Visualization of NMDA receptor-induced mitochondrial calcium accumulation in striatal neurons

Ca²⁺ influx through NMDA receptor-gated channels and the subsequent rise in intracellular Ca²⁺ concentration ([Ca²⁺](i)) have been implicated in cytotoxic processes that lead to irreversible neuronal injury. While many studies have focused on cytosolic Ca²⁺ homeostasis, much less is known about Ca²⁺ fluxes in subcellular organelles, such as mitochondria. The mitochondria play an important role in Ca²⁺ homeostasis by sequestering cytosolic Ca²⁺ loads. However, mitochondrial Ca²⁺ overload can impair AT synthesis, induce free radical formation, and lead to lipid peroxidation. Thus, it is also important to understand the mitochondrial Ca²⁺ fluxes induced by MDA. In this study, changes in mitochondrial Ca²⁺ concentration ([Ca²⁺](m)) in cultured striatal neurons were monitored with a Ca²⁺- binding fluorescent probe, rhod-2, and laser scanning confocal microscopy. The rhod-2 fluorescence signal was highly localized in mitochondrial areas of confocal images. A rapid increase of [Ca²⁺](m) was observed when neurons were treated with 100 μM NMDA. The increased [Ca²⁺](m) induced by NMDA could not be observed in the presence of ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, or CCCP, a protonophore that breaks down the mitochondrial membrane potential necessary for Ca²⁺ uptake. The magnitude and reversibility of changes in [Ca²⁺](m) induced by MDA were variable. In neurons receiving multiple pulses of NMDA, [Ca²⁺](m) did not return to baseline. The elevated [Ca²⁺](m) may persist indefinitely and may rise further after successive NMDA exposures. These data demonstrate that Ca²⁺ accumulates in mitochondria in response to NMDA receptor activation. This Ca²⁺ accumulation may play a role in the excitotoxic mitochondrial dysfunction induced by NMDA.