Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions

Abstract

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood-brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca²⁺ indicator GCaMP6f to study Ca²⁺ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two-photon microscopy. Ca²⁺ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca²⁺ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau-type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5-triphosphate receptor (IP3R2) severely suppressed the Ca²⁺ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca²⁺ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin-4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca²⁺ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca²⁺ signals only in subpial and perivascular endfeet.

Keywords: AQP4; TRPV4; aquaporin-4; astrocytes; glia; glymphatic; interstitial fluid.