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 Ca2+ 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 Ca2+ signal dynamics in healthy and diseased states. Using two-photon functional Ca2+ imaging in a novel transgenic reporter mouse line, combined with an event-based Ca2+ signaling analysis pipeline, we comprehensively characterized the Ca2+ 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 Ca2+ activity features, including event duration and signal frequency spectrum, distinct from those of interstitial non-perivascular macrophages. The Ca2+ activity of dural perivascular macrophages is uniquely coupled to behaviorally driven diameter fluctuation of their associated vessels. Most perivascular and non-perivascular meningeal macrophages displayed propagating intracellular Ca2+ activity. Both macrophage subtypes also exhibit synchronized intercellular Ca2+ 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 Ca2+ activity. Smaller subsets of both macrophage subtypes display either acute or persistent increases in Ca2+ activity, with the latter response mediated by CGRP/RAMP1 signaling. Collectively, our results highlight a previously unknown diversity of meningeal macrophage Ca2+ signaling at steady-state and in response to an aberrant brain hyperexcitability event.