REV-ERBα mediates complement expression and circadian regulation of microglial synaptic phagocytosis

The circadian clock has been shown to regulate various aspects of brain health including microglial and astrocyte activation. Here we report that deletion of the master clock protein BMAL1 induces robust increases in the expression of complement genes such as C3, C4b and C1q in the hippocampus. Loss of downstream REV-ERBα-mediated transcriptional repression led to increases in C4b in neurons and astrocytes as well as C3 protein in microglia and astrocytes. REV-ERBα deletion induced complement C3/C4b gene expression and increased microglial phagocytosis of synapses in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis in wild type mice which was abrogated by REV-ERBα deletion. This work uncovers the BMAL1-REV-ERBα axis as a regulator of complement expression and synaptic phagocytosis in the brain, thereby illuminating a novel mechanism of synaptic regulation by the circadian clock.


INTRODUCTION
The circadian clock orchestrates 24-hour rhythms in various cellular processes through transcriptional-translational feedback loops in most cells of the body (Takahashi, 2017). At the core of the clock's positive limb is the bHLH-PAS transcription factor BMAL1, which heterodimerizes with CLOCK or NPAS2 to drive the transcription of large sets of clockcontrolled genes (Mohawk et al., 2012). BMAL1 transcriptional targets include the negative limb feedback regulator CRY and PER proteins, as well as REV-ERB and , nuclear receptors which can also inhibit the actions of the positive limb (Preitner et al., 2002;Retnakaran et al., 1994). Disruption of this circadian machinery is associated with various pathophysiological states including cancer, diabetes and neurodegeneration (Everett and Lazar, 2014; Holtzman, 2016; Sulli et al., 2018). Deletion of BMAL1 abrogates circadian clock function and leads to an 85% decrease in REV-ERB expression in the brain (Musiek et al., 2013). REV-ERB functions as a transcriptional repressor in many tissues, and has been implicated in regulation of metabolism and inflammation (Everett and Lazar, 2014) Previous work from our group shows that deletion of BMAL1 or its downstream target REV-ERB causes neuroinflammation and impaired brain functional connectivity (Griffin et al., 2019;Musiek et al., 2013). Diminished BMAL1 and REV-ERB expression have also been described in mouse models of Alzheimer's Disease (AD) (Lee et al., 2020;Stevanovic et al., 2017). In AD, memoryassociated, synapse-rich regions such as the hippocampus are affected early in the disease course (Braak et al., 2006). Synaptic loss also precedes neuronal death in neurodegeneration (Selkoe, 2002). Circadian dysfunction is also a well-described symptom of AD and other neurodegenerative diseases (Musiek and Holtzman, 2016;Videnovic et al., 2014). Therefore, elucidating the how clock proteins regulate synaptic health is an important step in understanding the connection between circadian dysfunction and neurodegeneration.
A wealth of recent studies have emphasized the critical role of the complement system of the brain in regulating neuroinflammation and synaptic integrity. Synapses labeled with the opsonins C1q and C3 (Stevens et al., 2007) were first described to be pruned by microglia during development (Schafer et al., 2012). C4 protein, encoded by the mouse C4b gene, also contributes to synaptic pruning by microglia in vivo (Sekar et al., 2016). Complement-dependent microglial synaptic pruning has also been implicated in the pathogenesis of neurodegenerative and neuropsychiatric diseases (Hong et Musiek et al., 2013). Given the roles of the clock in neurodegeneration and microglial activation, we explored a potential role of the core clock in regulating synaptic health. Herein we establish a novel link between the BMAL1-REV-ERB axis, complement expression, and microglial synaptic pruning in the hippocampus.

Disruption of the BMAL1-REV-ERB axis induces complement upregulation in multiple brain cell types.
While analyzing our previously published transcriptomic dataset from Bmal1 KO (BMKO) hippocampal tissue, we observed a striking upregulation of several complement transcripts, in particular C4b and C3, two genes which are critical for synaptic phagocytosis (Fig 1A). Other complement related transcripts including C1qc, C1qb, C1qa C1ra, and C1rb were also significantly increased in BMKO hippocampus ( Fig 1A). C4b was similarly increased in cerebral cortex samples from 4mo tamoxifen-inducible global BMAL1 KO mice (CAG-Cre ERT2 ;Bmal1 f/f ) in which Bmal1 was deleted at 2mo, demonstrating that this is not a developmental phenomenon ( Fig S1).
To determine the cell type(s) in which BMAL1 deletion induces complement gene expression, we examined cerebral cortex tissue from pan-neuron-(CamK2a- Both astrocyte-and microglia-specific Bmal1 KO mice were treated with tamoxifen at 2mo and harvested 2 months later. Notably, C4b mRNA was strongly induced in the neuron-specific BMAL1 KO mice, while C3 was not ( Fig 1B). C4b was also induced in astrocyte-specific BMAL1 KO mice (Fig 1C). C4b, but not C3, was induced in global inducible Bmal1 KO mice (CAG-Cre ERT2 ;Bmal1 f/f ) 2mo after gene deletion (Fig. S1) Under basal conditions, neither C4b nor C3 was induced in microglia specific BMAL1 KO mice (Fig 1D). Deletion of BMAL1 in primary Bmal1 fl/fl neuron cultures via infection with an AAV8-Cre viral vector (versus AAV8-eGFP control) suppressed the BMAL1 transcriptional target REV-ERB (Nr1d1) by 85% and induced C4b expression but caused no increase in C3 (Fig 1E). BMAL1 deletion also increased expression of Fabp7 (Fig 1E), a known target of REV-ERB-mediated transcriptional repression (Schnell et al., 2014). This suggested that the upregulation of C4b gene expression observed with loss of BMAL1 could be mediated by de-repression as consequence of downstream REV-ERB loss. Accordingly, global deletion of REV-ERB caused striking increases in C4b, C3 and A. Relative expression of complement related transcripts taken from microarray analysis performed on hippocampus from 5mo RKO and littermate WT mice (N= 3/genotype) as well as BMAL1 KO mice (N=2). B. qPCR analysis of 11month old control (Cre-) and neuron-specific Bmal1 KO mice (Camk2a-iCre+;Bmal1 fl/fl ) for complement genes (N=3-4/group). C. qPCR analysis of control (Cre-) and astrocyte-specific Bmal1 KO mice (Aldh1l1-Cre ERT2 +;Bmal1 fl/fl ) for complement genes (N = 5 mice/group). D. qPCR analysis of control (Cre-) and microglia-specific Bmal1 KO mice (Cx3cr1-Cre ERT2 +;Bmal1 fl/fl ) for complement genes (N = 4-8/group). For C and D, all mice (Cre-or +) were treated with tamoxifen at 2mo and harvested at 4mo, mixed sexes, Cre-littermates were used as controls. E. qPCR analysis of mRNA from primary cortical neurons isolated from Bmal1 fl/fl mice, treated with AAV8-GFP (control) or AAV8-Cre (n = 4-5/group). F. qPCR analysis of WT or RKO mouse cortical tissue for complement genes (N= 5-10 mice/group). G. Representative 40X maximum intensity projections of hippocampus of 5mo WT or RKO mice stained for C3 and GFAP as well as the associated normalized volumes for C3-GFAP staining (n=8 mice, N=4/group). H. Representative 40X maximum intensity projections of 5mo WT or RKO mice stained for C3 and Iba1 as well as the associated normalized volumes for C3-Iba1 staining (n=8, N=4/group). In G and H, each point represents the average of 3 sections from a single mouse. *p<0.05 **p<0.01 ***p < 0.001 by 2-tailed T-test with Welch's correction.

FIGURES
other complement transcripts in the hippocampus as assessed by microarray analysis (Fig 1A) and confirmed in separate samples by qPCR ( Fig 1F). Increased C3 protein expression was observed in both activated astrocytes ( Fig 1G) and microglia (Fig 1H) in the hippocampus of 5mo REV-ERB KO (RKO) mice. The finding that Bmal1 deletion induces C4b mRNA early, but not C3, but that C3 mRNA and protein are increased at later ages, suggest that REV-ERB directly represses C4b expression in neurons and astrocytes, but the induction of C3 in both BMKO and RKO brain is likely secondary to inflammatory glial activation which occurs over time.

REV-ERB regulates microglial synaptic engulfment
We previously demonstrated that global REV-ERB deletion induced microglial activation in vivo (Griffin et al., 2019). Given those results and the observation of increased C4b and C3 expression in RKO brains, we examined the possibility that these changes would enhance synaptic phagocytosis in 4-6mo RKO mice. We primarily focused on the mossy fiber synapses in the CA2/3 region of the hippocampus, as these large synapses (thorny excrescences) can easily be stained and imaged using standard confocal microscopy. Triplelabeling of tissue sections was performed with antibodies against synaptophysin (a marker of presynaptic neuronal terminals - Fig 2A), CD68 (a microglial lysosome marker - Fig 2B) and Iba1 (to define microglial cell bodies and processes - Fig 2C). CD68 was used to ensure that the colocalized synaptic material was actually within the microglia phagosome. 3D reconstructions were made and total volumes of engulfed synaptic material were calculated. We noted a 10-fold increase in engulfed synaptic material in the hippocampus of RKO mice compared to their WT littermates at 6mo (Fig 2F). In the RKO microglia, we observed synaptic material in the microglial process and cell body (Fig 2Eii, Eiii), whereas WT microglia only had engulfed synaptic material in the cell body (Fig 2Ei).
To corroborate these results, we also performed large area scanning electron microscopy (SEM) experiments of the CA3 region of 6mo WT and RKO mice. Using this method, we visually confirmed an increased number of presynaptic terminals within or in contact with microglia in the hippocampus of RKO mice compared to WT (Fig 2G, 2H, 2H(i-v), 2I).
Interestingly, in our RKO mice we noted a downregulation in the expression of the Sirpa gene, which codes for the protein SIRP ( Fig 1A). SIRP was recently described as a surface receptor on microglia that serves as a "do-not-eat-me" signal (Lehrman et al., 2018). Taken together, our results suggest that REV-ERB deletion induces changes in microglial signaling that precipitates synaptic engulfment. Representative 3D surface rendering of microglia showing engulfed presynaptic material in lysosomes with zoomed in lysosomal content from WT mice in Ei and littermate RKO mice in Eii/Eiii. F. Quantification of the normalized Iba1-CD68-Synaptophysin volumes from microglia in the CA3 region of the hippocampus of 4-6mo WT and RKO mice (each point is average of 3 sections from one mouse, N = 15 and 12 RKO mice). G. Annotated, representative scanning electron micrographs of microglia in the CA3 of WT (Cyan) or RKO (Royal blue) mice sacrificed at 11AM with zoomed in pictures of engulfed presynaptic terminals in Hi and presynaptic terminals in contact with microglia in Hii, Hiii and Hiv. I. Quantification of presynaptic terminals in contact with or engulfed by microglia in the CA3 of WT or RKO mice. Each point represents one field of view, N=2 mice/genotype. **p < 0.01,**** p<0.0001 by 2-tailed T-test with Welch's correction.

CA3 synapses are reduced by REV-ERB deletion
Following our observations of synaptic pruning in the RKO mice, we investigated the status of the synapses in the CA3 region of the hippocampus. Synapses were double labeled by using the presynaptic marker synaptophysin and the postsynaptic marker homer1. In RKO mice, we observed a significant decrease in the synaptic volume in the CA3 region of the hippocampus by synaptophysin staining (Fig 3A), homer1 staining ( Fig 3B) and their colocalization ( Fig 3C) compared to their WT littermates. To further confirm these results, we counted synapses in large area SEM images from the stratum lucidum of WT or RKO mice.
Again, we noted a decrease in the number of synapses in the RKO mouse CA3 by SEM, as compared to their WT littermates ( Fig 3D).
To ensure that our observations were not due purely to changes in synaptophysin protein expression in the presynaptic terminal, synapses were also stained with a second presynaptic marker, synaptoporin, and quantified. Synaptoporin was used because it is enriched in the mossy fiber synapses of the hippocampus (Singec et al., 2002). Again, we observed a similar decrease in synaptic volume in CA3 of RKO mice compared to their WT littermates using synaptoporin staining ( Fig 3E). Interestingly, we did not observe significant differences in synaptic volumes between WT and RKO mice in the CA1 region of the hippocampus (Fig S2A-C), suggesting that some terminals may be more susceptible to loss than others. To determine whether the loss in synapses was due to a loss of neuronal cell bodies, we quantified the volume of neuronal nuclei of the dentate gyrus, which project to CA3, via NeuN staining. We found no significant difference between the neuronal nuclear volumes of the dentate gyrus between the WT and RKO mice ( Fig 3F). Taken together, our data suggest that deletion of REV-ERB results in robust synaptic loss in the CA3 region without obvious neuronal loss.

Time-of-day variation in microglial phagocytosis is regulated by REV-ERBα
In the brain, REV-ERB displays daily oscillation on the mRNA levelwith its peak at zeitgeber time (ZT) 8 (2pm) and its trough at ZT20 (2am) (Chung et al., 2014). We have also previously described oscillations in microglial activation, with low activation at ZT5 and high activation at ZT17 (Griffin et al., 2019). Consequently, we investigated microglial synaptic engulfment at different times of day. WT(C57BL/6) mice sacrificed at ZT5 (11AM) or ZT17 (11PM), and hippocampal sections were triple labeled with Iba1, CD68, and synaptophysin.
Microglia from the CA3 region of the hippocampus in mice sacrificed at ZT17 showed significantly more engulfed presynaptic protein than those at ZT5 (Fig 4A, Fig 4B, Fig 4Bi, Fig   4C). To further establish these findings, we used large area SEM to count the number of presynaptic terminals in contact with or within microglia at ZT5 or ZT22. In that experiment, we also noted a higher number of presynaptic terminals in contact with and within the microglia at ZT17 (Fig 4D, Fig 4E, Fig 4Ei, Fig 4F). As a final confirmation of this finding, we injected CX3CR1 GFP mice, which express GFP in microglia (Jung et al., 2000), with an AAV-CaMKII-mCherry viral vector in the retrospenial cortex. 4 weeks later, we collected brain samples at ZT5 and 17, and calculated the volume of mCherry+ material in individual GFP+ microglia. Here, we found that despite interactions between microglia and neurons at both timepoints, microglia at 11PM (CT17) engulfed more mCherry than those at 11AM (ZT5) (Fig 4G-I). RKO mice harvested at ZT5 and 17 showed a persistently increased level of synaptic engulfment in microglia, but with no time-of-day variation (Fig 4J), suggesting that daily oscillations in REV-ERBα may mediate rhythms in microglial synaptic phagocytosis. Overall, our data establish a REV-ERBα-dependent rhythm for the engulfment of neuronal materials in the brain parenchyma by microglia.

DISCUSSION
The current study shows that loss of the circadian protein BMAL1 causes upregulation of complement gene C4b in neurons and astrocytes, as well as increase astrocytic C3 expression, all via loss of downstream REV-ERB-mediated transcriptional repression. Deletion of REV-ERB leads to microglial activation, increase C4b and C3 expression, and increased microglial synaptic phagocytosis in CA3 region of the hippocampus. Finally, we demonstrate a time-of-day variation in synaptic phagocytosis in the hippocampus of WT mice, which is lost after REV-ERB deletion. Our finding suggests that the BMAL1-REV-ERBα axis regulates daily rhythms in synaptic phagocytosis, and that loss of REV-ERBα de-represses complement gene expression and locks the brain in a pro-phagocytic state.
The mechanisms by which Bmal1 deletion leads to increased complement gene expression are complex and multicellular but appear to depend on REV-ERBα. REV-ERB expression is decreased by ~85% following Bmal1 deletion, and REV-ERB deletion phenocopies the complement gene expression increases seen in Bmal1 KO brain. Indeed, it is well established that REV-ERB functions as a transcriptional repressor (Harding and Lazar, 1995  Bmal1 KO and RKO mice at 5-6mo also show an increase in C3 transcript and C3 protein in astrocytes. However, Tissue-specific Bmal1 deletion in neurons or astrocytes causes pronounced C4b increases but no C3 increases, suggesting that C3 expression in astrocytes and microglia is a secondary response to increased neuroinflammation in aged global Bmal1 KO or RKO mouse brain. Global inducible Bmal1 KO mice also do not show C3 induction at 2mo post-tamoxifen (despite high C4b expression), as these mice have not developed a full neuroinflammatory response at that age. Our data suggests that full induction of C3 in Bmal1 and RKO brain requires a time-dependent inflammatory response. We previously showed that expression can drive C3-dependent synapse pruning. Additionally, we noted that REV-ERB regulated the "do-not-eat-me" signal Sirpa (encoding SIRP) in our microarray data. Loss of inhibitory signaling from SIRP makes microglia more likely to prune synapses, since SIRP is primarily expressed on microglia in the CNS (Zhang et al., 2014). Therefore, our data suggest that de-repression of C4b in neurons and Traf2 in astrocytes and microglia, as well as diminished Sirpa expression in microglia, lead to a "perfect storm" of complement expression and microglial activation that promotes synaptic phagocytosis.

REV-ERB deletion increased
We focused on the CA2/3 mossy fiber synaptic boutons because they are large and easily labeled with 2 synaptic vesicle markerssynaptoporin and synaptophysin (Grosse et al., 1998). However, we did not observe synapse loss in the CA1 region, which may represent a regional variability in BMAL1-REV-ERB mediated synaptic pruning. Certainly, it is possible that certain synapses are more susceptible to pruning than others, and this should be addressed in the future. We cannot exclude the possibility that REV-ERBα deletion causes neuronal damage, which elicits microglial phagocytosis of dysfunctional synapses. Indeed, neuronal C4b upregulation could be a damage signal. However, the absence of neuronal cell body loss in the dentate gyrus of RKO mice shows that there is no overt neurodegenerative response. These dentate gyrus granule cells give rise to the mossy fiber boutons in the CA3 (Scharfman and Myers, 2012), which are clearly decreased in RKO mice. Thus, the effect of REV-ERB appears to be specific to the synapses.
Herein, we establish REV-ERB as a regulator of microglial synaptic phagocytosis.
However, we have previously reported that time-of-day changes in microglial morphology were abrogated by REV-ERB deletion (Griffin et al., 2019). Herein, we observed that microglia engulfed more synapses in the CA3 of the hippocampus at 11PM (ZT17) than at 11AM (ZT5).
This was evident by both immunofluorescence and by electron microscopy. To further confirm this striking finding, we used a viral approach in which the retrospenial cortex of mice with GFP  (Mang et al., 2016) .A study in the rat prefrontal cortex found that synaptic elimination was higher at ZT0 (Choudhury et al., 2020). Since these studies were done in the prefrontal cortex, they suggest potential brain region specific pruning patterns by time of day.
Heterogeneity in synaptic pruning has already been described, with areas such as the cerebellum exhibiting greater microglial phagocytic capacity (Ayata et al., 2018). Future work will have to explore the patterns in microglial phagocytosis across more brain regions. This work has clear implications for neurodegenerative and neuropsychiatric diseases, which have been linked to circadian disruption as well as complement dysregulation and synapse loss. Since REV-ERBα is a nuclear receptor with available small-molecule ligands (Solt et al., 2012), our findings suggest that it could be a therapeutic target for neurological and psychiatric disease. In previous studies in the brain, activation of REV-ERBs appears to suppress microglial cytokine production (Griffin et al., 2019) while inhibition of REV-ERBs can induce microglial amyloid-beta uptake and decrease plaque burden in mice (Lee et al., 2020).
Effects on synaptic engulfment should be considered as REV-ERB-based therapeutics are developed.

Mice:
Rev-erb +/-(Nr1d1 +/-) mice on C57bl/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at at Washington University. Heterozygous mice were bred together to generate Rev-erb +/+ (wt) and Rev-erb -/-(RKO) littermates which were used for experiments. Constitutive Bmal1 -/-(BMKO), CAG-Cre ERT2 , Aldh1L1-Cre ERT2 , Cx3cr1-Cre ERT2 , and Bmal1 fl/fl mice were obtained from the Jackson Laboratory, and bred so that mice were heterozygous for Cre and homozygous for the floxed Bmal1 allele. In all experiments, Cre-;Bmal1 f/f littermates were used as controls. All inducible knockout lines were were treated with tamoxifen (Sigma) dissolved in corn oil via oral gavage, 2.5mg/day for 5 days, at 2mo. Camk2a-iCre+;Bmal1 fl/fl mice were bred at University of Texas Southwestern. CX3CR1 GFP mice were obtained from The Jackson Laboratory. Mice were housed on a 12/12 light/dark cycle and fed ad libitum. All procedures performed on the mice were approved by the Washington University IACUC.

Immunohistochemistry:
The For other non-synaptic staining, 50m serial coronal sections were cut on a freezing sliding microtome and stored in cryoprotectant (30% ethylene glycol, 15% sucrose, 15% phosphate buffer in ddH2O). Sections were washed 3 times in Tris buffered saline (TBS), blocked for 30 mins in TBS containing 3% goat serum and 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO) then incubated in TBS containing 1% goat serum and 0.25% Triton X-100 with primary antibody overnight at 4˚C. Sections were then washed 3 times and incubated for 1 hour at room temperature with 1:1000 fluorescent secondary antibody and mounted on slides. Confocal images were taken on the Nikon Elements software on the Nikon A1Rsi scanning confocal microscope. Z-stacks were taken at a step size of 0.5-1µm from dark to dark through the tissue.
Microarray analysis: 5-6 mo Rev-erb -/and WT littermates were placed in constant darkness for 24 hours and then harvested with i.p. injection of pentobarbital in the dark, following pump perfusion. RNA was isolated from flash frozen hippocampus samples as above and submitted to the Genome Technology Access Center at Washington University for quality control, MessageAmp RNA library preparation and Agilent 4x44k mouse microarray. Raw data was normalized and analyzed using Partek Genomics suite v6.6. For Gene Ontology (GO) term analysis, a list of all genes which were upregulated at least 2-fold in KO with an uncorrected P value <0.05 by 2tailed T-test were uploaded to DAVID v6.8 (https://david.ncifcrf.gov/). Functional annotation analysis was performed for GO terms related to biological processes. Datasets are freely available on the Array Express Server: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-7590 Quantitative PCR: Flash-frozen brain tissue was homogenized with a mechanical handheld homogenizer for 20 seconds in RNA kit lysis buffer (PureLink TM RNA Mini Kit, Life Technologies, Carlsbad, CA) plus 1% -mercaptoethanol. RNA was then purified using the kit protocol. Cells well collected and lysed in Trizol (Life Technologies). The aqueous layer was collected following chloroform extraction (added at 1:5 then spun at 13000xg for 15 minutes) with RNA isolation protocol. RNA concentrations were then measured using the Nanodrop spectrophotometer and cDNA was made using a high capacity RNA-cDNA reverse transcription kit (Applied Biosystems/LifeTechnologies) with 1g RNA used per 20L reaction. Real-time quantitative PCR was performed with Taqman primers and PCR Master Mix buffer (Life Technologies) on the ABI StepOnePlus 12k Real-Time PCR thermocyclers. -actin (Actb) mRNA levels were used for normalization during analysis. The following primers were used (all from Life Technologies, assay number is listed): Actb: Mm02619580_g1, Nr1d1: Mm00520708_m1, C4b: Mm00437893_g1, C3: Mm01232779_m1, Fabp7: Mm00437838_m1.

Electron microscopy:
Animals were perfused with a fixative mix consisting of 2.5% glutaraldehyde + 2% paraformaldehyde (fresh, EM grade) in 0.15M cacodylate buffer with 2mM CaCl2 (final concentrations). Brains were extracted, blocked, and 100 µm coronal sections were made using a Leica 1200S vibratome. Tissues were then washed 3 x 10 minutes in cold cacodylate buffer containing 2mM calcium chloride, and then incubated in a solution of 1% OsO4 containing 3% potassium ferrocyanide in 0.3M cacodylate buffer with 4mM calcium chloride for 1 hour in the dark. Following incubation, tissues were incubated for 20 minutes in a 1% thiocarbohydrazide (TCH) solution, rinsed 3 x 10 minutes in ddH2O at room temperature and thereafter placed in 2% osmium tetroxide (NOT osmium ferrocyanide) in ddH20 for 30 minutes, at room temperature. Tissues were washed 3 x 10 minutes at room temperature in ddH2O then placed in 1% uranyl acetate (aqueous) and incubated at 4° overnight. Tissues were incubated in a lead aspartate solution at 60°C oven for 30 minutes, washed 5 x 3 minutes in ddH2O, and returned to the lead aspartate solution at 60°C for 30 minutes. Tissues were washed 3 x 10 minutes in room temperature ddH2O and dehydrated in 50%, 70%, 90%, 100%, 100% acetone (anhydrous), 10 minutes each. Samples were embedded in Durcupan ACM resin and polymerized 60°C oven for 48 hours. 70 nm slices were made using a Leica UC7 ultramicrotome, and sections were picked up on a Si wafer (Ted Pella, Redding, CA). Images were acquired on a Zeiss Merlin FE-SEM using a solid state backscatter detector (8kV, 900pA) at 7nm resolution with 5µs pixel dwell times and 4x line averaging. Large area scans of ~150µm x 150 µm field of view were acquired and stitched using Atlas 5.0 (Fibics, Ottawa, Canada)

Stereotactic surgery and Intracortical viral Injections
CX3CR1 GFP mice had their heads shaved and ear bars put in bilaterally. Iodine was then applied and the skin over the skull was cut open gently using surgical scissors. The skull was checked to make sure that it was level on both sides of the midline. A hole was drilled through the skull but not into the brain parenchyma. The coordinates used for the retrospenial cortex were 0.3mm mediolateral (M/L), -2mm anterior-posterior(A/P) and -1mm dorso-ventral(D/V) with bregma as a reference. The needle was placed in the target location and then allowed to rest for 2 mins before infusion. The AAV2-CaMKII-mCherry virus was obtained from the UNC viral vector core and infused at a rate of 0.2µL/min. In total, the mice were injected with 2L in the retrospenial cortex. After the infusion, we waited 5 mins for the virus to diffuse in the parenchyma and then the needle was slowly removed. The skin over the skull was then stitched up and antibiotic was applied to the area. The mice were then allowed to recover in an empty cage on a heating pad. After the surgery the mice were checked twice daily, 4 hours apart to ensure survival for 3 days. The mice were then allowed to age for 1 month before being sacrificed for sectioning and imaging.

Synaptic volume and engulfment analysis/3D reconstructions:
Imaris visualization and analysis software (Version 9, Bitplane, South Windsor, CT, USA) was used at the Washington University Center for Cellular Imaging. For all analyses Z-stacks were saved in the .nd2 file format and loaded into the software. 3-D surfaces had a surface detail ranging from 0.1-0.3m. To quantify the volume of the synapses, we generated 3-D surfaces from each of the synaptic markers (Synaptophysin, Synaptoporin and Homer1). We used the Batch colocalization function to colocalize the Synaptophysin and Homer1 volumes. The total volume for each Z-stack was summed. For the synaptic engulfment analyses, we first generated volumes from synaptophysin, CD68 and Iba1 staining. We then colocalized the synaptophysin volume with the CD68 volume. The resulting volume was then colocalized with the Iba1 volume to produce a final microglial synaptic engulfment volume. All values were normalized to the average values for the control group in each experiment.

Primary Cell Cultures:
WT Neuronal and astrocyte cultures were obtained from CD1 mice from Charles River Laboratories (Wilmington, MA). Neurons were isolated from E16-E18 pups and astrocytes were isolated from P0-2 pups. For experiments involving Rev-erb -/astrocytes or microglia, the cultures were made from mice on a C57BL/6J background. Cortices plus hippocampus were dissected and stripped of meninges in ice-cold DMEM (Life Technologies) and then incubated in 0.05% Trypsin-EDTA at 37˚C for 15 mins (Neurons) or 20 mins (Astrocytes). Tissue was gently triturated in 37˚C DMEM plus 10% FBS (Gibco). For astrocyte cultures cells were then plated in T75 flasks coated for 2 hours at 37˚C with 50µg/mL poly-D-Lysine (PDL -MP Biosciences, Santa Ana, CA) then rinsed with ddH2O. Astrocytes were then grown to confluence in DMEM, 10% FBS, and 1% Penicillin/Streptomycin (P/S) with or without 5 ng/mL GM-CSF. For primary microglia isolation from GM-CSF containing media, flasks were shaken at 225rpm at 37˚C for 2 hours and replated on plates coated with PDL. For neuronal cultures, triturated cells were transferred to a second tube to remove debris, then diluted in Neurobasal (Life Technologies) plus B27 (Life Technologies) prior to plating on a bed of astrocytes or a PDL-coated plate. For co-culture experiments, astrocytes were grown to confluence following replating in 24-well plates and neurons were added immediately after primary dissection at a concentration of 150,000 cells/mL in Neurobasal plus B27 supplement and glutamine (Life Technologies). After 48 hours, 50% of the media was changed to Neurobasal plus anti-oxidant free B-27 (AOF B-27,