Sleep-wake behavior and responses to sleep deprivation and immune challenge of protein kinase RNA-activated knockout mice

Abstract

Protein Kinase RNA-activated (PKR) is an enzyme that plays a role in many systemic processes, including modulation of inflammation, and is implicated in neurodegenerative diseases, such as Alzheimer's disease (AD). PKR phosphorylation results in the production of several cytokines involved in the regulation / modulation of sleep, including interleukin-1β, tumor necrosis factor-α and interferon-γ. We hypothesized targeting PKR would alter spontaneous sleep of mice, attenuate responses to sleep deprivation, and inhibit responses to immune challenge. To test these hypotheses, we determined the sleep-wake phenotype of mice lacking PKR (knockout; PKR^-/-) during undisturbed baseline conditions; in responses to six hours of sleep deprivation; and after immune challenge with lipopolysaccharide (LPS). Adult male mice (C57BL/6J, n = 7; PKR^-/-, n = 7) were surgically instrumented with EEG recording electrodes and an intraperitoneal microchip to record core body temperature. During undisturbed baseline conditions, PKR ^-/- mice spent more time in non-rapid eye movement sleep (NREMS) and rapid-eye movement sleep (REMS), and less time awake at the beginning of the dark period of the light:dark cycle. Delta power during NREMS, a measure of sleep depth, was less in PKR^-/- mice during the dark period, and core body temperatures were lower during the light period. Both mouse strains responded to sleep deprivation with increased NREMS and REMS, although these changes did not differ substantively between strains. The initial increase in delta power during NREMS after sleep deprivation was greater in PKR^-/- mice, suggesting a faster buildup of sleep pressure with prolonged waking. Immune challenge with LPS increased NREMS and inhibited REMS to the same extent in both mouse strains, whereas the initial LPS-induced suppression of delta power during NREMS was greater in PKR^-/- mice. Because sleep regulatory and immune responsive systems in brain are redundant and overlapping, other mediators and signaling pathways in addition to PKR are involved in the responses to acute sleep deprivation and LPS immune challenge.

Keywords: Alzheimer’s disease; Cytokines; Inflammation; Lipopolysaccharide; Neurodegeneration; PKR; Sleep; Sleep deprivation.

Fig. 1.

Experimental Protocol. After surgical recovery and habituation, C57BL/6J mice and PKR knockout (PKR^−/−) mice were enrolled into a six-day recording protocol. Baseline recordings from undisturbed mice started at light onset and continued for 48 h. After baseline recordings, all mice were sleep deprived for six hours beginning at light onset and then left undisturbed for an 18-h recovery sleep opportunity. All mice then were injected intraperitoneally with vehicle (PFS, pyrogenfree saline) at dark onset of protocol day four and with lipopolysaccharide (LPS) 24 h later, at dark onset of protocol day five. Recordings continued for the 24-h period following LPS administration, after which the protocol ended. In this protocol each animal served as its own control. (Figure created in Biorender.com).

Fig. 2.

PKR knockout mice exhibit a diurnal pattern of wakefulness, non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS) and core body temperature. Data are expressed as mean ± SEM percentage of recording time obtained from C57BL/6J mice (open symbols; n = 7) and PKR knockout mice (PKR^−/−, filled symbols; n = 6) during undisturbed baseline recordings. All sleep parameters are presented as hourly values whereas core body temperature is depicted in 30 min intervals. Statistical analyses were performed on sequential 3-h time blocks. Sample sizes for core body temperature measures are n = 6 (C57BL/6J) and n = 5 (PKR^−/−) due to failed temperature transponders. Statistically significant differences between C57BL/6Jmice and PKR^−/− mice are depicted as: *, p < 0.05; **, p < 0.01; *** p < 0.001). The shaded portion of each panel represents the dark period of the light:dark cycle.

Fig. 3.

Sleep deprivation reduces subsequent wakefulness and increases non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) in mice. Data are the mean ± SEM from C57BL/6J mice (left panels, n = 7) and PKR knockout mice (PKR^−/−; right panels, n = 6) mice during undisturbed baseline recordings (open symbols) and after sleep deprivation (filled symbols). All sleep parameters are presented as hourly values whereas core body temperature is depicted in 30 min intervals. Data for sleep parameters and core body temperature are not shown during the 6-h sleep deprivation period. Statistical analyses were performed on sequential 3-h time blocks. Sample sizes for core body temperature measures are n = 6 (C57BL/6J) and n = 5 (PKR^−/−) due to failed temperature transponders. Statistically significant differences between baseline and sleep deprivation are depicted as: *, p < 0.05; **, p < 0.01. The shaded portion of each panel represents the dark period of the light:dark cycle. BL=baseline undisturbed recordings; SD=sleep deprivation.

Fig. 4.

Loss of PKR function alters some aspects of sleep-wake responses to sleep deprivation. To determine the effect of genotype on responses to sleep deprivation, difference scores were calculated for each mouse for each hour by subtracting control values (undisturbed baseline) from experimental values obtained after sleep deprivation. Data are expressed as mean ± SEM from C57BL/6J mice (open symbols, n = 7) and PKR knockout mice (PKR^−/−; filled symbols, n = 6) during undisturbed baseline and after 6-h of sleep deprivation. All sleep parameters are presented as hourly values whereas core body temperature is depicted in 30 min intervals. Data are not depicted for the 6-h period of sleep deprivation. Statistical analyses were performed on sequential 3-h time blocks. Sample sizes for core body temperature measures are n = 6 (C57BL/6J) and n = 5 (PKR^−/−) due to failed temperature transponders. Statistically significant differences between mouse strains are: *, p < 0.05; **, p < 0.01. The shaded portion of each panel represents the dark period of the light:dark cycle.

Fig. 5.

Lipopolysaccharide increases non-rapid eye movement sleep (NREMS), reduces wakefulness and rapid eye movement sleep (REMS) and alters core body temperature of mice. Data are expressed as mean ± SEM from C57BL/6J mice (left panels) and PKR knockout mice (PKR^−/−; right panels, n = 6) after administration of vehicle (pyrogen-free saline; open symbols) and lipopolysaccharide (LPS; filled symbols). All sleep parameters are presented as hourly values whereas core body temperature is depicted in 30 min intervals. Statistical analyses were performed on sequential 3-h time blocks. Sample sizes for core body temperature measures are n = 6 (C57BL/6J) and n = 5 (PKR^−/−) due to failed temperature transponders. Statistically significant differences between mouse strains are: *, p < 0.05; **, p < 0.01; ***, p < 0.001 The shaded portion of each panel represents the dark period of the light:dark cycle.

Fig. 6.

Loss of PKR function reduces the impact of lipopolysaccharide on some facets of sleep-wake behavior and core body temperature. Data are expressed as mean ± SEM from C57BL/6J mice (open symbols, n = 7; core body temperature n = 7, thin line) and PKR knockout mice (PKR^−/−; filled symbols, n = 6; core body temperature n = 6, thick line). All sleep parameters are presented as hourly values whereas core body temperature is depicted in 30 min intervals. Statistical analyses were performed on sequential 3-h time blocks. Sample sizes for core body temperature measures are n = 6 (C57BL/6J) and n = 5 (PKR^−/−) due to failed temperature transponders. Statistically significant differences between mouse strains are: *, p < 0.05; **, p < 0.01; ***, p < 0.001. The shaded portion of each panel represents the dark period of the light:dark cycle.

Fig. 7.

Loss of PKR function does not alter lipopolysaccharide-induced changes in pro- and anti-inflammatory cytokines. Plasma concentrations of pro-inflammatory (IL-1β, IL-6, TNFα, IFNγ) and anti-inflammatory (IL-4, IL-10) cytokines following intraperitoneal administration of lipopolysaccharide (LPS; 0.4 mg/kg) did not differ between mouse strains. (C57BL/6J; n = 10; open bars; PKR^−/−; n = 7; filled bars).