Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons

Mitochondria are critical to cellular Ca²⁺ homeostasis via the sequestering of cytosolic Ca²⁺ in the mitochondrial matrix. Mitochondrial Ca²⁺ buffering regulates neuronal activity and neuronal death by shaping cytosolic and presynaptic Ca²⁺ or controlling energy metabolism. Dysfunction in mitochondrial Ca²⁺ buffering has been implicated in psychological and neurological disorders. Ca²⁺ wave propagation refers to the spreading of Ca²⁺ for buffering and maintaining the associated rise in Ca²⁺ concentration. We investigated mitochondrial Ca²⁺ waves in hippocampal neurons using genetically encoded Ca²⁺ indicators. Neurons transfected with mito-GCaMP5G, mito-RCaMP1h, and CEPIA3mt exhibited evidence of mitochondrial Ca²⁺ 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 Ca²⁺ waves. However, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptor blockade had no effect on mitochondrial Ca²⁺ wave were propagation. The Ca²⁺ 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 Ca²⁺ buffering and the molecular basis of mitochondrial Ca²⁺ waves in neurons in response to electrical stimulation.