DOI: 10.5061/dryad.tdz08kq2m
title: Data from: Microglia are necessary to regulate sleep after an immune challenge

description: 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.
description: 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;
 <em>E. coli</em> 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.