Spatio‐Temporal Diversity of Calcium Activity in Microglia

Microglia, the brain's innate immune cells, possess complex, highly motile branched processes. These act independently, enabling individual processes to carry out entirely distinct functions in parallel. Intracellular Ca²⁺ signaling is implicated in many of these distinct microglial functions. However, it has been difficult to quantify how such Ca²⁺ activity is compartmentalized in space and time to prevent unwanted cross-talk between signaling pathways. Previous studies have typically relied on manually drawn regions-of-interest (ROIs), which averages fluorescence within predefined compartments and therefore cannot resolve the fine-scale spatio-temporal propagation patterns that may be functionally relevant. To address this, we adopt an unbiased non-ROI-based analytical approach to comprehensively characterize the temporal, spatial and spatio-temporal dimensions of microglial Ca²⁺ activity in vivo. We find that microglial Ca²⁺ activity predominantly occurs in processes, tends to remain localized at its site of origin, and, when it propagates, often follows a well-defined direction (either toward or away from the soma) rather than spreading isotropically as would be expected under purely passive diffusion. The tendency of microglial Ca²⁺ activity to spread between intracellular regions does not correlate with peak amplitude, but appears to be limited by the branching points of the microglial processes. Finally, we show that Ca²⁺ activity can differ between the microglial soma and its processes in response to various pharmacological stimuli. These results suggest that Ca²⁺ signals are actively compartmentalized within microglia in a context dependent manner, rather than being synchronized across the entire cell.