Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability

The meninges, which envelop and protect the brain, host a dense network of resident macrophages with diverse roles in regulating homeostasis and neuroinflammation. Despite their importance, we have a limited understanding of their behavior in vivo. Many dynamic cellular functions of macrophages involve intracellular Ca2+ signaling. However, to our knowledge, virtually nothing is known about the spatiotemporal Ca2+ dynamics of meningeal macrophages. We developed a chronic intravital two-photon imaging approach and related computational analysis tools to interrogate meningeal macrophage Ca2+ dynamics, at a subcellular resolution, in a novel Pf4Cre:TIGRE2.0GCaMP6s/wt reporter mouse model. Using imaging in awake mice, we characterized the Ca2+ activity of meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD), an aberrant pro-inflammatory brain hyperexcitability event, implicated in migraine, traumatic brain injury, and stroke. In homeostatic meninges, macrophages in the dural perivascular niche exhibited several Ca2+ dynamic features, including event duration and signal frequency spectrum, distinct from those of in the interstitial, non-perivascular niche. Simultaneous tracking of meningeal macrophage Ca2+ dynamics and local vasomotion revealed a subset of dural perivascular macrophages whose activity was coupled to behaviorally-driven diameter fluctuations of their associated vessels. Most perivascular and non-perivascular meningeal macrophages displayed propagating intracellular Ca2+ activity as well as synchronized intercellular Ca2+ elevations, likely driven by extrinsic factors. In response to CSD, the majority of perivascular and non-perivascular meningeal macrophages showed a persistent decrease in Ca2+ activity, while a smaller subset displayed Ca2+ elevations. Mechanistically, CGRP/RAMP1 signaling mediated the increase but not the decrease in CSD-mediated Ca2+ signaling. Collectively, our results highlight a previously unknown diversity of meningeal macrophage Ca2+ dynamics at steady state and in response to an aberrant brain hyperexcitability event linked to neuroinflammation.