Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons

Mitochondria are critical to cellular Ca homeostasis via the sequestering of cytosolic Ca in the mitochondrial matrix. Mitochondrial Ca buffering regulates neuronal activity and neuronal death by shaping cytosolic and presynaptic Ca or controlling energy metabolism. Dysfunction in mitochondrial Ca buffering has been implicated in psychological and neurological disorders. Ca wave propagation refers to the spreading of Ca for buffering and maintaining the associated rise in Ca concentration. We investigated mitochondrial Ca waves in hippocampal neurons using genetically encoded Ca indicators. Neurons transfected with mito-GCaMP5G, mito-RCaMP1h, and CEPIA3mt exhibited evidence of mitochondrial Ca waves with electrical stimulation. These waves were observed with 200 action potentials at 40 Hz or 20 Hz but not with lower frequencies or fewer action potentials. The application of inhibitors of mitochondrial calcium uniporter and oxidative phosphorylation suppressed mitochondrial Ca waves. However, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptor blockade had no effect on mitochondrial Ca wave were propagation. The Ca waves were not observed in endoplasmic reticula, presynaptic terminals, or cytosol in association with electrical stimulation of 200 action potentials at 40 Hz. These results offer novel insights into the mechanisms underlying mitochondrial Ca buffering and the molecular basis of mitochondrial Ca waves in neurons in response to electrical stimulation.