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. 2011 Jan 11;108(2):846-51.
doi: 10.1073/pnas.1015217108. Epub 2010 Dec 27.

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Alexander S Thrane  1 , Phillip M Rappold, Takumi Fujita, Arnulfo Torres, Lane K Bekar, Takahiro Takano, Weiguo Peng, Fushun Wang, Vinita Rangroo Thrane, Rune Enger, Nadia N Haj-Yasein, Øivind Skare, Torgeir Holen, Arne Klungland, Ole P Ottersen, Maiken Nedergaard, Erlend A Nagelhus
Affiliations

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Alexander S Thrane et al. Proc Natl Acad Sci U S A. .

Abstract

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca(2+) signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca(2+) spikes and that deletion of Aqp4 reduces these signals. The Ca(2+) signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Aqp4 KO strategy and validation. (A) The targeted allele contained a flippase recognition target (FRT)-neomycin-FRT cassette after exon 3 and LoxP sites upstream and downstream of exons 1–3. Floxed mice were bred with Cre-expressing mice to produce mice with the Aqp4 KO allele. Western blot confirmed the absence of AQP4 in Aqp4−/− mice. (B) Immunofluorescence micrographs of mouse cortex probed with primary antibodies against AQP4 (green) and GFAP (red) with DAPI-labeled nuclei (blue) for orientation. The AQP4 immunofluorescence signal is absent in Aqp4−/− mice. Insets display perivascular AQP4 and GFAP labeling at higher magnification. (Scale bar: 25 μm; Inset, 5 μm.) (C) Experimental design for validating the effect of Aqp4 deletion on osmotically induced astrocyte swelling. Acute brain slices were prepared from WT and Aqp4−/− mouse pups. Slices were loaded with Texas red hydrazide and perfused with aCSF with normal (isotonic) or reduced osmolarity (−20% or −30% Osm). Sectional images were acquired for 3D volume analysis. (D) Two-photon imaging of Texas red hydrazide-loaded slices obtained from mice expressing GFP in astrocytes (Glt-1–EGFP BAC transgenic mice) confirmed that the dye was selectively taken up by astrocytes. (Scale bar: 5 μm.) (E) Exposure of acute cortical slices to 20% reduction in osmolarity (−20% Osm) induced more prominent swelling of astrocytic somata in WT (n = 37) than in Aqp4−/− mice (n = 26; P < 0.001 at 5 min, two-tailed Student t test). The initial swelling was followed by shrinkage reflecting regulatory volume decrease. More severe osmotic stress (−30% Osm) induced continuous swelling in both genotypes (n = 30 and 31). Error bars represent SEM. Lower shows representative images of astrocytes exposed to −20% Osm. The red ring marks the astrocyte soma circumference at baseline. (Scale bar: 5 μm.)
Fig. 2.
Fig. 2.
In vivo two-photon imaging of astroglial Ca2+ signals during hypoosmotic stress. (A) Diagram of experimental setup. Astrocytic Ca2+ transients were detected in anesthetized Glt-1–EGFP BAC transgenic mice after loading of Rhod2 AM onto the cortical surface. (B) Images show Rhod2 fluorescence in a GFP-expressing astrocyte. Because increase in Rhod2 signals were not associated with changes in GFP fluorescence, the ratio between the two signals provided a measure of the astrocytic Ca2+ signal. (C) During brain swelling, the ratio between Rhod2 and GFP fluorescent signal intensities (lower trace) was less sensitive to inadvertent small shifts in focal plane than Rhod2 signals (upper trace). Thus, Rhod2/GFP signals were used for reliably defining Ca2+ spikes [i.e., transients exceeding 20% (dashed line) of baseline]. (D) Pooled Ca2+ traces (measured as relative changes in Rhod2/GFP) for all astrocytes (n = 24) within an image field in a WT mouse subjected to i.p. water injection (indicated by arrow; 200 mL/kg) to induce osmotic brain swelling. Note increase in spike frequency and amplitude as brain edema develops. (E) In WT mice, relative spike amplitude and proportion of spikes lasting ≥30 s were higher in the last 15 min than in the first 15 min after water injection. (F) Traces of relative Ca2+ changes in WT astrocytes 30 min after i.p. water injection. The position of the respective astrocytes (confluent Rhod2 and GFP signals in yellow) is indicated in Left. FITC-dextran (green) was injected i.v. at the beginning of the experiment to outline the vasculature, and it confirmed vascular perfusion. Time-lapse sequence of the Ca2+ responses is shown in Right. Note the intense and long-lasting Ca2+ surge in the astrocytic soma and endfoot surrounding a vein. (Scale bar: 25 μm.) (G) In WT mice, the frequency of astrocytic Ca2+ spikes was higher already in the first 15 min after water injection compared with the control state, and it was even higher in the last 15 min of observation. In Aqp4−/− mice, water injection did not increase spike frequency. The percentage of astrocytes with more than or equal to one Ca2+ spike(s) per 15-min observation (active astrocytes) increased profoundly in WT mice after water injection. In Aqp4−/− mice, the number of active astrocytes increased only at the late phase of osmotic brain swelling. Error bars represent SEM. Mixed model analyses were performed using a binomial (Bernoulli) model with logit link for binary observations (passive or active cells), a Poisson model for count data (spike frequency), and a linear model for spike amplitudes (24).
Fig. 3.
Fig. 3.
Osmotically induced astrocytic Ca2+ responses and ATP release in vitro. (A) Two-photon images of Rhod2 AM-loaded acute cortical slices obtained from WT (Upper) and Aqp4−/− (Lower) mice. Traces shown are from astrocytes marked in Left. Exposure of slices from WT mice to aCSF with 20% reduction in osmolarity (−20% Osm) induced brisk astrocytic Ca2+ spikes. In contrast, this osmotic stress failed to elicit Ca2+ spikes in most AQP4-deficient astrocytes. (B) Quantitative analysis of astrocytic Ca2+ responses to osmotic stress. Deletion of Aqp4 or blocking P2 purinergic receptors with PPADS/suramin significantly reduced the number of astrocytes that responded with Ca2+ spikes during exposure to −20% Osm. When more severe hypoosmotic stress (−30% Osm) was applied, a larger fraction of the Aqp4−/− astrocytes responded. The amplitude of the Ca2+ spikes differed between the genotypes for both types of stress. Image shows intense Ca2+ signals in Rhod2-loaded astrocytes after microinjection of ATP and FITC-dextran (green; to verify injection) into the slice. Representative traces from WT and Aqp4−/− mice are shown. (C) Cultured astrocytes exposed to hypoosmotic media (−20% Osm, 15 min) released significantly more ATP than those kept in isotonic media. Astrocytes from Aqp4−/− mice did not show osmotically induced ATP release. P values were obtained by two-tailed Student t test. Error bars represent SEM. (Scale bar: 25 μm.) (D) Diagram showing proposed involvement of AQP4 in astrocyte signaling cascades during hypoosmotic stress. AQP4-mediated water influx triggers Ca2+ transients, partly by promoting release of ATP and activation of P2 purinergic receptors.
See this image and copyright information in PMC

Comment in

  • Glia: aquaporin: not so swell?
    Welberg L. Welberg L. Nat Rev Neurosci. 2011 Feb;12(2):66. doi: 10.1038/nrn2984. Nat Rev Neurosci. 2011. PMID: 21309098 No abstract available.

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