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 ATP synthesis, induce free radical formation, and lead to lipid peroxidation. Thus, it is also important to understand the mitochondrial Ca2+fluxes induced by NMDA. 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+]mwas observed when neurons were treated with 100 μMNMDA. The increased [Ca2+]minduced 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+]minduced by NMDA were variable. In neurons receiving multiple pulses of NMDA, [Ca2+]mdid not return to baseline. The elevated [Ca2+]mmay 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.