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. 2024 Sep 2;76(12):1209–1222. doi: 10.1002/iub.2903

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

Ting Yang 1, Yan Tang 2, Xinghua Liu 3,4, Song Gong 3,4,✉, Ensheng Yao 1,✉
PMCID: PMC11580365  PMID: 39223969

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: chemogenetics, circadian rhythms, glymphatic system, microglia, P2Y12

1. INTRODUCTION

The glymphatic system is a unique fluid transport system formed by perivascular spaces around arteries, capillaries, and veins, enveloped by the endfeet of astrocytes. 1 The cerebrospinal fluid (CSF) that circulates within the perivascular spaces around blood vessels can exchange substances with the surrounding interstitial fluid, thus playing a role in monitoring and clearing metabolic waste from the brain. 2 , 3 , 4 Impairment of the glymphatic system results in a reduction in the rate of clearing interstitial solutes (including Aβ), and consequently, may initiate neurodegenerative conditions like Alzheimer's disease (AD). 5 So far, research on factors influencing the glymphatic system has found that aquaporin‐4 (AQP4), a water channel protein localized on the endfeet of astrocytes, is a key factor affecting the exchange of substances within the perivascular spaces and interstitial fluid. 3 However, the impact of microglia on the glymphatic system remains unclear.

Microglia are one of the most widely functional cell types in the brain, primarily serving as the brain's immune cells. 6 Microglia also contribute to other physiological processes, such as brain development, synaptic plasticity, learning and memory. 7 , 8 However, there has been limited research on whether microglia are involved in the glymphatic system. In a study by Eszter Császár in 2022, it was discovered that P2Y12‐positive microglia around brain arteries and capillaries have close interactions with vascular endothelial cells and astrocyte endfeet. Approximately 93% of astrocytes indirectly contact vascular endothelial cells through P2Y12‐positive microglia and regulate blood flow. 9 These studies indirectly confirm that microglia and astrocytes collectively form the perivascular spaces. Additionally, research by Ehud Lavi and colleagues revealed that astrocytes and microglia jointly constitute the perivascular glymphatic system channels surrounding blood vessels. 10 Hence, we hypothesize that microglia play a role in regulating the glymphatic system.

The morphological characteristics of microglia are remarkably diverse and regulated by various factors. ATP induces the elongation of microglial processes and the chemotaxis of microglia through the P2Y12 receptor on microglia. Microglia lacking the P2Y12 receptor in mice lose their ability to extend toward nucleotide in vitro and in vivo. 11 Activated microglia release neurotrophic factors, supporting neurons, and enhancing their survival and function. Moreover, microglia dynamically regulate the microenvironment surrounding neurons, ensuring appropriate ion concentrations and pH values. Can altering microglia activity affect the function of the glymphatic system?

In our research, we explored the relationship between the morphological changes in microglia and the circadian rhythm of glymphatic system function in physiological conditions. Furthermore, we utilized chemogenetic method to modulate microglia, and correlated the function of microglia and glymphatic system.

2. MATERIALS AND METHODS

2.1. Animals and groups

2.1.1. Animals

The experiments were performed according to the ARRIVE guidelines and approved by the Experimental Animal Ethics Committee of First Affiliated Hospital, Shihezi University School of Medicine (KJ2022‐214‐01). Male wild‐type C57 mice, aged 6–8 weeks, and P2Y12 knockout mice of the same age were sourced from Huafukang Biotechnology Co., Ltd. (China). The mice were kept in a controlled environment with a temperature maintained between 22 and 24°C and humidity ranging from 45% to 60%. They were provided with ad libitum access to both water and food. We maintained a 12‐h light/dark cycle throughout the experiment. Before study, the mice were given 1 week to acclimate to their surroundings.

2.1.2. Groups

The experiment consists of three parts. In the first part, the diurnal rhythm of microglia was explored. Twenty‐four 6–8‐week‐old male wild‐type C57 mice were divided into four groups: ZT0 (8 am, light), ZT6 (2 pm, light), ZT12 (8 pm, dark), and ZT18 (2 am, dark), with six mice in each group. In the second part, the effect of microglia on glymphatic system was explored. We used seven male wild‐type C57 mice aged 6–8‐week‐old were injected with PLX5622 to delete microglia, seven P2Y12 knockout mice aged 6–8‐week‐old, and seven mice aged 6–8‐week‐old male wild‐type C57 as the control group. In the third part, we utilized chemogenetics to manipulate microglia. Thirty male wild‐type C57 mice aged 6–8‐week‐old were divided into six groups, with five mice in each group. The groups included the HM3D group, HM4D group, and control group for the cortical region (virus injected into the cortex), and the HM3D group, HM4D group, and control group for the hippocampal region (virus injected into the hippocampus).

2.2. Cisterna magna cannulation

Mice were anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital (0.05 mL/10 g) and secured in a stereotaxic frame. The hair on the back of the neck was shaved, and a midline incision was made in the skin. Forceps were used to lift the surface muscles, and a 30‐gauge needle connected to a polyethylene tube (PE10, NO.427401, Intramedic) was slowly and gently inserted into cisterna magna. The tube was filled with artificial CSF, and the distal end was sealed. Upon sensing that the needle had entered the cisterna magna, ionomer cement was used to fix the needle in place. Carprofen (5 mg/kg, subcutaneous) was given on five consecutive days after each surgery for pain relief.

2.3. Intracisternal CSF tracer infusion

The fluorescent CSF tracers (in the first part: dextran, fluorescein, 40 kMW; in the second and the third part: dextran, Texas Red, 70 kMW) were dissolved as 50 mg/mL with artificial CSF to create a storage solution. The working solution required diluting the storage solution with artificial CSF at a ratio of 1:5. Subsequently, the working solution was slowly infused into the cannula at a rate of 1 μL/min using a micro pump, with each infusion delivering 10 μL. At the end of the infusion, the needle was kept in place to prevent CSF reflux. In the first part, four groups of mice at four different time points (ZT0, ZT6, ZT12, and ZT18) were injected with 40 kMW tracer. In the second part, to avoid the influence of time factors, the three groups of mice underwent cisterna magna injection at ZT12 on the same day. In the third part, to avoid the influence of time factors, the six groups of mice underwent cisterna magna at ZT12 on the same day. The tracer was circulated for 30 min before cardiac perfusion.

Mice were anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital (0.05 mL/10 g) and perfused with 0.9% saline through cardiac perfusion. The brain tissue was then carefully extracted and placed in paraformaldehyde for more than 24 h to ensure proper fixation. After fixation, macroscopic whole brain was photographed by laser zoom‐stereo microscope (Nikon, SMZ18, Japan). For brain slicing, the tissue was sequentially transferred to 20% sucrose solution overnight, followed by 30% sucrose solution to ready for cryoprotection. The brain was then sectioned into 30 μm thick coronal sections at −20°C using a cryostat (Leica CM, 1860 UV). The brain sections were imaged by automated section scanner (VS120, Olympus) and a confocal microscope imaging system (Olympus, Japan). The density of whole brain fluorescence was analyzed by ZEN blue software, and the density of fluorescence in the brain sections was quantified by Fiji software. Three slices were selected from each brain, and the fluorescence of the cortical and the hippocampal of each slice was quantified, the mean values of three slices were then taken to represent the fluorescence of the cortex and hippocampus of sample.

2.4. Stereotaxic injection

Mice were anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital (0.05 mL/10 g). Once fully anesthetized with no limb response, the mice were securely positioned on a stereotaxic apparatus. Incise the skin on the mouse's head to expose the surface of the skull. The coordinates for the right cortical injection were −2.18 mm anteroposterior relative to Bregma, +1.5 mm mediolateral, and −0.75 mm dorsoventral. The coordinates for hippocampal injection were −2.18 mm anteroposterior relative to Bregma, +1.5 mm mediolateral, and −2 mm dorsoventral. In each mouse, a total volume of 0.4 μL of the virus solution was injected at a rate of 0.1 μL/min. The virus solution included 0.1 μL rAAV‐hCD68‐HM4D(Gi)‐EGFP (titer: 5.14E+12 vg/mL) or rAAV‐hCD68‐HM3D(Gq)‐EGFP (titer: 5.09E+12 vg/mL) and 0.3 μL saline. After the injection, the needle tip was kept still for 10 min before slowly withdraw. The incision on the scalp was sutured, local antibiotics were applied. Carprofen was given on five consecutive days after each surgery for pain relief.

2.5. Drugs

A 25 mg quantity of DCZ (MedChemExpress, HY‐42110) was dissolved in 2.5% DMSO, divided into aliquots, and stored at −80°C. For intraperitoneal injection, the stock solution was diluted in saline to achieve a final dose of 100 μg/kg. DCZ was administered 50 min before the infusion of the tracer. PLX5622 (MedChemExpress, HY‐114153), prepared as a stock solution with 50 mg/mL in DMSO, was stored at −20°C. PLX5622 (50 mg/kg) was intraperitoneally injected into the animals every day for 7 days. 12

2.6. Immunofluorescence

The sections underwent three washes in phosphate‐buffered saline (PBS), each lasting for 15 min. Subsequently, the sections were incubated in a PBS solution containing 0.3% Triton X‐100 for 30 min to enhance permeability. Afterward, the sections were incubated in a 1% bovine serum albumin solution for 1 h to minimize nonspecific binding. For primary antibody labeling, the sections were incubated with a diluted Iba‐1 (1:200, Celling Signaling, Cat: 17198), GFAP (1:200, Servicebio, Cat: GB12096‐100), and AQP4 (1:200, Servicebio, Cat: GB11311‐1‐100) at 4°C for 24 h. After three washes in PBS, the sections were incubated with goat anti‐rabbit secondary antibody (1:200, ABclonal, AS014) for 4 h. Following secondary antibody incubation, the sections were washed three times with PBS for 15 min each. Finally, the sections were carefully mounted onto slides, covered with coverslips, and sealed using a 50% glycerol solution (PBS:glycerol = 1:1). Images were captured using a microscope (Mshot, China), and the Iba‐1 positive cells or GFAP positive cells were quantified using ImageJ software.

2.7. Sholl analysis

Microglia images were acquired using an automated section scanner. The microglia were analyzed with the Neuroanatomy plugin within the Fiji software. Concentric circles were centered on the soma, starting at a radius of 0.1 μm and in cementing by 0.1 μm for each successive circle. Sholl analysis was manually performed for each cell by counting the number of intersections between microglia branches and each increasing circle to create a Sholl plot. The area under the curve (AUC) of the sholl plot was also created using Prism9 (GraphPad, San Diego, CA).

2.8. AQP4 polarization analysis

AQP4 polarization analysis was calculated from DAPI, GFAP, and AQP4 immunostaining as previously described. 13 In brief, a donut‐shaped area was drawn within five pixels from a capillary, which encompassed the void surrounded by zones enriched in DAPI and GFAP expression. The ratio of AQP4 immunofluorescence within the void to the global AQP4 signal indicated AQP4 polarization: AQP4 polarization = donut‐shaped area AQP4/global AQP4.

2.9. Circadian rhythm analysis

We conducted circadian rhythm analysis utilizing the ‘circacompare’ R package 14 to evaluate the fluorescence intensity, the number of astrocytes, the number of microglia, and the AUC of the daily results. The code is available on GitHub at https://github.com/RWParsons/circacompare/. In summary, the fluorescence intensity data and microglial data at each time point were fit using a cosine curve. A circadian rhythm was indicated by a rhythmic p‐value below .05.

2.10. Statistical analysis

All statistical analyses were performed using Prism9 (GraphPad, San Diego, CA). The Shapiro–Wilk test was first applied to confirm the normality of the data. F‐test was used to compare variances between different groups. When data followed a normal distribution, we adopted the Unpaired t‐test for the comparation of two groups, and one‐way analysis of variance (ANOVA) with Tukey's post‐hoc test for comparisons between multiple groups. The results were expressed as mean ± SD. p‐Values less than .05 were considered as statistically significant differences.

3. RESULTS

3.1. The microglial morphology exhibits a diurnal rhythm that corresponds to their coverage area and the functions of the glymphatic system

The glymphatic system demonstrates a circadian rhythm, reaching its peak activity during the sleep period. 15 To investigate the relationship between the rhythmic patterns of microglia and the glymphatic system, we utilized the CircaCompare method to assess the activity of the glymphatic system at four distinct time points: ZT0 (8 am), ZT6 (2 pm), ZT12 (8 pm), and ZT18 (2 am). To analyze the function of the glymphatic system, we injected a tracer via the magna and circulated it for 30 min After injecting the tracer, we conducted whole‐brain imaging as well as fluorescent imaging of brain sections (Figure 1A). Fluorescence images were captured from both the top and bottom of the intact brain (Figure 1B) and the brain slice (Figure 1C). The results suggested that the glymphatic system exhibited its peak activity during the ZT0–ZT6 period. Subsequently, the glymphatic system showed a gradual decline in activity during the ZT12–ZT18 period, such as the cortex and hippocampus (Figure 1D).

FIGURE 1.

FIGURE 1

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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.

We next analyzed the quantity and morphology of Iba1‐positive microglia at the four time points (Figure 2A). The results indicated that the number of microglia in the hippocampus corresponded to the diurnal rhythm of glymphatic system function, with the highest number of microglia during ZT0–ZT6 when glymphatic system function was most active (Figure 2C,E). However, the number of microglia in the cortex did not exhibit a significant diurnal rhythm. In contrast to the less pronounced rhythmicity in microglial numbers, microglial morphology showed significant periodicity. Sholl analysis of microglial processes revealed that during ZT0–ZT6, microglia exhibited increased branching and more complex morphology, while during ZT12–ZT18, microglial processes showed decreased branching (Figure 2B).

FIGURE 2.

FIGURE 2

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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.

To evaluate the extent of microglial coverage and the rhythmicity in this coverage, we utilized the AUC of branch interaction numbers. Subsequently, we compared its rhythmic pattern with the glymphatic system function using CircaCompare. The outcomes revealed that the coverage areas of microglia in both the hippocampus and cortex follow a diurnal pattern (Figure 2F). These patterns aligned with the rhythm of the glymphatic system, with the maximum coverage of microglial occurring at ZT0–ZT6 when glymphatic system function was at its peak. Consistent with previous studies, 16 , 17 , 18 the number of the main components of the glymphatic system, GFAP‐positive astrocytes, exhibited changes that align with the diurnal rhythm of the glymphatic system, increasing during ZT0–ZT6 and decreasing during ZT12–ZT18 (Figure 2D).

3.2. Deleting microglia enhances the function of the glymphatic system

The activated microglia exhibited a rounded cell body with short branches, whereas the non‐activated microglia typically displayed long branches. In our findings, when microglia were in a non‐activated state, the glymphatic system functioned most robustly. Conversely, when microglia were in an activated state, the glymphatic system operated at its lowest level. To investigate the impact of microglia on glymphatic system function, we employed PLX5622 to deplete microglia. 19 The results revealed that, in comparison to the control group of mice, inhibiting microglia led to an improvement in glymphatic system function (Figure 3A,C). Likewise, glymphatic system function in P2Y12 KO mice was also enhanced when compared to wild‐type mice (unpaired t‐test, p = .0180) (Figure 3B). Compared to the control group, PLX5622 reduced the number of microglia by over 50%, while the P2Y12 group's microglial count fell between that of the control group and the PLX5622‐treated group (Figure 3D–F).

FIGURE 3.

FIGURE 3

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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.

3.3. The impact of chemogenetic regulation of microglia on the function of the glymphatic system

The excitatory G‐protein coupled receptor HM3D(Gq) and the inhibitory G‐protein coupled receptor HM4D(Gi) are DREADDs (designer receptor exclusively activated by designer drugs), that upon binding of the drug deschloroclozapine (DCZ), lead to silencing or activation of transfected neurons, respectively. The morphology of microglia can also be regulated by chemogenetics. 20 , 21 , 22 Expressing HM4D and ‘silencing’ in microglia can lead to an increase in ramification. 23 , 24 In our study, we injected rAAV‐hCD68‐HM3d(Gq)‐EGFP or rAAV‐hCD68‐HM4d(Gi)‐EGFP into the cortex or hippocampus of wild‐type mice and monitored microglia using DCZ (Figure 4A,G).

FIGURE 4.

FIGURE 4

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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.

At ZT12, corresponding to the weakest activity of the glymphatic system and the lowest number of microglial processes, mice received an intraperitoneal injection of DCZ. After 50 min, tracer was injected into the cisterna magna (Figure 4A). The findings indicated that, compared to the control group, the activation of the HM3D receptor on microglia in the cortex or hippocampus following DCZ administration resulted in an improvement in glymphatic system function (Figure 4B,C,E). Specifically, fluorescence intensity analysis of the slices revealed a significant increase in fluorescence intensity in the cortex of the HM3D cortical injection group compared to the control group (Figure 4O). However, no significant differences were observed in the fluorescence intensity across the entire slice (p = .2338) and in the hippocampal region (p = .7484) (Figure 4N,P). Additionally, expressing HM3D in hippocampal microglia and activating it with DCZ did not alter the function of the hippocampal glymphatic system (Figure 4T). These results indicated that activating microglia in the cortex improved both local and global glymphatic system function, whereas activating microglia in the hippocampus enhanced global glymphatic system function (Figure 4I,K), with less significant effects on the local.

The application of DCZ to ‘inhibit’ microglia expressing HM4D in the cortex or hippocampus did not lead to substantial alterations in the glymphatic system's function (Figure 4D,F,J,L,N–P,R–T).

3.4. The impact of chemogenetic regulation on the morphology of microglia

We next conducted Sholl analysis on microglial morphology. The results showed that activating HM3D in microglia using DCZ led to a significant increase in branch number and a more complex morphology compared to the control group (p = .0160) (Figure 5A–I). Unexpectedly, the inhibition of HM4D in microglia using DCZ led to a noteworthy rise in branch number and a more intricate morphology compared to the control group (p_cortex = .0108, p_hippocampus = .0032) (Figure 5A–I). Sholl analysis of the surrounding uninfected microglia in the presence of activated HM3D or inhibited HM4D revealed no significant changes in their morphology, similar to the control group (Figure 5C,E,J,I).

FIGURE 5.

FIGURE 5

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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.

We finally analyzed the total number of microglia. In the brains expressing HM3D or HM4D in cortical microglia, there was no significant difference in the number of Iba‐1 positive microglia in the hippocampus and cortex compared to the control group (Figure 5J,K). In the brains expressing HM3D or HM4D in the hippocampus, both cases led to a significant increase in the number of microglia in both the hippocampus and cortex compared to the control group (Figure 5J,K).

3.5. Chemogenetic activation of microglia increases the polarized distribution of AQP4

Finally, we analyzed the polarized distribution of AQP4 after chemogenetic manipulation of microglia. The results showed that activating the HM3D receptor on microglia in the cortex or hippocampus using DCZ significantly enhanced the polarization of AQP4 (p = .0228), while the application of DCZ to ‘inhibit’ microglia expressing HM4D in cortex or hippocampus did not alter the polarized distribution of AQP4 (Figure 6B–E).

FIGURE 6.

FIGURE 6

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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.

4. DISCUSSION

The function of the glymphatic system is influenced by circadian rhythms, with an enhanced functionality observed during sleep. 17 , 25 The morphology and dynamics of microglia are also regulated by the endogenous circadian clock, with fewer branches and lower dynamics observed during wakefulness. 26 , 27 , 28 Hence, we speculate that there is a connection between the rhythmicity of the glymphatic system and the rhythmicity of microglia. In our study, we observed diurnal variations in the morphology of microglia, with fewer branches during wakefulness and increased branching during sleep, resulting in a larger coverage area. The number of microglia in the hippocampus also exhibited diurnal changes, decreasing during wakefulness and increasing during sleep. However, rhythmic variations in the number of microglia were not observed in the cortex.

The efficiency of metabolite clearance by the glymphatic system during sleep is seven times higher than during wakefulness. 17 It is conceivable that, during the transition from wakefulness to sleep, the concentration of metabolic waste (or harmful substances) in the brain reaches its peak, prompting the shift of microglia from a dormant state to an activated state. Alternatively, the transition of microglia could also be activated by changes in ATP concentration. By examining the rhythmic patterns in the morphology of both the glymphatic system and microglia, we noticed a parallel daily pattern: during sleep, the glymphatic system is active, and microglia are at rest (exhibiting increased branching); during wakefulness, the glymphatic system is inactive, and microglia become activated (resulting in decreased branching).

The rhythmicity of glymphatic system function is primarily regulated by the quantity of astrocytes and AQP4, with the potential involvement of microglia numbers to some extent. In our study, the number of microglia showed rhythmicity in the hippocampus but lacked rhythmicity in the cortex. Using PLX5622 to deplete microglia and P2Y12 knockout mice, the results consistently indicated that a reduction in microglia numbers led to an enhancement of glymphatic system function. The phenomenon may be attributed to the decrease in the number of microglia, resulting in a relative reduction in interstitial fluid volume, an increase in perivascular space volume, and a decrease in CSF flow resistance. 17 Microglia are components of the perivascular space. 10 They have direct contact with 85% of blood vessels and extensive direct contact with endothelial cells and astrocytes. 9 P2Y12‐positive microglia surrounding blood vessels exhibit chemotaxis in response to ADP released by neurovascular unit cells. When microglia are depleted using PLX5622 or when there is a disruption in P2Y12 receptors, microglia cannot undergo chemotaxis to the perivascular space, leading to a decrease in perivascular space resistance. Consequently, with the depletion of microglia or absence of P2Y12 receptors, cerebral blood flow is compromised, resulting in a relative increase in perivascular space, and a decrease in CSF flow resistance.

The morphology of microglia exhibited a consistent circadian rhythm with glymphatic system function. To further elucidate the impact of the activation and inhibition states of microglia on the glymphatic system, we employed chemogenetics to locally activate or inhibit microglia in the cortex or hippocampus. Activation of microglia expressing HM3D in the cortex or hippocampus using DCZ resulted in enhanced glymphatic system function. Sholl analysis of cell morphology revealed that, during this activation, microglial morphology became more complex. A possible reason is that chemogenetic modulation of microglia may lead to their hyperpolarization or depolarization, affecting their normal dynamics and reducing their responsiveness to environmental ADP and ATP. This, in turn, prevents the recruitment of microglia to the perivascular space, resulting in a decrease in perivascular space resistance and, consequently, an enhancement of glymphatic system function. 9 Previous studies have shown that AQP4 serves as a crucial link in the glymphatic system, 29 facilitating the exchange of CSF and interstitial fluid (ISF). In physiological conditions, the glymphatic system aids in clearing metabolic waste from the brain, contributing to the maintenance of central nervous system homeostasis. 30 Our study found that chemogenetic activation of microglia enhance glymphatic system function, accompanying with an increased polarized distribution of AQP4 in cortex and hippocampus.

Due to the limited availability of adeno‐associated virus vectors that effectively target microglia, there has been relatively little research on chemogenetic or optogenetic interventions in microglia. Following the pioneering work by The Watkins's lab in 2016, where they were the first to utilize Gi and Gq DREADDs in rat microglia via viral expression, more researches have been incorporating chemogenetics in the study of microglia. 20 , 21 In 2021, Anthony and colleagues achieved successful expression of AAV‐HM3D and AAV‐HM4D in microglia of wild‐type mice. They utilized CNO to activate DREADDs, allowing control over microglia. Whether activating microglia through the Gq system (HM3D) or inhibiting them through the Gi system (HM4D), both approaches resulted in an increased extension of microglial processes. 31 This aligns with our experimental findings. The phenomenon may be attributed to rapid alterations in microglial activity, whether decreasing or increasing, significantly enhance calcium activity in microglial processes. 31 Moreover, using chemogenetics to manipulate microglia in CX3CR1creER/+:R26LSL‐hM4Di/+ mice can reverse the reduction in microglial processes and enlargement of cell induced by spinal nerve transection, leading to an increase in microglial processes. 24 Furthermore, Rouven Schulz and his team achieved successful engineering of a hybrid protein involving GPCR and HM3D, which they introduced into microglia through transfection. When the DREADD receptor was activated with CNO, they noted an augmentation in microglial processes. 32

In conclusion, our study reveals a dynamic relationship between microglia and the glymphatic system. Employing chemogenetic techniques, we demonstrated the modulatory capacity of microglia in regulating the glymphatic system.

FUNDING INFORMATION

The authors acknowledge the financial support by Financial Science and Technology Plan Project of Shihezi (2023BX01‐3) and Bingtuan Guiding Science and Technology Program (2022ZD072).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Yang T, Tang Y, Liu X, Gong S, Yao E. Microglia synchronizes with the circadian rhythm of the glymphatic system and modulates glymphatic system function. IUBMB Life. 2024;76(12):1209–1222. 10.1002/iub.2903

Ting Yang and Yan Tang contributed equally to this work.

Contributor Information

Song Gong, Email: gongsong@tjh.tjmu.edu.cn.

Ensheng Yao, Email: yes1219@126.com.

DATA AVAILABILITY STATEMENT

The data used in this work is available from the corresponding author on reasonable request.

REFERENCES

  • 1. Simard M, Arcuino G, Takano T, Liu QS, Nedergaard M. Signaling at the gliovascular interface. J Neurosci. 2003;23(27):9254–9262. https://pubmed.ncbi.nlm.nih.gov/14534260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, et al. Brain‐wide pathway for waste clearance captured by contrast‐enhanced MRI. J Clin Invest. 2013;123(3):1299–1309. 10.1172/JCI67677 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra111. 10.1126/scitranslmed.3003748 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Jessen NA, Munk ASF, Lundgaard I, Nedergaard M. The glymphatic system: a Beginner's guide. Neurochem Res. 2015;40(12):2583–2599. 10.1007/s11064-015-1581-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Kress BT, Iliff JJ, Xia M, Wang M, Wei HS, Zeppenfeld D, et al. Impairment of paravascular clearance pathways in the aging brain. Ann Neurol. 2014;76(6):845–861. 10.1002/ana.24271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Colonna M, Butovsky O. Microglia function in the central nervous system during health and neurodegeneration. Annu Rev Immunol. 2017;35:441–468. 10.1146/annurev-immunol-051116-052358 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Thion MS, Ginhoux F, Garel S. Microglia and early brain development: an intimate journey. Science (New York, N.Y.). 2018;362(6411):185–189. 10.1126/science.aat0474 [DOI] [PubMed] [Google Scholar]
  • 8. Wolf SA, Boddeke HWGM, Kettenmann H. Microglia in physiology and disease. Annu Rev Physiol. 2017;79:619–643. 10.1146/annurev-physiol-022516-034406 [DOI] [PubMed] [Google Scholar]
  • 9. Császár E, Lénárt N, Cserép C, Környei Z, Fekete R, Pósfai B, et al. Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions. J Exp Med. 2022;219(3):e20211071. 10.1084/jem.20211071 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Lavi E, Cong L. Type I astrocytes and microglia induce a cytokine response in an encephalitic murine coronavirus infection. Exp Mol Pathol. 2020;115:104474. 10.1016/j.yexmp.2020.104474 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W‐B, et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci. 2006;9(12):1512–1519. https://pubmed.ncbi.nlm.nih.gov/17115040 [DOI] [PubMed] [Google Scholar]
  • 12. Brown FN, Iwasawa E, Shula C, Fugate EM, Lindquist DM, Mangano FT, et al. Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus. Fluids Barriers CNS. 2023;20(1):42. 10.1186/s12987-023-00433-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Cao J, Yao D, Li R, Guo X, Hao J, Xie M, et al. Digoxin ameliorates glymphatic transport and cognitive impairment in a mouse model of chronic cerebral hypoperfusion. Neurosci Bull. 2022;38(2):181–199. 10.1007/s12264-021-00772-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Parsons R, Parsons R, Garner N, Oster H, Rawashdeh O. CircaCompare: a method to estimate and statistically support differences in mesor, amplitude and phase, between circadian rhythms. Bioinformatics (Oxford, England). 2020;36(4):1208–1212. 10.1093/bioinformatics/btz730 [DOI] [PubMed] [Google Scholar]
  • 15. Hablitz LM, Plá V, Giannetto M, Vinitsky HS, Stæger FF, Metcalfe T, et al. Circadian control of brain glymphatic and lymphatic fluid flow. Nat Commun. 2020;11(1):4411. 10.1038/s41467-020-18115-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Ma Q, Ries M, Decker Y, Müller A, Riner C, Bücker A, et al. Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain. Acta Neuropathol. 2019;137(1):151–165. 10.1007/s00401-018-1916-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep drives metabolite clearance from the adult brain. Science (New York, N.Y.). 2013;342(6156):373–377. 10.1126/science.1241224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Yao D, Li R, Hao J, Huang H, Wang X, Ran L, et al. Melatonin alleviates depression‐like behaviors and cognitive dysfunction in mice by regulating the circadian rhythm of AQP4 polarization. Transl Psychiatry. 2023;13(1):310. 10.1038/s41398-023-02614-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Elmore MRP, Najafi AR, Koike MA, Dagher NN, Spangenberg EE, Rice RA, et al. Colony‐stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron. 2014;82(2):380–397. 10.1016/j.neuron.2014.02.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Grace PM, Strand KA, Galer EL, Urban DJ, Wang X, Baratta MV, et al. Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation. Proc Natl Acad Sci U S A. 2016;113(24):E3441–E3450. 10.1073/pnas.1602070113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Grace PM, Wang X, Strand KA, Baratta MV, Zhang Y, Galer EL, et al. DREADDed microglia in pain: implications for spinal inflammatory signaling in male rats. Exp Neurol. 2018;304:125–131. 10.1016/j.expneurol.2018.03.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Parusel S, Yi M‐H, Hunt CL, Wu L‐J. Chemogenetic and optogenetic manipulations of microglia in chronic pain. Neurosci Bull. 2023;39(3):368–378. 10.1007/s12264-022-00937-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Gu N, Eyo UB, Murugan M, Peng J, Matta S, Dong H, et al. Microglial P2Y12 receptors regulate microglial activation and surveillance during neuropathic pain. Brain Behav Immun. 2016;55:82–92. 10.1016/j.bbi.2015.11.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Yi M‐H, Liu YU, Liu K, Chen T, Bosco DB, Zheng J, et al. Chemogenetic manipulation of microglia inhibits neuroinflammation and neuropathic pain in mice. Brain Behav Immun. 2021;92:78–89. 10.1016/j.bbi.2020.11.030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Eide PK, Vinje V, Pripp AH, Mardal K‐A, Ringstad G. Sleep deprivation impairs molecular clearance from the human brain. Brain. 2021;144(3):863–874. 10.1093/brain/awaa443 [DOI] [PubMed] [Google Scholar]
  • 26. Liu YU, Ying Y, Li Y, Eyo UB, Chen T, Zheng J, et al. Neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling. Nat Neurosci. 2019;22(11):1771–1781. 10.1038/s41593-019-0511-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Stowell RD, Sipe GO, Dawes RP, Batchelor HN, Lordy KA, Whitelaw BS, et al. Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex. Nat Neurosci. 2019;22(11):1782–1792. 10.1038/s41593-019-0514-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Sun W, Suzuki K, Toptunov D, Stoyanov S, Yuzaki M, Khiroug L, et al. In vivo two‐photon imaging of anesthesia‐specific alterations in microglial surveillance and photodamage‐directed motility in mouse cortex. Front Neurosci. 2019;13:421. 10.3389/fnins.2019.00421 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Rasmussen MK, Mestre H, Nedergaard M. The glymphatic pathway in neurological disorders. Lancet Neurol. 2018;17(11):1016–1024. 10.1016/S1474-4422(18)30318-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Nedergaard M, Goldman SA. Glymphatic failure as a final common pathway to dementia. Science (New York, N.Y.). 2020;370(6512):50–56. 10.1126/science.abb8739 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Umpierre AD, Bystrom LL, Ying Y, Liu YU, Worrell G, Wu L‐J. Microglial calcium signaling is attuned to neuronal activity in awake mice. Elife. 2020;9:e56502. 10.7554/eLife.56502 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Schulz R, Korkut‐Demirbaş M, Venturino A, Colombo G, Siegert S. Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses. Nat Commun. 2022;13(1):4728. 10.1038/s41467-022-32390-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data used in this work is available from the corresponding author on reasonable request.

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