+](i) within different stimulation protocols. 2. For a 15 s stimulus, the recovery time doubled as the glutamate concentration was raised 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 concentration 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 caused a rapid fall in [Ca2+](i) followed by a slow rise after the drug was washed out. A similar pattern was seen with the 5 min, 3 mu M glutamate stimulus. The effects of CGP-37157 are consistent with the release of substantial mitochondrial Ca2+ stores during recovery from an intense glutamate stimulus. 4. These studies suggest that mitochondria become 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+-Ca2+ exchange. The data highlight a previously under-appreciated role for [Na+](i) in the regulation of [Ca2+](i) in central neurones.