Imaging Mitochondrial Ca²⁺ Uptake in Astrocytes and Neurons using Genetically Encoded Ca²⁺ Indicators (GECIs)

Mitochondrial Ca²⁺ plays a critical role in controlling cytosolic Ca²⁺ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca²⁺ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca²⁺ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca²⁺ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca²⁺ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca²⁺ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca²⁺ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca²⁺ signaling, as well as astrocyte-neuron interactions.