Figure 5: Purinergic signalling mediates astrocytic hyperactivity.

(a) The connexin channel blocker carbenoxolone (Cbx, 100 μM), the P2 receptor blocker PPADS (500 μM) and the cell-permeable IP3 receptor blocker 2-APB (500 μM) all significantly reduced the frequency of hyperactive astrocytes in APPPS1 mice (Cbx: n=90 astrocytes from n=4 mice; PPADS: n=228 astrocytes from n=3 mice; 2-APB: n=134 astrocytes from n=3 mice; CPA: n=148 astrocytes from n=4 mice; P<0.05, χ²-test for all groups). In contrast, the P2X receptor blocker TNP-ATP had no effect (n=219 astrocytes from n=3 mice; P>0.05, χ²-test; 100 μM). Baseline activity was first recorded during topical application of artificial cerebrospinal fluid (Ctl) and again after drug application. (b) Cbx, PPADS, 2-APB and CPA, but not TNP-ATP, also reduced the frequency of astrocyte calcium transients in all active astrocytes (P<0.05, Kruskal–Wallis test followed by Dunn’s multiple comparisons test). (c,d) Inhibition of IP3 receptors (2-APB) significantly reduced the rate and velocity of calcium waves, while inhibition of P2 receptors (PPADS) or depletion of intracellular calcium stores (CPA) completely inhibited calcium waves (P<0.05, Kruskal–Wallis test followed by Dunn’s multiple comparisons test for all). There was a trend for reduction of calcium wave parameters after inhibition of connexin channels with Cbx. (e) Cbx, 2-APB and CPA significantly reduced the amplitude of astrocytic calcium transients. 2-APB and CPA also significantly increased their duration (P<0.05, Kruskal–Wallis test followed by Dunn’s multiple comparisons test for both). (f) Cbx and PPADS had no effect on calcium transients in wild-type littermates, while 2-APB and CPA significantly reduced astrocytic calcium transient frequency in these mice (Kruskal–Wallis test followed by Dunn’s multiple comparisons test; carbenoxolone, n=178 astrocytes from n=3 mice; PPADS, n=151 astrocytes from n=3 mice; 2-APB, n=127 astrocytes from n=3 mice; CPA, n=105 astrocytes from n=3 mice).