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Despite their importance, we have a limited understanding of their behavior in vivo. Many dynamic cellular functions of macrophages involve intracellular Ca\n                    <jats:sup>2+<\/jats:sup>\n                    signaling. However, virtually nothing is known about the spatiotemporal Ca\n                    <jats:sup>2+<\/jats:sup>\n                    dynamics of meningeal macrophages in vivo. We developed a chronic intravital two-photon imaging approach and related computational analysis tools to interrogate meningeal macrophage Ca\n                    <jats:sup>2+<\/jats:sup>\n                    dynamics, at subcellular resolution, in a novel Pf4-Cre:Ai162 conditional GCaMP6s reporter mouse model. Using imaging in awake mice, we characterized Ca\n                    <jats:sup>2+<\/jats:sup>\n                    activity in 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 Ca\n                    <jats:sup>2+<\/jats:sup>\n                    dynamic features, including event duration and signal frequency spectrum, distinct from those localized to the interstitial, non-perivascular niche. Simultaneous tracking of macrophage Ca\n                    <jats:sup>2+<\/jats:sup>\n                    dynamics and local vasomotion revealed a subset of dural perivascular macrophages whose activity was coupled to locomotion-driven diameter fluctuations of their associated vessels. Most perivascular and non-perivascular meningeal macrophages displayed propagating intracellular Ca\n                    <jats:sup>2+<\/jats:sup>\n                    activity and synchronized intercellular Ca\n                    <jats:sup>2+<\/jats:sup>\n                    elevations, potentially driven by extrinsic factors. In response to CSD, the majority of perivascular and non-perivascular meningeal macrophages showed a persistent decrease in Ca\n                    <jats:sup>2+<\/jats:sup>\n                    activity, while a smaller subset displayed Ca\n                    <jats:sup>2+<\/jats:sup>\n                    elevations. Mechanistically, calcitonin gene-related peptide receptor signaling mediated the increase but not the decrease in CSD-mediated Ca\n                    <jats:sup>2+<\/jats:sup>\n                    signaling. Collectively, our results highlight a previously unknown diversity of Ca\n                    <jats:sup>2+<\/jats:sup>\n                    dynamics in meningeal macrophages at steady state and in response to an aberrant brain hyperexcitability event linked to neuroinflammation.\n                  <\/jats:p>","DOI":"10.7554\/elife.109888","type":"journal-article","created":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T17:30:12Z","timestamp":1771003812000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.109888","source":"Crossref","is-referenced-by-count":1,"title":["Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability"],"prefix":"10.7554","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6141-6080","authenticated-orcid":true,"given":"Simone","family":"Carneiro-Nascimento","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04drvxt59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School","place":["Boston, United States"]}],"role":[{"role":"author","vocabulary":"crossref"},{"role":"data-curation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Chao","family":"Wei","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04drvxt59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School","place":["Boston, United States"]}],"role":[{"role":"author","vocabulary":"crossref"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Anna","family":"Gutterman","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04drvxt59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School","place":["Boston, United States"]}],"role":[{"role":"author","vocabulary":"crossref"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0630-6660","authenticated-orcid":true,"given":"Dan","family":"Levy","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04drvxt59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School","place":["Boston, United States"]}],"role":[{"role":"author","vocabulary":"crossref"},{"role":"corresponding-author","vocabulary":"crossref"},{"role":"conceptualization","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]}],"member":"4374","published-online":{"date-parts":[[2026,5,13]]},"reference":[{"key":"bib1","doi-asserted-by":"publisher","DOI":"10.1126\/sciimmunol.adh1129","type":"journal-article","article-title":"Extrasinusoidal macrophages are a distinct subset of immunologically active dural macrophages","volume":"9","author":"Amann","year":"2024","journal-title":"Science Immunology"},{"key":"bib2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-23482-5","type":"journal-article","article-title":"Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing","volume":"12","author":"Atcha","year":"2021","journal-title":"Nature Communications"},{"key":"bib3","doi-asserted-by":"publisher","DOI":"10.64898\/2025.12.08.693074","type":"preprint","article-title":"The circadian clock regulates scavenging of fluid-borne substrates by brain border-associated macrophages","volume-title":"bioRxiv","author":"Barr","year":"2025"},{"key":"bib4","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1038\/s41577-025-01186-x","type":"journal-article","article-title":"The spatial and temporal activation of macrophages during fibrosis","volume":"25","author":"Behmoaras","year":"2025","journal-title":"Nature Reviews. 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