Meningeal macrophages exhibit diverse calcium signaling at steady state and in response to aberrant cortical hyperexcitability in awake mice

The meninges, which envelop and protect the brain, host a large number of resident macrophages that play a crucial role in regulating homeostasis and neuroinflammation. Intracellular Ca ²⁺ signaling mediates a variety of macrophage functions. While diverse, ontogeny, transcriptional profiles, and phenotypes of meningeal macrophages have been described, little is known about their spatiotemporal Ca ²⁺ signal dynamics in healthy and diseased states. We used two-photon Ca ²⁺ imaging in a novel reporter mouse line, combined with an event-based signaling analysis pipeline, to comprehensively characterize the Ca ²⁺ activity of meningeal macrophages both at steady state and in response to cortical spreading depolarization (CSD) - a pathophysiological brain hyperexcitability event, implicated in migraine, traumatic brain injury, and stroke. In homeostatic meninges of behaving mice, perivascular macrophages exhibit several Ca ²⁺ activity features, including event duration and signal frequency spectrum, distinct from those of interstitial non-perivascular macrophages. The Ca ²⁺ activity of dural perivascular macrophages is uniquely coupled to behaviorally driven diameter fluctuation of their associated vessels. Most perivascular and non-perivascular meningeal macrophages display propagating intracellular Ca ²⁺ activity. Both macrophage subtypes also exhibit synchronized intercellular Ca ²⁺ elevations, likely driven by an external factor. In response to CSD, the majority of perivascular and non-perivascular meningeal macrophages show a persistent decrease in their Ca ²⁺ activity. Smaller subsets of both macrophage subtypes display either acute or persistent increases in Ca ²⁺ activity, with the latter response mediated by CGRP/RAMP1 signaling. Collectively, our results highlight a previously unknown diversity of meningeal macrophage Ca ²⁺ signaling at steady-state and in response to an aberrant brain hyperexcitability event.