NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture

Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca²⁺ loads and regulate the shape of intracellular Ca²⁺ concentration ([Ca²⁺]_i) responses in neurons. When isolated mitochondria are exposed to high [Ca²⁺], Ca²⁺ enters the matrix via the uniporter and returns to the cytosol by Na⁺/Ca²⁺ exchange. Released Ca²⁺ may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca²⁺ recycling, the continuous uptake and release of Ca²⁺ by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca²⁺]_i and mitochondrially targeted aequorin to measure matrix Ca²⁺ concentration ([Ca²⁺]_mt) to determine whether Ca²⁺ recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na⁺/Ca²⁺ exchange, respectively. As predicted by the Ca²⁺ recycling hypothesis, blocking the uniporter immediately following challenge with 200 μM NMDA produced a rapid and transient increase in cytosolic Ca²⁺ without a corresponding increase in matrix Ca²⁺. Blocking mitochondrial Ca²⁺ release produced the opposite effect, depressing cytosolic Ca²⁺ levels and prolonging the time for matrix Ca²⁺ levels to recover. The Ca²⁺ recycling hypothesis uniquely predicts these reciprocal changes in the Ca²⁺ levels between the two compartments. Ca²⁺ recycling was not detected following treatment with 20 μM NMDA. Thus Ca²⁺ recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca²⁺-dependent neurotoxicity.