Microglia, as immune cells in the central nervous system, possess the ability to adapt morphologically and functionally to their environment. Glymphatic system, the principal waste clearance system in the brain, exhibits circadian rhythms. However, the impact of microglia on the glymphatic system function remains unknown. In this study, we explored the intricate relationship between microglia and the glymphatic system. Examining diurnal patterns, we identified synchronized behaviors in glymphatic activity and microglial morphology, peaking during sleep and exhibiting distinct changes in branching complexity. Depleting microglia using PLX5622 or in P2Y12 knockout mice enhanced glymphatic function. Chemogenetic manipulation of microglia demonstrated that activating HM3D improved glymphatic function, while inhibiting HM4D unexpectedly increased microglial complexity. These findings highlight the dynamic influence of microglia on the glymphatic system.

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.

The glymphatic system is a brain-wide fluid and solute transport pathway that contributes to cerebrospinal fluid-interstitial fluid exchange and the removal of metabolites, inflammatory mediators, and aggregation-prone proteins. Glymphatic activity is shaped by sleep state, vascular pulsatility, respiration, vasomotion, astrocytic aquaporin-4 (AQP4) polarization, meningeal lymphatic drainage, and circadian timing. However, key aspects of brain fluid transport remain debated, including the relative roles of perivascular spaces, periarterial basement membrane pathways, intramural periarterial drainage, diffusion, convection, and anesthesia-dependent physiology. These uncertainties are central to neuroimmune disease, where inflammation, sleep disruption, vascular dysfunction, astrocytic disorganization, and microglial state changes frequently coexist. Here, we review evidence that microglia may coordinate circadian regulation of glymphatic brain clearance. Microglia are circadian, purinergic, and morphologically dynamic immune cells positioned near synapses, vessels, astrocytes, and perivascular spaces. Microglial activity appears to be temporally coupled to physiological states and clearance pathways relevant to glymphatic function: microglial surveillance varies across natural sleep-wake states, microglial phagocytosis can compensate for impaired AQP4-dependent clearance, and meningeal lymphatic drainage shapes microglial inflammatory responses. Recent work further shows that microglial morphology, signaling through the P2Y12 receptor (encoded by P2RY12), and glymphatic tracer influx are linked across Zeitgeber time, and that acute P2Y12 blockade can phase-shift glymphatic rhythms and alter sleep architecture. We propose that microglia may act as neuroimmune chronomodulators that influence the timing and spatial organization of clearance rather than as simple enhancers or inhibitors of flow; however, microglial changes may also represent adaptive responses to altered sleep, vascular physiology, astrocytic state, or glymphatic transport. In neuroimmune disease, glymphatic dysfunction may reflect temporal misalignment among microglial state, sleep architecture, vascular dynamics, astrocytic AQP4 polarity, and fluid transport routes. This framework supports temporally resolved experimental designs and translational studies integrating glymphatic imaging, sleep physiology, circadian biomarkers, inflammatory markers, vascular measures, and cognitive outcomes.

Xu Zhang, Lihan Su, Shijie Zhang, Jing Shen and Ensheng Yao. Exp Neurobiol -0001;35:351-72. https://doi.org/10.5607/en26043