Astrocyte aquaporin mediates a tonic water efflux. (A) In vivo chemical labelling of astrocytes. Sulforhodamine B (SRB, 10 mg/ml) was intraperitoneally injected in awake mice (10 µl/g). Monochrome images show representative astrocyte labeling in living acute brain slices from cortex under low (×20; scale bar, 50 µm) and high magnification (×63; scale bar, 20 µm) by epifluorescence. Below, SRB labeling was confirmed to be astrocyte-specific in acute brain slices of the astrocyte reporter line GFAP-EGFP, where light sheet imaging was used to gain optical sectioning (Materials and methods; Fig. S1). Scale bar, 50 µm. (B) Optical imaging of astrocyte water transport in acute brain slices. Transmembrane water transport was triggered with hypo- and hypertonic solution, inducing water inflow and outflow that were respectively reflected by SRB fluorescence decrease and increase (expressed as dF/F0). The hypo- or hypertonic solution was applied to slices over different lengths of duration displayed in colors corresponding to the time courses of SRB fluorescence (n = 52 astrocytes, 4 mice). (C) Acutely blocking astrocyte AQP4 with TGN-020 caused intracellular water accumulation and swelling. Left, while no effect was seen under CTR condition (vehicle only, n = 23 astrocytes), TGN-020 (20 µM) significantly decreased astrocyte SRB fluorescence (n = 30, 6 mice). Imaging was performed in acute brain slices of layer II/III S1 cortex. Middle, the downward slope was compared between the periods before and after the application of TGN-020. Right, illustration shows astrocyte aquaporin sustaining a tonic water efflux. Its blockade causes water accumulation and cell swelling. (D) In vivo validation of the effect of TGN-020 application on astrocyte water homeostasis. Left, fiber photometry was used for real-time recording of SRB fluorescence of astrocyte population in S1 cortex in freely moving mice, with saline (CTR) or TGN-020 being intraperitoneally injected when SRB was trapped in astrocytes. Fiber photometry recording shows that in vivo SRB injection resulted in rapid entry into mouse cortex and, in about one hour, led to astrocyte labeling (inset scale bar, 50 µm). Middle, example response to saline and TGN-020. Relative to CTR, TGN caused a decrease in astrocyte SRB fluorescence and its oscillation range (n = 8 recordings per condition, 5 mice).

Acutely blocking astrocyte aquaporin induces swelling-associated Ca2+ oscillation. (A) In vivo expression in astrocytes of the genetically encoded fluorescent Ca2+ indicator GCaMP6 for imaging astrocyte Ca2+ in acute brain slices. Light sheet microscopy was used to capture transient Ca2+ signals of local regions. As a positive control, astrocyte swelling was induced by hypotonic solution (100 mOsM) that caused Ca2+ changes from their homeostatic level. Left, representative time courses of swelling-induced Ca2+ changes in detected response regions; middle to left, the histogram distribution and bar representation showing the signal strengths that were derived from the temporal integral of individual Ca2+ time courses normalized per minute, before and after cell swelling (n = 15 astrocytes of three mice). (B) Astrocyte Ca2+ oscillation caused by acute aquaporin blocking with TGN-020 (n = 31 astrocytes of 5 mice), due to the inhibition of the tonic water efflux that led to astrocyte swelling as illustrated. Time scale and Ca2+ rise scale (dF/F0) are the same as in A. Scale bar, 50 µm. (C) Intercellular effect on SST interneurons of blocking astrocyte aquaporin water efflux. TGN-020 (20 µM) or the equal molar vehicle (CTR) was bath applied to acute cortical slices of SST-GCaMP6 mice (n = 7-12 measurements from 4 mice). Scale bar, 50 µm.

Tonic water efflux via aquaporin modulates phasic transmembrane water transport and astrocyte volume response. (A) Left, in basal condition astrocyte aquaporin mediates a tonic water efflux. Right, protocol to induce water outflow from astrocytes, therefore their shrinking, by hypertonic extracellular solution (400 mOsM) in either control condition (CTR) or in the presence of AQP4 inhibitor TGN-020 (20 µM). (B) Time courses of astrocyte SRB fluorescence increase upon the phasically induced water outflow, reflecting the occurrence of shrinking. The histograms and bar charts compare the start time, namely the delay between hypertonic solution application and rise in SRB fluorescence, the time to reach the peak of shrinking, and the absolute amplitude of water outflow-induced SRB increase (n = 58 astrocytes for CTR, and 47 astrocytes for TGN-020, four mice). (C) Right, protocol to trigger water inflow into astrocytes by hypotonic extracellular solution (100 mOsM) in either CTR solution or in the presence of TGN-020 (20 µM). (D) Time courses of astrocyte SRB fluorescence decrease caused by water inflow, which also reflects concomitant cell swelling. In contrast to the observation with hypertonicity-induced water outflow and astrocyte shrinking, a reduction was observed for both the start time and the time-to-peak with TGN-020 (n = 12 astrocytes for CTR, and 12 astrocytes for TGN-020, four mice). TGN-020 led to a decrease in the absolute amplitude of astrocyte swelling.

AQP4-mediated tonic water outflow regulates global swelling in cortical parenchyma. CSD-associated general swelling was induced by photostimulating ChR2-expressing pyramidal cells in acute cortical slices, and recorded by imaging intrinsic optical signal (IOS) with infrared illumination. (A) Representative recording. The pia surface is on the upper side; green, blue and red squares correspond to regions of interest in Layers I, II-III and IV, respectively. Transmittance signals are represented in pseudocolor images at different time points post photostimulation. Scale bar, 100 µm. (B-C) Kymograph and time courses showing the IOS changes following photoactivaiton, derived from the radial line of interest indicated by a black arrow in A. The light blue line indicates the 10-s photostimulation that increases the infrared transmittance signal (dT/T0) across cortical layers, as also illustrated by the time courses; a, b, c and d correspond to the time points depicted in A. After the onset delay (a), the first (b) and second (c) peak of IOS are characteristic of the CSD and a prolonged general swelling, respectively. (D-E) TGN-020 (20 µM) inhibition of AQP4 reduced the initial onset and the maximal amplitude of both the CSD and general swelling (n = 6 - 18 measurements from 13 mice per condition) in layer II/III cortex. Inhibiting spiking activity with TTX (1 µM) only affected the amplitude of the initial CSD response. (F) Astrocytes swelling, reflected by SRB fluorescence decrease, monitored in control condition (n = 75 astrocytes) and in the presence of TGN-020 (n = 17 astrocytes) in layer II/III cortex.

Perturbing the tonic water efflux via astrocyte aquaporin alters brain water homeostasis. In vivo DW-MRI (7T) was employed to map water diffusion in the entire brain scale following the acute inhibition of astrocytic AQP4 (TGN-020, intraperitoneal injection, 200 mg/kg), with paralleled control experiments performed with saline injection. (A) Experimental protocol for DW-MRI. Saline or TGN020 was injected at 10 min after the start of acquisition. DWI was acquired every 5 min. (B) Representative image obtained at three different b values to derive the water diffusion rates. (C) Left, brain water diffusion rate was mapped by the calculation of apparent diffusion coefficient (ADC). Right, representative images illustrating the relative changes of ADC at 60 min after injection of saline or TGN-020. (D) Time courses depicting the temporal changes in ADC in the cortex, striatum, and hippocampus, revealing the regional heterogeneity. Arrowhead indicates the injection of saline or TGN-020 (n = 10 mice for saline injection, 9 mice for TGN-020).