He, C.; Grubb, S.; Tao, L.; Kılıc, K.; Zhang, X.; Devor, A.; Sheikh, N. Z.; Cai, C.

The leptomeninges, composed of the arachnoid mater, and pia mater, contains distinct subgroups of fibroblasts that differ in location and transcriptomic profiles. These fibroblasts contribute to the blood cerebrospinal fluid barrier under physiological conditions, participate in fibrosis, and support blood brain barrier integrity during injury and disease. However, their Ca2+ signaling profiles and underlying mechanisms in health and disease remain poorly understood. In this study, we divided leptomeningeal fibroblasts into three subgroups based on their locations: arachnoid fibroblasts, pia mater fibroblasts and perivascular fibroblasts. We employed two-photon microscopy in awake transgenic mice expressing Ca2+; indicators in leptomeningeal fibroblasts to investigate spontaneous and behaviorally evoked Ca2+; transients across different fibroblast subgroups. We found that each subgroup exhibits a distinct Ca2+; activity profile, with pia mater fibroblasts showing the highest-amplitude Ca2+; transients. Moreover, these fibroblasts displayed unique responses to both whisker air-puff stimulation and locomotion. We further demonstrated, using a chronically implanted cannula beneath the cranial window, that locomotion-associated vasodilation is followed by TRPV4 channel-mediated fibroblast Ca2+; elevations. Finally, systemic inflammation induced by lipopolysaccharide (LPS) reduced spontaneous Ca2+; transients in pia mater fibroblasts, likely due to macrophage infiltration following the inflammatory response. For the first time, this study characterizes spontaneous and behaviorally evoked Ca2+; dynamics in distinct leptomeningeal fibroblast subgroups in awake animals, providing novel insights into the functional roles of leptomeningeal fibroblasts in the healthy and diseased brain.