{"doi":"10.1101/2025.10.01.679335","title":"Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability","abstract":"Abstract 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 2+ signaling. However, virtually nothing is known about the spatiotemporal Ca 2+ 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 2+ dynamics, at subcellular resolution, in a novel Pf4 Cre :TIGRE2.0 GCaMP6s/wt reporter mouse model. Using imaging in awake mice, we characterized Ca 2+ 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 2+ 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 2+ 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 2+ activity and synchronized intercellular Ca 2+ 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 2+ activity, while a smaller subset displayed Ca 2+ elevations. Mechanistically, CGRP/RAMP1 signaling mediated the increase but not the decrease in CSD-mediated Ca 2+ signaling. Collectively, our results highlight a previously unknown diversity of Ca 2+ dynamics in meningeal macrophages at steady state and in response to an aberrant brain hyperexcitability event linked to neuroinflammation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":576788,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9515,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1485626,"name":"Chao Wei","orcid":"0000-0002-2560-7517","position":1,"is_corresponding":false},{"id":1486048,"name":"Anna Gu","orcid":null,"position":2,"is_corresponding":false},{"id":562255,"name":"Dan Levy","orcid":"0000-0003-0630-6660","position":3,"is_corresponding":false},{"id":1486047,"name":"Silvana Gomes Nascimento","orcid":null,"position":0,"is_corresponding":true}],"reference_count":62,"raw_metadata":null,"created_at":"2026-07-19T02:58:00.620755Z","pmid":"41256472","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}