Microglia play a critical role in the neuroimmune response, but little is known about the role of microglia in sleep following an inflammatory trigger. Nevertheless, decades of research have been predicated on the assumption that an inflammatory trigger increases sleep through microglial activation. We hypothesized that mice (n = 30) with depleted microglia would sleep less following administration of lipopolysaccharide (LPS) to induce inflammation. Brains were collected and microglial morphology was assessed using quantitative skeletal analyses, and physiological parameters were recorded using non-invasive piezoelectric cages. Mice fed PLX5622 (PLX) diet for 3 weeks had a transient increase in sleep that dissipated by week 2. Subsequently, following a first LPS injection (0.4 mg/kg), mice with depleted microglia slept more than mice on control diet. All mice were returned to normal rodent chow to repopulate microglia in the PLX group (10 days). Nominal differences in sleep existed during the microglia repopulation period. However, following a second LPS injection, mice with repopulated microglia slept similarly to control mice during the dark period but with longer bouts during the light period. Comparing sleep after the first LPS injection to sleep after the second LPS injection, controls exhibited temporal changes in sleep patterns with no change in cumulative minutes slept, whereas in mice with repopulated microglia cumulative sleep decreased during the dark period across all days. Microglia repopulated after PLX also had a reactive morphology with fewer branch endpoints per cell. We conclude that microglia are necessary to regulate sleep after an immune challenge.

Adult (8-10 weeks-old) male C57BL/6J mice (20-25 g, Jackson Laboratories, Bar Harbor, ME) were used for all experiments (n = 30). Mice were singly housed and were maintained on a 14 hour light (200 lux, cool white fluorescent light; zeitgeber time [ZT] 0): 10 hour dark (ZT 14) cycle at an ambient temperature of 23°C ± 2°C. Mice were acclimated to non-invasive piezoelectric sleep cages and fed a normal diet of standard rodent chow for 7 days. Following the acclimation period, mice were randomly assigned to control diet (AIN-76A) or PLX5622 (PLX) diet (1200 mg/kg) formulated in AIN-76A rodent chow for a 21-day microglia depletion period. Mice continued to receive control or PLX diet and were subjected to an inflammatory challenge using lipopolysaccharide (LPS; E. coli 0111:B4, Sigma-Aldrich). LPS was made as a stock solution in sterile saline (0.9%) and injected intraperitoneally (i.p.) at 0.4 mg/kg in a volume of 0.05 ml between ZT3 and ZT4 (first LPS administration; LPS1). At 4 days post-LPS injection, all mice were switched back to a normal diet to allow repopulation of microglia in the PLX group. Following a 10-day repopulation period, mice were subjected to a second LPS injection (LPS2) and all mice remained on normal diet until tissue was collected 7 days after the second LPS injection. Throughout the study, access to food and water remained ad libitum. Following LPS1, 1 mouse died, and 2 mice were euthanized for excessive weight loss and dehydration. Following LPS2, 1 mouse died. There was a technical error in tissue collection that resulted in one brain not being processed for immunohistochemistry and microglial analyses. Final sample sizes for each experiment are included in the figure legends.