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Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle

Abstract Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor‐1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1 GFP/GFP mice recapitulate what found in PLX5622‐treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase‐dependent manner.


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CX3CR1 plays a relevant role in these effects, because cx3cr1 GFP/GFP mice recapitulate what found in PLX5622-treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase-dependent manner.
K E Y W O R D S cx3cr1, electroencephalography, long-term potentiation, microglial depletion, miniature excitatory post-synaptic currents, sleep, spontaneous excitatory post-synaptic currents

| INTRODUCTION
The environmental light changes caused by the 24-h period of Earth's rotation around its axis modulate the internal clock in our brain, triggering cycles of alertness and sleepiness in the circadian rhythm. The role of glial cells in the modulation of the biological circadian clock is a matter of intense research (Ingiosi et al., 2013;Garofalo et al., 2020). Several studies point to astrocytes as major determinants of the glial contribution to synaptic homeostasis during the sleep/wake cycle, through the IP 3 /Ca 2+ signaling pathway (Foley et al., 2017), modulating the extracellular levels of ATP and adenosine (Halassa et al., 2009;Nadjar et al., 2013;Porkka-Heiskanen et al., 1997) and participating to the glymphatic system for the clearance of metabolites and chemicals from the extracellular space of the brain (Xie et al., 2013). However, key roles have been hypothesized also for microglia during normal sleep and upon sleep disturbances Choudhury et al., 2020) as well as in neural plasticity processes associated with synaptic remodeling during sleep (Yang et al., 2014;Niethard et al., 2017;de Vivo et al., 2017;Tuan and Lee, 2019;Stanhope et al., 2020;Zhou et al., 2020). In particular, microglial cells undergo molecular alterations along the sleep and wake phases, expressing different patterns of cytokines upon inflammatory challenge (Fonken et al., 2015). Microglia are modulated by the arousal state (Stowell et al., 2019;Liu et al., 2019) and by sleep deprivation, with effects on their expression of receptors for neurotransmitters, hormones and cytokines involved in regulating process motility and phagocytic activity (Wisor et al., 2011). Most studies performed to investigate the effect of sleep and wake on synapses and glial cells used models of sleep deprivation where the wake phase differs for the level and quality of stimuli used to avoid sleep (Havekes and Aton, 2020). To limit possible confounding interference induced by animal manipulation, we used light as a zeitgeber cue, exposing mice to 12:12 light/dark cycles (light: Zeitgeber Time [ZT] 0-ZT12, dark: ZT12-ZT0). We investigated the role of microglial cells by depletion studies, taking advantage of the role exerted by colony stimulating factor-1 receptor (CSF-1R) signaling on myeloid lineage cells (Ginhoux et al., 2010) and of previous reports demonstrating that CSF-1R deletion or inhibition leads to a near complete elimination of microglial cells (Erblich et al., 2011, Elmore et al., 2014. We treated mice with PLX5622, a CSF-1R inhibitor that drastically reduces the density of microglial cells in different brain regions, and we analyzed different endpoints: (1) the time spent in sleep or active states during the light and dark phases of the day; (2) the basal excitatory synaptic transmission; and (3) neuronal plasticity processes in the hippocampal region at ZT4 (light) and ZT16 (dark).
These two time points were selected for being far from the light changes and thus better representing constant behavioral conditions. We described that, in the nearly complete absence of microglia, mice spent more time sleeping during the dark phase. The depletion of microglia also affected excitatory synaptic transmission in a phase-dependent way: (1) abolishing the differences in spontaneous excitatory postsynaptic currents (sEPSC) and miniature EPSC (mEPSC) amplitude and sEPSC frequency between the dark and light phases and (2) increasing hippocampal long-term potentiation (LTP) exclusively during the light phase.
The chemokine fractalkine (CX3CL1) receptor (CX3CR1) is highly expressed in microglia and its genetic deletion in mice disclosed multiple regulatory roles of CX3CR1 on brain development, synapsis maturation, and neuronal function (Paolicelli et al., 2011;Bol os et al., 2017), as well as on neuronal survival in models on neuropathology (Limatola and Ransohoff, 2014). To investigate whether the effects induced by microglial depletion were at least in part dependent on microglial CX3CR1 signaling, the sleep/wake cycle and the excitatory synaptic transmission along the light/dark cycle were also investigated in cx3cr1 GFP/GFP mice, where CX3CR1 is deleted (Jung et al., 2000), with outcomes comparable to microgliadepleted mice. Furthermore, we report that microglial cx3cr1 expression is lower during the light phase and that, in vitro, it is modulated by the sleep-dependent metabolite ATP, thus suggesting that CX3CR1 plays a central role in regulating microglial responses during the light/dark cycle.
In conclusion, we demonstrated that under physiological conditions, microglia affect the duration of sleep and are necessary for synaptic changes occurring during the wake phase, disclosing also a key role for CX3CR1 in sleep-wake cycle.

| Housing and light conditions
Mice were housed in standard breeding cages at a constant temperature (22 ± 1 C) and relative humidity (50%). Food and water were available ad libitum.
Before any experimental intervention, all mice were housed for at least 7 days upon arrival with a 12:12 h light/dark cycle (light 7:00, ZT0; dark 19:00, ZT12). The same light conditions were kept for all experimental phases, except for the electrophysiology experiments performed in hippocampal slices during the dark phase (see below).
PLX5622 and control chow administration started at this moment and continued until the end of the experiment. After 1 week of full recovery following the electrodes implantation, mice experienced 1 week of handling and plugging-unplugging operations every day (2-5 min per day). The day before the experiment, the mice were placed in the recording cage for acclimatization.
Grass Technologies ©2011 (Twin software 4.5.3.23) was used for simultaneous EEG, EMG, and video recordings performed on pairs of mice for 24 h (12 h lights and 12 h dark). Each pair was formed by one PLX5622 or cx3cr1 GFP/GFP and one control mouse. The mice were simultaneously connected to a system consisting of AS40 Amplifier by a flexible cable. The EEG and EMG data were collected with a sampling rate of 200 Hz and an antialiasing band pass analogue filter.
The video-EEG-EMG analysis was initially performed in five controls and five PLX5622-treated mice for the whole dark and light period, only excluding the first hour after light change (22 h in total).
The results of this exploratory analysis are reported in the supplementary results ( Figure S2) and revealed that comparable information can be obtained by analyzing shorter periods (7 h each phase). In consideration of the time-consuming procedure of analysis, the EEG-EMG analysis was limited from ZT4 to ZT11 in the light period and from ZT13 to ZT20 in the dark period.
Each video and EEG-EMG recording epoch lasting 8 s has been classified into the following behavioral classes: 1. Active behavior (movement condition). The mice performed overt movements in the cage for most of the given epoch of 8 s. The movements were characterized by ample displacements of body parts such as trunk, head, and forelimbs. Significant EMG activity was expected in this behavioral condition.
2. Passive behavior (passive wake condition). The mice showed no movement (i.e., substantial immobility) periods intermingled with small movements of the trunk, head, and forelimbs for the majority of 8 s. The maximal duration of immobility considering two contiguous epochs was 8 s. This criterion was expected to minimize the risk that "passive condition" could be misclassified as sleep and vice-versa.
3. Immobility condition. The mice performed no movement of the trunk, head, and forelimbs for at least 20 s across three or more epochs. This condition was associated with a low EMG activity and required the analysis of the ongoing EEG and EMG waves for a discrimination between a condition of passive behavior and sleep (see

| Slice preparation for electrophysiology
For electrophysiology, mice were sacrificed during the light phase at ZT4 and during the dark phase at ZT16. In order to perform all the electrophysiological recordings during the working time of the day, mice sacrificed at ZT16 received a light/dark cycle inversion (light on at 19:00) for at least 14 days prior to start the control chow or PLX5622 treatment, for microglia depletion in wild-type mice. In this way, slices were harvested at 11:00 for all groups. To verify that light/dark cycle inversion did not affect the results of the experiments, a group of control mice receiving the normal light/dark cycle was sacrificed at 23:00. No significant differences were observed between the ZT16 groups in normal and inverted cycles and data were pooled.
Transverse 350 μm slices were cut at 4 C with a vibratome (Thermo Scientific, USA) and then placed in a chamber containing oxygenated ACSF. After their preparation, slices were allowed to recover for at least 1 h at 30 C.
Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded in ACSF by holding the cell at the reversal potential of GABA current (À70 mV). Miniature excitatory post-synaptic currents (mEPSCs) were isolated by adding Tetrodotoxin (0.5 mM, TOCRIS) to ACSF in the perfusion line for at least 10 min before starting acquisition. Signals were acquired (sampling 10 kHz, low-pass filtered 2 kHz) with DigiData-1440A using pCLAMP-v10 software (Molecular Devices, USA).

Analysis of sEPSCs and mEPSCs was performed offline using
MiniAnalysis software (Mini Analysis, Synaptosoft Fort Lee, NJ, USA) with the threshold for detection set at 5 pA. Cumulative probability curves were constructed by considering 100 consecutive events each cell.
Slices were transferred to the slice-recording chamber interface (BSC1, Scientific System Design Inc), maintained at 30-32 C and constantly superfused at the rate of 2.5 ml/min with oxygenated ACSF.
At the beginning of each recording, a concentric bipolar stimulating electrode (SNE-100X 50 mm long Elektronik-Harvard Apparatus GmbH) was placed in the hippocampus CA1 stratum radiatum for stimulation of Shaffer collateral pathway projection to CA1. Stimuli consisted of 100 μs constant current pulses of variable intensities, applied at 0.05 Hz. A glass micropipette (0.5-1 MΩ) filled with ACSF was placed in the CA1 region, at 200-600 μm from the stimulating electrode, in order to measure orthodromically evoked fEPSP. The paired-pulse ratio (PPR) was measured from responses to two synaptic stimuli at 50 ms interstimulus interval. PPR was calculated as the ratio between the field excitatory post synaptic potential (fEPSP) amplitude evoked by the second stimulus (A2) and that by the first (A1; A2/A1). Only the slices that showed stable fEPSP amplitudes were included in the experiments. Field EPSP was recorded and filtered (low pass at 1 kHz) with an Axopatch 200A amplifier (Axon Instruments, CA) and digitized at 10 kHz with an A/D converter (Digidata 1322A, Axon Instruments). Data acquisition was stored on a computer using pClamp 9 software and analyzed offline with Clampfit 10 software (both from Axon Instruments).
2.8 | Isolation of CD11b + cells from adult mice from brain areas C57BL6/N mice (ZT4 n = 15, ZT16 n = 13) were taken from cages and rapidly anesthetized, in no more than 1 min. Animals were intracardially perfused with PBS, brains were extracted, and the hip-

| ATP treatment of microglial primary cultures from pups
Microglial cells were obtained from mixed glia cultures derived from the cerebral cortices of post-natal day 0-2 (P0-P2) C57BL6/J mice.

| ATP treatment of microglial primary cultures from adult mice
Sixty-day-old mice were deeply anesthetized and intracardially perfused with ice cold PBS. Brains were removed, each hemisphere was cut into small pieces and disrupted in a glass-teflon homogenizer. Cell suspension was applied to a 30-μm cell strainer, labeled with CD11b + Microbeads and passed through MACS Columns (Miltenyi Biotec).
2.11 | Analysis of cx3cr1 mRNA expression CD11b+ cells isolated from tissue and cultured microglial cells were lysed in Trizol reagent (Invitrogen) for isolation of total RNA and purified by isopropanol precipitation; quality and yield were verified using the NANODROP One system (Thermo Scientific Scientific).

| Statistical analyses
Statistical significance was assessed by Student's t-test, one-way or two-way ANOVA for parametrical data, as indicated. In particular, for PCR, statistical analyses were conducted using one-way ANOVA followed by Dunn's and Tukey post hoc test (Sigmaplot software).
Electrophysiological experiments were analyzed by two-way ANO-VAs, considering subject (controls, PLX5622 and cx3cr1 GFP/GFP mice) and environmental condition (ZT4 and ZT16) as the between-subject variables (Sigmaplot software). Post hoc comparisons were performed using Holm-Sidak test. For the cumulative distribution analyses, we applied the Kolmogorov-Smirnov two-sample test (KS) using the MiniAnalysis software (Synaptosoft Fort Lee, NJ, USA). All mean differences were considered statistically significant when p < .05.
For EEG experiments, a statistical analysis of the epochs lasting 8 s data was performed by Statistica 10.0 packages (StatSoft Inc., www. statsoft.com). The primary analysis of those epochs was performed on the sleep duration, whereas the secondary analysis was focused on the duration of the exploratory movements (the latter was quantitatively tested in parallel experiments using the DVC cages). Due to the sample size, non-parametric tests were used. The Wilcoxon test was used for a within-group analysis of the sleep (NREM + REM sleep) and movement duration between the light and the dark periods. The Mann-Whitney U test was used for the between-group analysis.
For the primary analysis of REM and NREM sleep duration, the p value was corrected (Bonferroni) for 8 statistical comparisons, namely p < .05/8 = .006 = p < .05 corrected. For exploratory purposes, a statistical threshold of p < .05 uncorrected was also used. The statistical comparisons were (1) 4 within-group comparison light vs dark for the C57BL/6, PLX5622, C57BL/6J and cx3cr1 GFP/GFP groups; (2) 2 between-group comparisons for the light period for the C57BL/6 versus PLX5622 groups and the C57BL/6J versus cx3cr1 GFP/GFP groups; and (3) 2 between-group comparisons for the dark period for the C57BL/6 versus PLX5622 groups and C57BL/6J versus cx3cr1 GFP/GFP group. Note that almost all sleep periods were spent in the NREM sleep. The same design was used for the secondary analysis of movement durations.
Sample size (n/N) refers to the number of: (1) slices/mice, for filed cell recording analysis; (2) cells/mice for patch clamp recordings; (3) wells/cultures for in vitro analysis. N refers to the number of mice for EEG and motor activity analysis. All data are expressed as mean ± standard error of the mean (SEM).

| Effect of microglial depletion on sleep duration in the light/dark cycle
To investigate the effect of microglial cell depletion on the wakesleep cycle, we treated mice with the CSF-1R inhibitor PLX5622. This treatment is reported to efficiently and transiently deplete microglia in several brain regions (Huang et al., 2018), and we confirm this result in three different brain regions involved in sleep regulation such as the hypothalamus (the whole structure) (92.81 ± 3.37% depletion), the prefrontal cortex (PFC) (95.28 ± 2.32% depletion) and the hippocampus (93.89 ± 2.76% depletion) (Ono and Yamanaka, 2017;Durán et al., 2018) (Figure S1). Microglia-depleted mice were analyzed for video, fronto-parietal EEG and back-muscle EMG recordings in 24 h and the data analysis was performed for 7 h (from ZT4 to ZT11) in the light phase and 7 h (from ZT13 to ZT20) in the dark phase. As expected, both control and PLX5622 treated mice showed longer sleep (total and NREM sleep) duration during the light phase (Table 1; Figure 1a,d), and longer movement durations during the dark phase (Table 1; Figure 1c,f) and REM sleep was shorter in the dark phase for control mice (Table 1; Figure 1b,d).
Interestingly, when compared to the control, the PLX5622 group spent a higher amount of time in sleep (total and NREM) during the dark phase (Table 1, Figure 1a,d). Note that mice spent most of the sleep duration in the NREM phase (see Figure 1a,d versus b,e).
The latency of the sleep onset was shorter in the PLX5622 group both in light and dark phases (p < .05 corrected, Table 1). Detailed results of the analyses within and between groups are reported in Tables S1, S2, and S3.
Similar results for NREM sleep were obtained when EEG data were analyzed for longer periods (11 h in the light phase and 11 h in the dark phase), as shown in Figure S2. All the successive analyses were performed on shorter periods of 7 h.
To better understand the effects of microglial depletion on sleep, we performed analyses on circadian variables after PLX5622 treatment in 12 h light/12 h dark and in 12 h dark/12 h dark cycles. For these analyses, we took advantage of an innovative system, the digital ventilated cages (DVC), that automatically records motor activity for prolonged periods (24 h per day) and with minimal operator intervention, thus avoiding interfering with animal rhythms. Results shown in Figures S3 and S4 show that no overt variations of circadian variables were observed between control and PLX5622 treated mice. Figure S3 shows the comparison of average activity, 24 h activity pattern, acrophase, and diurnality between the control and PLX5622-treated mice along the light/dark cycle. To study the effect of microglial depletion on circadian time (CT), we investigated the effect of PLX5622 treatment in the dark/ dark experimental setting: Figure S4 demonstrates that no significant variation in the average activity, activity onset, and phase shift were observed upon PLX5622 treatment in mice housed in 12 h dark 12 h dark condition, indicating no effects on mouse inner CT.
Analysis of time spent in movement (Figure 1c,f) did not reveal significant differences among control and microglial-depleted mice, while total wakefulness phase was decreased both in light and dark periods (Table 1). Interestingly, comparable results on NREM sleep duration were obtained using another CSF1R antagonist, PLX3397, that partially depleted microglial cells (around 88%, data not shown) (Jin et al. 2017) with an independent set of analyses ( Figure S5 Figure S5B, from about 1% to 6%, possibly due to some experimental differences described in Supplementary Materials.

| Microglial depletion affects hippocampal synaptic transmission in a daily-phase dependent manner
In several brain regions, the number and the morphology of dendritic spines on neurons along with the expression levels of AMPA receptors can change across the light/dark cycle, resulting in an increased frequency and/or amplitude of the mEPSC during the dark phase (Vyazovskiy et al., 2008;Liu et al., 2010;Maret et al., 2011;Yang et al., 2014;Bridi et al., 2020). To investigate whether microglia play a role in the synaptic scaling of miniature events along the light- We then measured spontaneous synaptic activity performing the recordings in the absence of TTX, to better approximate the physiological condition. We found a main effect of phase and treatment on the sEPSC amplitude (F[1,38] = 6.959, p = .012; F(1,38) = 11.374, p = .002, respectively (Figure 2b, left). Post hoc analysis showed that at ZT16 the mean amplitude of sEPSC was higher (ZT16: 14.91 ± 1.59 pA, n/N = 10/4 cells; ZT4: 11.68 ± 0.82 pA, n/N = 10/4 cells, T A B L E 1 Time spent by each group in the different behavioral conditions. Mean ± SEM and percentages of the time (h) spent by each mouse group (i.e., C57BL/6, PLX5622, C57BL/6J, and CX3CR1) in the following behavioral conditions: sleep (non-rapid eye movement + rapid eyes movement [NREM + REM]), NREM, REM, wakefulness (movement + passive wake), movement, and passive wake durations. The mean values were calculated considering 7 h of the light phase (of the day) and 7 h of the dark phase, which were considered as representative of 24 h based on an exploratory analysis carried out at early stage of the study. In those hours, the 8-s epochs classified as "undefined" were very few (i.e., < 1 on 1000), so we did not consider them in reporting the percentages of the time spent in sleep and wakefulness. Asterisks in bold indicate the statistically significant differences between the experimental (i.e., PLX5622 or CX3CR1) and control (i.e., C57BL/6 or C57BL/6J) mouse groups (p < .05 uncorrected)  These results further indicate that microglia, in the dark phase, contribute to the modulation of glutamate release.
These results suggest that in basal condition LTP amplitude do not differ between ZT4 and ZT16. By contrast, microglia depletion uncovers an inhibitory role of microglia during the light phase, being the LTP level increased by PLX5622 treatment. 0.58 ± 0.14, n/N = 8/3, p = .17; ATP 10 μM: 0.85 ± 0.19; n/N = 8/3, p = .99; one-way ANOVA followed by Dunn's post hoc test). We also investigated CX3CR1 receptor expression upon extracellular ATP stimulation in cultured microglial cells by FACS analysis. Figure 3c shows that ATP reduces the expression of CX3CR1, confirming the reduction of cx3cr1 mRNA expression (C: 7822 ± 171 vs. ATP 1 mM: 7029 ± 83, p = .006; ATP 100 μM: 8043 ± 144; n/N = 3/3 per condition; p = .27, one-way ANOVA followed by Holm-Sidak post hoc test). LPS was again used as positive control (LPS 100 ng/ml: 5745 ± 122, n = 3/3; C vs. LPS, p < .001). Moreover, we confirmed the reduction of cx3cr1 expression due to ATP stimulation on microglial cells obtained from adult mice, as shown in Figure 3d (C: 1 ± 0.1; ATP 100 μM: 0.28 ± 0.07; n/N = 3/3; p < .001, Student's t-test). These results led us to speculate that the increased levels of ATP present in the brain in the first hours of sleep, due to reduced use of energy during the NREM phase (Dworak et al., 2010), could reduce cx3cr1 expression in microglia, thus affecting their response to neuronal CX3CL1 signaling.

| CX3CR1-CX3CL1 signaling underlies microglia effects along the light/dark cycle
To investigate the role played by CX3CR1 signaling in mediating microglial effects on the light/dark cycle, we extended our study to the cx3cr1 GFP/GFP mice. For these experiments, C57BL/6J mice were used as control, due to the similarity in the genetic background.
Altogether, these data indicate that cx3cr1 participates in regulating the phase-dependent microglial alterations.

| DISCUSSION
In this study, we investigated the role of microglia in the changes associated to sleep/wake phases throughout the 12:12 light/dark cycle, focusing on the modulation of hippocampal synaptic functions and on behavioral states upon microglial depletion with CSF-1R inhibitor. We demonstrate for the first time that the microglial depletion with PLX5622 increases the time spent by mice in the NREM sleep during the dark phase. Upon microglia depletion, we also reported a reduction in the hippocampal excitatory neurotransmission in the dark and an increased long-term potentiation during the light phase indicating that microglia cells play a role in shaping neuronal functions along the daily cycle.
In line with previous data reporting post-synaptic changes occurring during wakefulness, such as increased levels of hippocampal AMPA receptor subunits and ultra structural spine modifications (Vyazovskiy et al., 2008Liu et al., 2010Yang et al., 2014;de Vivo et al., 2017), we found an increased amplitude of both miniature and sEPSC events recorded in the CA1 hippocampal region of mice at ZT16. In addition, at ZT16, we measured an increase in sEPSC frequency and a parallel reduction in the PPR, indicating that also presynaptic changes occur along the daily cycle that can be ascribed to modification of the probability of glutamate release (Zucker, 1989).
Overall, our data support the idea of a net synaptic potentiation taking place during wakefulness in the hippocampus, in accordance with the synaptic homeostasis hypothesis (SHY) that proposes sleep as a period necessary to maintain a proper control of synaptic strength in the brain (Tononi and Cirelli, 2003).
Our data that microglial depletion with PLX5622 reduced the amplitude of both mEPSC and sEPSC at ZT16, abolishing the phasedependent differences in basal synaptic transmission, suggest that microglial cells predominantly control the synaptic strength during the wake phase. Previous studies demonstrated that the direct contact between microglial processes and synapses in awake mice reflects enhanced synaptic activity and that partial microglial ablation decreased the synchronization of evoked neural activity (Akiyoshi et al., 2018). In accordance with our findings, microglial depletion in rats has been associated with decreased total hippocampal synaptic transmission and impaired cognitive function (Yegla et al., 2021). On the other hand, it has been also reported that chronic microglial depletion increases both excitatory and inhibitory synaptic connections to excitatory cortical neurons (Liu et al., 2021). This evidence, together, concurs to highlight a prominent role of microglia in sculpting neuronal circuit connectivity and regulating subsequent functional activity in a region-specific manner.
Microglial depletion with PLX5622 is an efficient and reversible event (Elmore et al., 2014;Dagher et al., 2015) but recent evidence showing that, in addition to depleting microglia, PLX5622 also induces long-term changes in the peripheral myeloid and lymphoid cells must be considered (Lei et al., 2020). Furthermore, it has been shown that not the entire population of microglia responds to CSF-1R antagonists and a small percentage persists with depletion, which could be responsible for some of the observed effects (Elmore et al., 2014).The fractalkine receptor CX3CR1 has a key role in mediating the communication among neurons and microglial cells, in physiological and pathological conditions Limatola and Ransohoff, 2014).
We report that, similarly to what observed upon microglial depletion, cx3cr1 GFP/GFP mice had no phase-dependent differences in sEPSC amplitude, indicating that the fractalkine/CX3CR1 signaling between neurons and microglia is necessary for the wake-dependent increase of excitatory synaptic transmission. Similarly, we observed a reduction in the frequency of sEPSC and a corresponding increase of the PPR at ZT16 both in microglial-depleted and in cx3cr1 GFP/GFP mice, suggesting that, during wake, microglia positively modulate presynaptic glutamate release probably trough the CX3CR1 signaling.
Several evidence demonstrated that sleep is relevant for initial memory encoding and subsequent long-term memory consolidation, exerting effects on molecular, cellular and network mechanisms of plasticity (Abel et al., 2013, Klinzing et al. 2019. Hippocampal synaptic plasticity may vary with the time of day: diurnal variations in the incidence and magnitude of LTP in the CA1 region have been reported, with the largest responses occurring in the day (Harris and Teyler, 1983;Raghavan et al., 1999) or in the night (Chaudhury et al. 2005, Jilg et al., 2019Dana and Martinez Jr, 1984;Leung et al., 2003). The variability of these results can be related to the different experimental conditions, such as stimulation protocol and animal model used, but also to the strong dependence of plasticity to the phase of sleep (NREM vs. REM) which are difficult to assess in ex vivo preparations. For these reasons, our results on the alterations of hippocampal LTP upon microglial alterations in the dark and light phase must be interpreted considering these experimental limitations. In control conditions, we report no differences in LTP magnitude at ZT4 and ZT16. However, both microglial depletion and CX3CR1 deletion, induce an increase of LTP amplitude at ZT4, as previously reported . It can be speculated that interfering with microglia during this period affects the production of cytokines such as interleukin-1 and tumor necrosis factor-α which have been associated to NREM induction (Hide et al., 2000;Shieh et al., 2014) and LTP inhibition (Prieto et al., 2019). Although further experiments are necessary to finely define microglial function on long term synaptic plasticity along the circadian cycle, our data demonstrate that microglia differentially affect LTP and synaptic strength in the light/ dark phases.
Previous data demonstrated that hampering microglial activities with minocycline in aged rats (Griffin et al., 2006) or impairing fractalkine signaling in the cx3cr1 knockout mice Milior et al., 2016) potentiated the hippocampal synaptic plasticity.
Together, these data indicate that the phase-dependent microglial effects on hippocampal neurotransmission are mediated via the expression of cx3cr1. We observe that the depletion of microglia did not affect REM sleep. Even if we cannot provide experimental explanations for this specific effect of microglial cell depletion, we could speculate that microglial cells mostly impact on circuits involved in wake during the active phase, such as the hypothalamic orexin and MHC neurons (Lee et al., 2005;Hassani et al., 2009). Further experiments are needed to better understand these differences.
We hypothesize that the reduction of cx3cr1 expression in microglial cells isolated from three different brain regions of mice at ZT4 could be explained by a direct effect of ATP accumulated in the brain during sleep (Dworak et al., 2010;Krueger et al., 2010;Marpegan et al., 2011). This hypothesis is supported by data showing that, in vitro, ATP administration decreases cx3cr1 expression in microglia.
The accumulation of ATP during sleep has been associated with the need to replenish the brain energy sources (Burkeen et al., 2011). ATP is also released during neuronal activity and it is a recognized danger signal for brain parenchyma (Rodrigues et al., 2015). Extracellular ATP can reach high local concentrations, especially at regions of cell-to-cell contact (Pankratov et al., 2006), but it must be considered that ATP is rapidly degraded with the production of adenosine, which is a very well described sleep regulator (Porkka-Heiskanen et al., 1997;Stenberg et al., 2003). A recent report describes an ATP-dependent regulation of adenosine-mediated microglial depression of neuronal firing (Badimon et al., 2020). We hypothesize that the ATP-induced reduction of cx3cr1 expression during the light phase represents the activation of coordinated communication mechanisms among astrocytes-microglia and neuronal cells to re-tune synaptic activities and the strength of circuitry in the different day phases to allow for learning, memory and environmental adaptation.
We show that PLX5622 treatment had minor effects on motor activity and only during the light phase. Previous studies reported that microglial depletion did not alter behavior, locomotor, or cognitive activity in mice, but improved cognition in the 3xTg-AD mouse model of Alzheimer's disease and in model of cranial irradiation (Elmore et al., 2014;Dagher et al., 2015;Acharya et al., 2016). Other studies report that altered cerebellar CSF-1R signaling affects motor learning and social interaction (Kana et al., 2019). The different outcomes on motor functions could be explained by the different experimental conditions and by the long-versus short-term analysis performed to detect alterations of basal motor activities.
In this study, we did not observe specific effects of microglial depletion on circadian internal rhythms, as demonstrated by the absence of alteration of specific behavioral parameters such as the mean activity, the activity onset, and the phase shift in a dark/dark housing condition. This led us to speculate that the effects of microglial depletion on total sleep and NREM sleep duration are due to modulation of homeostatic processes involved in the control of the sleep/wake phase rather that circadian mechanisms.
To conclude, several studies investigating the role of microglial cells in the sleep/wake cycle focused on the effects induced by conditions of sleep deprivation, with different experimental approaches, from gentle handling to forced stimulation of laboratory animals Wadhwa et al., 2017;Tuan and Lee 2019;Hall et al., 2020). Keeping animals awake involves the activation of the hypothalamic-pituitary-adrenal axis, thus contributing to different levels of stress; this must be considered as additional variable together with sleep loss to decipher the observed effects (Meerlo et al., 2002;Havekes and Aton, 2020). In this study, we demonstrate that microglia participate in the regulation of sleep/wake cycle and synaptic transmission in a phase-dependent manner. Previous studies reported microglial alteration upon chronic sleep deprivation protocols , hypothesizing that prolonged sleep loss, priming microglial cells, could increase the brain susceptibility induced by a secondary insult. The variations in cx3cr1 expression across different zeitgeber times suggest that the functional interactions of microglia with hippocampal synapses vary along the light/dark cycle.
The observation that the lack of cx3cr1 parallels the depletion of microglia on sleep and synaptic transmission strongly indicates that the CX3CR1/CX3CL1 signaling pathway is crucial for the homeostatic role of microglia in the sleep/wake cycle. Our data led us to speculate that microglial cells, through CX3CR1 signaling, participate in reshaping the glutamatergic synaptic transmission with pre-and postsynaptic mechanisms, contributing to the synaptic up scaling taking place during wake. Further investigations will be necessary to understand how CX3CR1 mediates these effects.
Additional studies targeting the sleep pressure and the circadian processes will be also necessary to clarify the detailed neurophysiological mechanisms underlying these effects.