Fig. 6. Attenuation of aTRN neuronal excitability is responsible for changes in wakefulness stability following microglial depletion.

a aTRN neuronal activity and sleep architecture were recorded at night, before and after microglial depletion. b Averaged firing rates (FR) of aTRN neurons before and after DT or vehicle administration (n = 6 DT-injected mice, n = 6 vehicle-injected mice). Each line represents change in mean firing rate of aTRN neuron from one mouse. W wakeful, N NREM sleep. DT 0 vs DT 4, p = 0.007 for wake state. c, e Differences in neuronal firing rates between wakefulness and NREM sleep during transitions from wakefulness to NREM sleep (c DT 0, n = 63 neurons from 6 mice; DT 4, n = 81 neurons from 6 mice; p = 0.028 for DT 0 vs DT 4) and from NREM sleep to wakefulness (e DT 0, n = 69 neurons from 6 mice; DT 4, n = 81 neuron from 6 mice s; p = 0.017 for DT 0 vs DT 4) before/after microglial depletion. Spike events occurring 5–20 s before/after transition onsets were included. d, f aTRN activity during transitions from wakefulness to NREM sleep (d) and from NREM sleep to wakefulness (f) before/after microglial depletion. Neuronal activities within ±20 s from transition onsets were normalized to their minimum firing rates. Right panels, averaged aTRN neuronal activities during transitions. g Experimental procedure for chemogenetic activation of aTRN in microglia-depleted mice. Top, validation of hM3Dq expression. Scale bar = 1 mm. h, i The number of state transition (h left: WN wakefulness to NREM sleep; right: NW NREM sleep to wakefulness; GFP, DT 0 vs DT 4: p = 0.015 for WN, p = 0.020 for NW) and % total duration of wake and NREM sleep (i left, total duration of wake; right, total duration of NREM sleep; GFP, DT 0 vs DT 4: p = 0.005 for wake, p = 0.007 for NREM sleep) were compared before/after microglial depletion in GFP-expressing (n = 7) or hM3Dq-expressing (n = 7) mice. Each line represents change in transition number from one mouse. j Left panel: in control mice expressing GFP, microglial depletion significantly shortened the bout durations of stable wakefulness (n = 7 mice). Right panel: chemogenetic activation of the aTRN abolished the effect of microglial depletion on wakefulness stability (n = 7 mice). k A proposed model for ceramide-mediated microglia-neuron interactions in wakefulness/NREM sleep regulation. Microglia regulate the diurnal variation in ceramide levels, which determines the neuronal activity of the aTRN and, in turn, controls transition states between wakefulness and NREM sleep. Green cells: aTRN microglia; blue cells: aTRN neurons; red molecules: ceramide; black raster plot, action potentials produced by aTRN neurons. *p < 0.05; two-sided unpaired t-tests for c and e; two-sided paired t-tests for b, h, and i. two-sided Kolmogorov–Smirnov test for j. Data are reported as mean ± SEM. See also Supplementary Fig. 9.