Extended Data Fig. 7: Distance of adrenergic axon boutons to microglia, GRAB_ADO expression, and effect of local Gi-DREADD activation on sleep.

a, Example images of Iba1-labeled microglia (magenta) and Dbh-eYFP-labeled axons (cyan) in prefrontal cortex. Left and top right, 3D rendering images of a 50-μm-thick slice; scale bar, 20 μm; middle right, high-magnification view of the boxed region; scale bar, 5 μm; bottom right, further enlarged view of the region in dashed box and automatically detected axon buttons (yellow); scale bar, 2 μm. b, Percentage of buttons within 2 μm from the nearest microglia. Bars, mean ± s.e.m. Each circle represents data from one mouse (n = 3 mice; black circle, TH-labeled axon boutons detected by immunohistochemistry; cyan circle, Dbh-eYFP-labeled boutons). Dist., distance. c, Image showing the expression of the GRAB_ADO in the prefrontal cortex. Scale bar, 100 μm. d, e, Coronal diagram (d) and image (e) showing local infusion site in the prefrontal cortex of Tmem119-CreERT2; R26-LSL-Gi-DREADD mice. Scale bar, 200 μm. f-h, Percentage of time (f), mean episode duration (g), and episode number per hour (h) for each brain state within 2.5 h after CNO or ACSF infusion. Each circle indicates data from one mouse (mean ± s.e.m.; n = 5 mice). ^*P < 0.05, ^**P < 0.01 (f, paired two-tailed t-test, ^*P = 0.024; g, paired two-tailed t-test, ^*P = 0.022; h, paired two-tailed t-test; NREM, ^*P = 0.016; Wake, ^*P = 0.020). i-m, Similar to (d-h), but for basal forebrain infusion (n = 5 mice; k, paired two-tailed t-test; NREM, ^**P = 0.0067; Wake, ^**P = 0.0028; REM, ^**P = 0.0025; l, paired two-tailed t-test, ^*P = 0.030; m, paired two-tailed t-test, ^*P = 0.020). n-r, Similar to (d-h), but for dorsal striatum infusion (n = 4 mice).

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