Microglial P2Y12 signaling phase-shifts glymphatic circadian rhythms and alters sleep architecture in mice

Microglia regulate neural circuits and vascular-glial interfaces, yet whether microglial purinergic signaling coordinates glymphatic function and brain state remains unclear. We tested the role of the microglial Gi-coupled receptor P2Y12 using acute pharmacological inhibition with MRS2395 in adult mice. Glymphatic influx was quantified across circadian time by cisterna-magna tracer infusion and whole-brain fluorescence imaging, and related to microglial density and morphology assessed by Iba1 immunofluorescence and Sholl analysis in cortex, hippocampus, and amygdala. Sleep-wake architecture and cortical oscillations were evaluated by continuous 24-h EEG/EMG. In controls, microglial density and process complexity showed robust circadian rhythms that aligned with glymphatic timing. P2Y12 blockade reduced microglial density, flattened or rephased structural rhythms, and shifted glymphatic influx across the day-night cycle while preserving overall rhythmicity. Regionally, amygdala tracer signal correlated with Iba1⁺ cell density in controls and this coupling was lost after P2Y12 inhibition. Across regions, glymphatic readouts associated more strongly with microglial morphology than with cell number. EEG/EMG revealed no change in total sleep time with MRS2395, yet sleep architecture was reorganized: stage composition shifted toward NREM at the expense of REM, transitions between REM and NREM increased, bout durations shortened, and spectral power shifted toward slower activity, with higher gamma and lower alpha/theta/beta at multiple time points. These results identify microglial P2Y12 signaling as a temporal organizer that links microglial structural dynamics to perivascular fluid exchange and to sleep-wake organization. Disruption of this pathway alters the phase and stability of glymphatic and sleep rhythms, suggesting a neuroimmune target for conditions with circadian misalignment, sleep fragmentation, or impaired metabolic clearance.