Figure 2: Astrocytic hyperactivity correlates with reactive astrogliosis around plaques and involves astrocytic endfeet.

(a) Astrocytosis (GFAP staining, green) predominantly occurs <50 μm around plaques (dashed circles). Plaques were double-stained with methoxy-XO4 (blue) and 6E10 antibody (red). Representative image from fixed brain sections from n=4 analysed mice. Nuclei are stained with Hoechst 33258 (blue). Scale bar, 250 μm. (b) There was a negative correlation between astrocyte activity and distance to plaque (Pearson’s r: −0.25). Overall, significantly more astrocytes were hyperactive and less astrocytes were inactive at a plaque distance <50 μm compared with >50 μm (pie charts; hyperactivity, 38.2% versus 14.0%; inactivity, 26.5% versus 45.8%; n=501 astrocytes from n=22 mice; P<0.05, χ²-test). (c,d) The relative calcium-evoked fluorescence increase of OGB-1 was similar in APPPS1 mice compared with age-matched wild-type littermates. Similarly, there were no significant differences in the temporal kinetics (total duration, time to peak and peak to baseline) of the calcium signals in both groups (n=175 astrocytes from n=22 APPPS1 mice versus n=137 astrocytes from n=18 wild-type littermates; P>0.05, Mann–Whitney test). (e) The fraction of spontaneously active endfeet was higher in APPPS1 mice than in wild-type littermates (35.0±2.7% versus 13.7±2.2%; P<0.05, Mann–Whitney test; the fraction of labelled endfeet was equal in both groups). (f) In astrocytes with active endfeet, we observed calcium elevations that propagated from astrocytic somata into endfeet, as well as calcium elevations that appeared exclusively in endfeet. In this representative time-lapse series, OGB-1 fluorescence (pseudo-coloured) in SR101-positive astrocytes was monitored. In one astrocyte, spontaneous calcium activity occurs first in the soma (1) and subsequently propagates into an endfoot (2). In another astrocyte, calcium elevations appear exclusively in perivascular endfeet (3). Dashed vertical lines indicate onset of signal increase in regions of interest 1–3. Scale bar, 20 μm.