Astrocytes govern critical aspects of brain function via Ca2+ signals, the majority of which associate with mitochondria. However, little is known with regard to
in situsources, kinetics or mechanisms of Ca2+ influx in astrocytic mitochondria. To address this knowledge gap, we expressed the genetically encoded calcium indicator, GCaMP6f within the mitochondrial matrix of adult mouse astrocytes in the dorsolateral striatum (DLS) and hippocampus (HPC). We found spontaneous Ca2+ events in astrocytic mitochondria with subcellular differences between somatic, branch, and branchlet mitochondria, as well as inter-regional differences between astrocytes in the DLS and HPC. We also found a strong dependency of spontaneous mitochondrial Ca2+ fluxes on endoplasmic reticulum stores, the surprising lack of a major role for the mitochondrial calcium uniporter, MCU, and dual mitochondrial Ca2+ responses with multiple neurotransmitter agonists. Together, our findings provide a foundational understanding of mechanisms for Ca2+ influx in astrocytic mitochondria within disease-relevant brain regions.