Study of Calcium Signaling in Astrocytes with a Novel Endoplasmic Reticulum–Targeted GCaMP Sensor

The endoplasmic reticulum (ER), the major organelle for the storage of Ca²⁺, maintains a concentration of Ca²⁺ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca²⁺ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca²⁺ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca²⁺ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains.