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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.
Highlights
Being encased in the rigid skull the brain is critically dependent on mechanisms for volume control, both at the cellular and organ level
AQP4 increases the kinetics of osmotic swelling and boosts intracellular Ca2+ signals that may be necessary for efficient volume regulation (Benfenati et al, 2011; Jo et al, 2015; Mola et al, 2016; Thrane et al, 2011), possibly through interaction with the transient receptor potential vanilloid isoform 4 (TRPV4), a swellingsensitive cation channel (Benfenati et al, 2011; Jo et al, 2015; Toft-Bertelsen, Krizaj, & MacAulay, 2017)
We characterized astrocytic Ca2+ signaling in acute cortical slices exposed to artificial CSF (aCSF) with 20% reduction in osmolarity
Summary
Being encased in the rigid skull the brain is critically dependent on mechanisms for volume control, both at the cellular and organ level. AQP4 increases the kinetics of osmotic swelling and boosts intracellular Ca2+ signals that may be necessary for efficient volume regulation (Benfenati et al, 2011; Jo et al, 2015; Mola et al, 2016; Thrane et al, 2011), possibly through interaction with the transient receptor potential vanilloid isoform 4 (TRPV4), a swellingsensitive cation channel (Benfenati et al, 2011; Jo et al, 2015; Toft-Bertelsen, Krizaj, & MacAulay, 2017). We lacked technology for studying Ca2+ signals in the distal astrocytic processes and endfeet of adult animals. Studies using synthetic Ca2+ dye loading mainly reported on Ca2+ signals in cell bodies and the most proximal processes, leaving ~90% of the astrocytic territory unsampled (Tong, Shigetomi, Looger, & Khakh, 2013). We report that hypoosmotic stress elicits robust astrocytic Ca2+ responses, whose signature and underpinning vary within the astrocytic territory
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