Microglia synchronizes with the circadian rhythm of the glymphatic system and modulates glymphatic system function

Abstract

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.

Keywords: P2Y12; chemogenetics; circadian rhythms; glymphatic system; microglia.

FIGURE 1

The glymphatic system exhibits circadian rhythm. (A) Schematic diagram of the experimental procedure. (B) Representative images of whole‐brain CSF tracers (top view and bottom view). (C) Representative images of CSF tracers in the coronal slices. (D) Fit the fluorescence density of the whole brain top, whole brain bottom, whole slice, hippocampus, and cortex at ZT0, ZT6, ZT12, and ZT18 into a cosine curve. p < .05 represents the presence of a circadian rhythm in the fluorescence density of this fraction. *p < .05, **p < .01, ***p < .001. ZT0, n = 6; ZT6, n = 6; ZT12, n = 6; ZT18, n = 6.

FIGURE 2

The microglial morphology exhibits a diurnal rhythm that corresponds to their coverage area and the functions of the glymphatic system. (A) Representative images of cortex and hippocampus Iba‐1 immunostaining at ZT0, ZT6, ZT12, and ZT18. The arrows point representative microglia. (B) Statistical results of Sholl analysis of microglia in the cortex at ZT0, ZT6, ZT12, and ZT18. (C) Statistical results of Sholl analysis of microglia in the hippocampus at ZT0, ZT6, ZT12, and ZT18. (D) Fit the number of astrocytes in the cortex and hippocampus at ZT0, ZT6, ZT12, and ZT18 into a cosine curve. (E) Fit the number of microglia in the cortex and hippocampus at ZT0, ZT6, ZT12, and ZT18 into a cosine curve. (F) Cosine fitting of the whole‐brain fluorescence intensity at four time points (red), and cosine fitting of the AUC of microglial Sholl analysis interactions in cortex and hippocampus at the four time points (blue and green). *p < .05, **p < .01, ***p < .001. ZT0, n = 6; ZT6, n = 6; ZT12, n = 6; ZT18, n = 6.

FIGURE 3

Deleting microglia enhances the function of the glymphatic system. (A) Representative images of CSF tracer in the whole brain in the control group, PLX5622 group, and P2Y12 KO group. (B and C) Quantification of whole‐brain tracer fluorescence density in the control group, PLX5622 group, and P2Y12 KO group. (D) Representative images of cortex and hippocampus Iba‐1 immunostaining at ZT0, ZT6, ZT12, and ZT18 in the control group, PLX5622 group, and P2Y12 KO group. (E and F) Quantification of the number of microglia in the hippocampus and cortex. *p < .05, **p < .01, ***p < .001. Control, n = 6; PLX5622, n = 6; P2Y12, n = 6. A.U., arbitrary units.

FIGURE 4

The impact of chemogenetic regulation of microglia on the function of the glymphatic system. (A) Schematic diagram of the cortex virus injection experimental procedure. (B) The image of whole brain fluorescence. (C and D) Quantification of the fluorescence density at the top of the whole brain in the control group, HM3D group, and HM4D group. (E and F) Quantification of the fluorescence density at the bottom of the whole brain in the control group, HM3D group, and HM4D group. (G) Schematic diagram of the hippocampus virus injection experimental procedure. (H) The image of whole brain fluorescence. (I and J) Quantification of the fluorescence density at the top of the whole brain in the control group, HM3D group, and HM4D group. (K and L) Quantification of the fluorescence density at the bottom of the whole brain in the control group, HM3D group, and HM4D group. (M) Representative images of the cortex virus injection and tracer injection. (N–P) Quantification of the fluorescence density in slices, hippocampus, and cortex in the control group, HM3D group, and HM4D group for cortical virus injection. (Q) Representative images of the hippocampal virus injection and tracer injection. (R–T) Quantification of the fluorescence density in slices, hippocampus, and cortex in the control group, HM3D group, and HM4D group for hippocampal virus injection. *p < .05, **p < .01. Cortex virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5. Hippocampal virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5. A.U., arbitrary units.

FIGURE 5

The impact of chemogenetic regulation on the morphology of microglia. (A) Representative images of virus expression and Iba‐1 immunostaining in the control group, HM3D group, and HM4D group. (B and D) In mice receiving cortex virus injection, statistical results of Sholl analysis for microglia in the cortex and hippocampus, respectively. (C and E) In mice receiving cortex virus injection, statistical analysis of the AUC of the number of interactions in the cortex and hippocampus, respectively. (F and H) In mice receiving hippocampal virus injection, statistical results of Sholl analysis for microglia in the cortex and hippocampus, respectively. (G and I) In mice receiving hippocampal virus injection, statistical analysis of the AUC of the number of interactions in the cortex and hippocampus, respectively. *p < .05, **p < .01, ***p < .001, # PLX5622 group has significant differences with other group. Cortex virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5. Hippocampus virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5. A.U., arbitrary units.

FIGURE 6

The impact of chemogenetic regulation of microglia on the polarized distribution of AQP4. (A) Immunostaining of GFAP (green), AQP4 (red), and DAPI (blue) in the control group, HM3D group, and HM4D group. (B and C) In mice receiving cortex virus injection, quantification of polarized distribution of AQP4 in cortex and hippocampus in the control group, HM3D group, and HM4D group. (D and E) In mice receiving hippocampal injection, quantification cortex, and hippocampus part of the polarized distribution of AQP4 in astrocyte foot processes in the control group, HM3D group, and HM4D group. *p < .05, **p < .01, Cortex virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5. Hippocampus virus injection: Control, n = 5; HM3D, n = 5; HM4D, n = 5.