{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:04:21Z","timestamp":1782281061344,"version":"3.54.5"},"posted":{"date-parts":[[2025,10,2]]},"group-title":"Neuroscience","reference-count":71,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T00:00:00Z","timestamp":1759363200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["R21NS130561"],"award-info":[{"award-number":["R21NS130561"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["R01NS115972"],"award-info":[{"award-number":["R01NS115972"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["R01NS133625"],"award-info":[{"award-number":["R01NS133625"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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 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\n                  <jats:sup>Cre<\/jats:sup>\n                  :TIGRE2.0\n                  <jats:sup>GCaMP6s\/wt<\/jats:sup>\n                  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 of 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, CGRP\/RAMP1 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":"10.1101\/2025.10.01.679335","type":"posted-content","created":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T03:15:17Z","timestamp":1759461317000},"source":"Crossref","is-referenced-by-count":0,"title":["Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability"],"prefix":"10.64898","author":[{"given":"Simone","family":"Carneiro-Nascimento","sequence":"first","affiliation":[{"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao","family":"Wei","sequence":"additional","affiliation":[{"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Gutterman","sequence":"additional","affiliation":[{"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0630-6660","authenticated-orcid":false,"given":"Dan","family":"Levy","sequence":"additional","affiliation":[{"name":"Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026031001001004000_2025.10.01.679335v3.1","doi-asserted-by":"publisher","DOI":"10.1038\/ni.3320"},{"key":"2026031001001004000_2025.10.01.679335v3.2","doi-asserted-by":"publisher","DOI":"10.1016\/J.IMMUNI.2020.02.015"},{"key":"2026031001001004000_2025.10.01.679335v3.3","first-page":"563","article-title":"Tissue-specific macrophages: how they develop and choreograph tissue biology","volume":"23","year":"2023","journal-title":"Nature reviews"},{"key":"2026031001001004000_2025.10.01.679335v3.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41583-019-0201-x"},{"key":"2026031001001004000_2025.10.01.679335v3.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.12.040"},{"key":"2026031001001004000_2025.10.01.679335v3.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05397-3"},{"key":"2026031001001004000_2025.10.01.679335v3.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04596-2"},{"key":"2026031001001004000_2025.10.01.679335v3.8","doi-asserted-by":"publisher","DOI":"10.1126\/sciimmunol.adh1129"},{"key":"2026031001001004000_2025.10.01.679335v3.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2018.01.011"},{"key":"2026031001001004000_2025.10.01.679335v3.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-019-0393-4"},{"key":"2026031001001004000_2025.10.01.679335v3.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06993-7"},{"key":"2026031001001004000_2025.10.01.679335v3.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2025.04.005"},{"key":"2026031001001004000_2025.10.01.679335v3.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-019-0344-y"},{"key":"2026031001001004000_2025.10.01.679335v3.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2022.10.005"},{"key":"2026031001001004000_2025.10.01.679335v3.15","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1016\/j.immuni.2022.09.005","article-title":"Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation","volume":"55","year":"2022","journal-title":"Immunity"},{"key":"2026031001001004000_2025.10.01.679335v3.16","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05753-x"},{"key":"2026031001001004000_2025.10.01.679335v3.17","doi-asserted-by":"crossref","unstructured":"B. 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