Intracellular Ca2⁺ dynamics are required for microglial process motility during surveillance and in response to injury

Microglia, the resident immune cells of the central nervous system, play pivotal roles in brain surveillance and injury responses through highly dynamic process motility. Here, we investigated the role of Ca 2 ⁺ signaling in mediating microglial process motility under both physiological and pathological conditions in vivo. Using dual-laser two-photon microscopy in a transgenic mouse model expressing red fluorescent protein (mCherry) and the Ca 2 ⁺ indicator (GCaMP6m) specifically in microglia, we observed autonomous Ca 2 ⁺ microdomains that operate asynchronously within individual processes, exhibiting significantly more frequent and localized Ca 2 ⁺ transients compared to non-motile structures. Upon focal laser-induced cortical injury, we observed rapid Ca 2 ⁺ waves in microglia, with processes extending toward the lesion site displaying correlated Ca 2 ⁺ activity. Attenuation of Ca 2 ⁺ transients by BAPTA-AM, a Ca 2 ⁺ chelator, impaired both homeostatic and laser-induced motility. Importantly, microglia-specific genetic overexpression of plasma membrane Ca 2 ⁺ ATPase 2 in vivo markedly reduced both homeostatic surveillance and injury-induced motility. Together, our findings provide evidence that microglial intracellular Ca 2 ⁺ signaling is required for process motility in vivo, supporting a model in which local Ca 2 ⁺ dynamics enable surveillance and injury-directed remodeling.