Extended Data Fig. 3: Effect of P2Y12 agonist on sleep, control experiments for Ca²⁺ imaging in mice without Gi-DREADD, comparison of microglia Ca²⁺ activity before and after CNO-induced Gi activation within each brain state, and Ca²⁺ activity during baseline period.

a-c, Percentage of time (a), mean episode duration (b), and episode number per hour (c) for each brain state within 2 h after i.c.v. infusion of P2Y12 agonist (2MeSADP) or ACSF. Each circle indicates data from one mouse; error bar: ± s.e.m. (n = 9 mice). ^*P < 0.05, ^**P < 0.01 (a, two-tailed Wilcoxon signed rank test, ^*P = 0.039; b, paired two-tailed t-test, ^**P = 0.0030; c, paired two-tailed t-test; NREM, ^*P = 0.012; Wake, ^*P = 0.015). d, Schematic for two-photon (2 P) Ca²⁺ imaging in head-fixed mice without Gi-DREADD expression. e, Z-scored Ca²⁺ activity averaged across all microglia (CNO: n = 25; saline: n = 28; from 4 mice). Dashed line, time of injection. f, Population summary of the change in mean Ca²⁺ level. g, Diagram illustrating the comparison of Ca²⁺ activity before and after CNO or saline injection within each brain state. First, Ca²⁺ activity in NREM or wakeful episodes are averaged separately for before and after injection; second, the difference between the averaged Ca²⁺ activity before and after injection are calculated for each brain state. h, CNO-induced change in microglia Ca²⁺ for each brain state (difference between before and after injection). CNO, n = 47; saline: n = 40; from 5 mice. Bars, mean ± s.e.m. (two-tailed Mann-Whitney U test; NREM, ^****P < 0.0001; Wake, ^****P < 0.0001). i, j, Amplitude (i) and frequency (j) of Ca²⁺ events during baseline period (before saline or CNO injection). CNO: n = 47; saline: n = 40; from 5 mice. Data are presented as mean ± s.e.m.

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