Abstract

Mitochondrial Ca²⁺ uptake responds dynamically and sensitively to changes in cytosolic Ca²⁺ levels and plays a crucial role in sequestering the large Ca²⁺ load induced byN-methyl-d-aspartate (NMDA) receptor activation. However, the precise interrelationships between NMDA receptor activation, cytosolic Ca²⁺ increase, and mitochondrial Ca²⁺ uptake remain obscure. To reliably, independently, and simultaneously detect cytosolic and mitochondrial Ca²⁺ concentration changes in the same cell, we loaded primary striatal neurons with two Ca²⁺ indicators, calcium green 1N and rhod-2, and visualized the fluorescence signals from single neurons with laser scanning confocal fluorescence microscopy. In kinetic data analysis, only calcium green signals from predefined cytosolic areas and rhod-2 signals from predefined mitochondrial regions were used, and attention was focused on the initial rapid rising phase of the responses. When neurons were treated with 100 μm NMDA, increases of cytosolic and mitochondrial Ca²⁺ showed similar time courses and rates of change, and seemed to be time-locked. In contrast, when neurons were treated with 100 μm kainate, 50 mm KCl, or 0.3 μm ionomycin, mitochondrial Ca²⁺ increases lagged behind cytosolic Ca²⁺ increases. These data suggest that mitochondrial Ca²⁺ uptake in response to an increase of cytosolic Ca²⁺ is faster and more tightly coupled during NMDA receptor activation than during non-NMDA receptor or voltage-dependent Ca²⁺ channel activation. This proficient mitochondrial Ca²⁺ uptake may avert a large rise in cytosolic Ca²⁺ concentration in response to NMDA receptor activation. Yet, it may lead to excessive Ca²⁺ accumulation inside mitochondria and render mitochondria susceptible to Ca²⁺ mediated injury.