Thesis title: Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle
The internal clock in our brain is modulated by the light/dark cycle caused by Earth’s rotation around its axis. In turn, organisms undergo cycles of alertness and sleepiness regulated by the circadian rhythms. The role of glial cells in the modulation of the circadian clock has recently become a matter of intense research (Ingiosi et al., 2013; Garofalo et al., 2020). Different studies show how astrocytes are important contributor to synaptic homeostasis during the sleep/wake cycle, mainly through the IP3/Ca2+ signalling 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). However, key roles have been suggested also for microglia during normal sleep and upon sleep disturbances (Bellesi et al., 2017; Choudhury et al., 2020) as well as in neural plasticity processes associated with synaptic remodelling 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 show 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, 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-ZT24). We investigated the role of microglial cells by depletion studies, taking advantage of the role exerted by colony stimulating factor-1 receptor (CSF-1R) signalling 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). In the present work, we treated mice with the CSF-1R inhibitor PLX5622, which drastically reduces the density of microglial cells in different brain regions, and analysed animals for the time spent in sleep or active states during the light and dark phases of the day. Mice were also analysed for the effect of microglial depletion on synaptic activity and 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 behavioural 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: i) abolishing the differences in sEPSC amplitude and frequency between the dark and light phases and ii) 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 and synapsis maturation (Paolicelli et al., 2011; Bolòs 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 signalling, the sleep/wake cycle and the excitatory synaptic transmission were also investigated in cx3cr1GFP/GFP mice, where is deleted (Jung et al., 2000), with outcomes similar to microglia-depleted 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, further suggesting that CX3CR1 plays a central role in determining 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 happening between the wake and sleep phases, disclosing also a key role for CX3CR1 in sleep-wake cycle.