Selective contributions of neuronal and astroglial interleukin-1 receptor 1 to the regulation of sleep

Interactions between sleep and immune function are bidirectional. Although the mechanisms that govern these interactions are not fully elucidated, the pro-inflammatory cytokine, interleukin-1β (IL-1), is a known regulator of sleep and mediator of immune responses. To further clarify the underlying substrates of sleep and immune interactions, we engineered two transgenic mouse lines that express interleukin-1 receptor 1 (IL1R1) only in the central nervous system (CNS) and selectively on neurons (NSE-IL1R1) or astrocytes (GFAP-IL1R1). During spontaneous sleep, compared to wild type (WT) animals, NSE-IL1R1 and GFAP-IL1R1 mice have more rapid eye movement sleep (REMS) that is characterized by reduced theta power in the electroencephalogram (EEG) spectra. The non-REM sleep (NREMS) EEG of each of the IL1R1 transgenic mouse strains also is characterized by enhanced power in the delta frequency band. In response to 6 h of sleep deprivation, sleep of both IL1R1 transgenic mouse strains is more consolidated than that of WT animals. Additionally, the NREMS EEG of NSE-IL1R1 mice contains less delta power after sleep deprivation, suggesting astroglial IL1R1 activity may modulate sleep homeostasis. Intracerebroventricular injection of IL-1 fails to alter sleep or brain temperature of NSE-IL1R1 or GFAP-IL1R1 mice. These data suggest that selective IL1R1 expression on neurons or on astrocytes is not sufficient for centrally-administered IL-1 to induce sleep or fever. Lack of sleep and febrile responses to IL-1 in these IL1R1 transgenic mouse strains may be due to their inability to produce IL-6 in brain. Overall, these studies demonstrate, through the use of novel transgenic mice, that IL1R1 on neurons and astrocytes differentially mediates aspects of sleep under physiological conditions and in response to central IL-1 administration.     

Nitric Oxide Donors SIN-1 and SNAP Promote Nonrapid-Eye-Movement Sleep in Rats

We previously showed that inhibition of brain NO production suppresses sleep in rats and rabbits. In the present experiments we studied the effects of stimulation of NO-receptive brain mechanisms on sleep. Male rats were injected intracerebroventricularly with the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 400 μg) or molsidomine (SIN-1, 7 and 70 μg). Seven micrograms of SIN-1 did not affect sleep, but increased the delta wave activity of the electroencephalogram (EEG) during nonrapid-eye-movement sleep (NREMS) and suppressed EEG alpha and beta activities in NREMS and delta, theta, and beta activities during wakefulness. Seventy micrograms of SIN-1 significantly increased NREMS after a latency of ≈9 h. EEG power was suppressed in each frequency band during rapid-eye-movement sleep (REMS) and wakefulness, whereas during NREMS, delta activities were increased after the injection of 7 μg SIN-1, and higher frequencies were suppressed after both doses. On the recovery day sleep remained elevated, but EEG power returned to baseline. The effects of SNAP on NREMS were similar to those of SIN-1, but REMS was decreased and slight increases in brain temperature accompanied the sleep changes. The EEG theta, alpha, and beta activities were suppressed in both wakefulness and REMS. Collectively, these results are consistent with the hypothesis that NO plays a role in the regulation of vigilance.     

Sleep in spontaneous dwarf rats

Spontaneous dwarf rats (SDRs) display growth hormone (GH) deficiency due to a mutation in the GH gene. This study investigated sleep in SDRs and their somatotropic axis and compared to Sprague-Dawley rats. SDRs had almost undetectable levels of plasma GH. Hypothalamic GH-releasing hormone (GHRH) mRNA was increased, whereas GHRH-receptor (GHRH-R) and somatostatin mRNAs were decreased in SDRs. Hypothalamic GHRH and somatostatin peptide content decreased in SDRs. Quantitative immunohistochemistry for GHRH and GHRH-R corroborated and extended these findings. In the arcuate nucleus, the number of GHRH-positive cells was significantly higher, whereas GHRH-R-positive perikarya were diminished in SDRs. Cortical GHRH and GHRH-R measurements showed similar expression characteristics as those found in the hypothalamus. SDRs had less rapid eye movement sleep (REMS) and more non-REMS (NREMS) than the control rats during the light period. The electroencephalogram (EEG) delta and theta power decreased during NREMS in the SDRs. After 4-h of sleep deprivation, SDRs had a significantly reduced REMS rebound compared to the controls, whereas NREMS rebound was normal in SDRs. The enhancement in delta power was significantly less than in the control group during recovery sleep. Intracerebroventricular (icv) administration of GHRH promoted NREMS in both strains of rats; however, increased REMS and EEG delta activity was observed only in control rats. Icv injection of insulin-like growth factor 1 increased NREMS in control rats, but not in the SDRs. These results support the ideas that GHRH is involved in NREMS regulation and that GH is involved in the regulation of REMS and in EEG slow wave activity regulation during NREMS.     

Sleep-wake behavior and responses of interleukin-6-deficient mice to sleep deprivation

Interleukin (IL)-1 and tumor necrosis factor (TNF) are involved in the regulation of non-rapid eye movements sleep (NREMS). Accumulating evidence suggests IL-6 modulates sleep under some pathophysiologic conditions. We used mice lacking a functional IL-6 gene to investigate further a potential role for IL-6 in the regulation of sleep. IL-6 knockout mice (B6.129S6-Il6tm1Kopf; n=10) and C57BL/6J mice (n=10) were purchased from the Jackson Laboratory (Bar Harbor, ME). Twenty-four-hour baseline recordings were obtained from mice in the absence of any experimental manipulation. Mice were then subjected to 6-h sleep deprivation beginning at light onset. Recordings were obtained during the deprivation period and for 18 h thereafter. During baseline conditions there were no differences between mouse strains with respect to the duration, timing or intensity of NREMS. However, across the 24-h recording period IL-6 knockout mice spent approximately 30% more time in rapid eye movements sleep (REMS) than did C57BL/6J mice. Relative to C57BL/6J mice, core body temperatures of IL-6 knockout mice were higher during the light period of the light:dark cycle. Both strains responded to sleep deprivation by spending more time in NREMS and REMS. Although the total increase in the amount of NREMS after sleep deprivation was the same in both strains, IL-6 knockout mice took 6h longer to accumulate this additional sleep. Under the conditions of this study, IL-6 does not appear necessary for the full manifestation of NREMS, although this cytokine may influence the dynamics of responses to sleep deprivation. That mice lacking IL-6 spend more time in REMS suggests that interactions between IL-6 and REMS regulatory mechanisms may differ from those of IL-1 and/or TNF.     

Induction of Mutant Sik3Sleepy Allele in Neurons in Late Infancy Increases Sleep Need

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3^Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3^Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1^CreERT2 and Sik3^ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1^CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1^CreERT2; Sik3^ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3^Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.     

Restoring the Molecular Clockwork within the Suprachiasmatic Hypothalamus of an Otherwise Clockless Mouse Enables Circadian Phasing and Stabilization of Sleep-Wake Cycles and Reverses Memory Deficits

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2-null controls expressing EGFP (SCN^Con) was arrhythmic, whereas Cry1-complemented mice (SCN^Cry1) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCN^Con mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCN^Cry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS δ power and expression of REMS within total sleep (TS). Wakefulness in SCN^Con mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCN^Cry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS δ power, although the SCN^Con mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCN^Cry1 mice were comparable to WTs. Finally, SCN^Con mice exhibited poor sleep-dependent memory but this was corrected in SCN^Cry1mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep.SIGNIFICANCE STATEMENT The circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but the presence of multiple local brain and peripheral clocks mean the respective roles of SCN and other clocks in regulating sleep are unclear. We therefore used virally mediated genetic complementation to restore molecular circadian functions in the suprachiasmatic hypothalamus, focusing on the SCN, in otherwise genetically clockless, arrhythmic mice. This initiated circadian activity-rest cycles, and circadian sleep-wake cycles, circadian patterning to the intensity of non-rapid eye movement sleep (NREMS) and circadian control of REMS as a proportion of total sleep (TS). Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the suprachiasmatic hypothalamus, alone, can direct circadian regulation of sleep-wake.     

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1β or an IL-1β fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ. Cells were also tested under identical conditions with an IL-1β synthetic peptide fragment (IL-1β_208–240) previously shown to possess biological activity. IL-1β, TNFα and IFNγ potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1β_208–240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1β, rabbit IL-1β_208–240 and human IL-1β_208–240 was blocked by the IL-1ra, while TNFα- and IFNγ-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1β and human IL-1β_208–240 was also blocked by the sIL-1R. Our findings suggest that IL1β_208–240 acts as an IL-1β agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.     

Intermittent hypoxia causes REM sleep deficits and decreases EEG delta power in NREM sleep in the C57BL/6J mouse

BACKGROUND AND PURPOSE: Obstructive sleep apnea (OSA) severely impairs sleep architecture. We hypothesized that both intermittent hypoxia (IH) and non-hypoxic arousals of OSA result in significant disruption of non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS). PATIENTS AND METHODS: Polysomnography was performed in C57BL/6J mice (n=5) exposed to IH (cycling of FIO2 from 20.9 to 5.0%) or sleep fragmentation (SF: high flow air blasts) throughout the 12-h light phase over 5 consecutive days. RESULTS: Both IH and SF induced arousals from sleep. On Day 1 of exposure, total NREMS during the light phase decreased comparably during IH (44.1+/-7.8%/12h, P<0.05) and SF (43.7+/-3.3%/12h, P<0.05) but returned to baseline levels of 62.0+/-7.8%/12h by Day 5 of exposure under both conditions. During IH, however, the electroencephalographic (EEG) delta power of NREMS remained impaired throughout the 5-day period of IH with a nadir of 65.4+/-5.6% relative to baseline (P=0.01), and REMS was effectively abolished during the light phase. In contrast, SF did not cause a significant reduction in either EEG delta power or REMS during the light phase. CONCLUSIONS: Thus, hypoxic exposure, but not arousals, caused overall deficits in the EEG delta power of NREMS and marked deficits in the total amount of REMS. We propose that hypoxic arousals may have a more severe impact on sleep architecture in patients with OSA than non-hypoxic arousals.     

The time course of sigma activity and slow-wave activity during NREMS in cortical and thalamic EEG of the cat during baseline and after 12 hours of wakefulness.

The extrapolation from recent neurophysiological findings concerning the dependency of spindle and slow-wave oscillations of thalamocortical neurons on membrane potential to macroscopic EEG events, predicts a reciprocal relation between spindle activity and slow-wave activity (SWA) in thalamic and cortical EEG during non-rapid-eye-movement sleep (NREMS). To test this hypothesis, the EEG recorded in 8 cats, from the nucleus centralis lateralis of the thalamus and from the skull during a 12-h baseline dark period and during a 12-h recovery dark period, following a 12-h sleep deprivation, were analyzed. Per 12-s epoch, sleep-wake behaviour was determined and spectral power density was computed in the slow-wave frequency range (0.5-4.0 Hz) and in the spindle frequency region (sigma activity: 11.0-14.5 Hz). To analyze the development of EEG power densities in the course of NREMS and during the transition from NREMS to REMS, the last epoch of wakefulness and the first 15 epochs of NREMS, as well as the last epochs of NREMS and the first epoch of REMS were selected from the NREM-REM cycles. For each animal the values were averaged over 4-h intervals. In the cortical EEG, SWA was minimal at NREMS onset and increased progressively in the course of NREMS. SWA declined sharply prior to REMS. sigma Activity increased gradually towards a uniform level after NREMS onset. During the transition to REMS, sigma activity initially increased and then decreased rapidly. In the thalamic EEG, the time course of SWA paralleled that of the cortex. However, the development of sigma activity during the first part of NREMS differed: in the thalamic EEG, sigma activity was maximal during the beginning of NREMS and slightly decreased thereafter. After sleep deprivation, SWA within NREMS was markedly enhanced in both the cortical and the thalamic EEG. Sigma activity was attenuated in the thalamic EEG, whereas in the cortical EEG it was temporarily elevated. The present data show that, in the thalamic EEG, an inverse relation exists between spindle and slow-wave activity during baseline NREMS. This relation is preserved after sleep deprivation. In the cortical EEG, a reciprocal relation between spindling and SWA is less evident.     

The neuron-specific interleukin-1 receptor accessory protein is required for homeostatic sleep and sleep responses to influenza viral challenge in mice

Interleukin-1β (IL1) is involved in sleep regulation and sleep responses induced by influenza virus. The IL1 receptor accessory protein (AcP) and an alternatively spliced isoform of AcP found primarily in neurons, AcPb, form part of the IL1 signaling complex. IL1-induced sleep responses depend on injection time. In rat cortex, both IL1 mRNA and AcPb mRNA peak at Zeitgeber Time (ZT) 0 then decline over the daylight hours. Sleep deprivation enhances cortical IL1 mRNA and AcPb mRNA levels, but not AcP mRNA. We used wild type (WT) and AcPb knockout (KO) mice and performed sleep deprivation between ZT10 and 20 or between ZT22 and 8 based on the time of day expression profiles of AcPb and IL1. We hypothesized that the magnitude of the responses to sleep loss would be strain- and time of day-dependent. In WT mice, NREMS and REMS rebounds occurred regardless of when they were deprived of sleep. In contrast, when AcPbKO mice were sleep deprived from ZT10 to 20 NREMS and REMS rebounds were absent. The AcPbKO mice expressed sleep rebound if sleep loss occurred from ZT22 to 8 although the NREMS responses were not as robust as those that occurred in WT mice. We also challenged mice with intranasal H1N1 influenza virus. WT mice exhibited the expected enhanced sleep responses. In contrast, the AcPbKO mice had less sleep after influenza challenge compared to their own baseline values and compared to WT mice. Body temperature and locomotor activity responses after viral challenge were lower and mortality was higher in AcPbKO than in WT mice. We conclude that neuron-specific AcPb plays a critical role in host defenses and sleep homeostasis.     

Short light-dark cycles influence sleep stages and EEG power spectra in the rat

To investigate the influence of light on sleep and the electroencephalogram (EEG), chronically implanted rats were continuously recorded during a baseline day under 12-h light-12-h dark (LD 12:12) conditions, and an experimental day with short LD (LD 1:1) cycles. The percentage of non-REM sleep (NREMS) was higher and the percentage of REM sleep (REMS) lower in the 1-h light [corrected] intervals than in the 1-h dark intervals. The maximum of NREMS induction by 1-h light occurred in the habitual 12-h dark period (activity period), while the largest enhancement of REMS by 1-h darkness occurred in the second half of the habitual 12-h light period (rest period). The EEG of waking, NREMS and REMS was subjected to spectral analysis to determine the power density of the frequency components in the range of 0.25-25.0 Hz. The overall 24-h time course of the EEG-spectra in NREMS was similar under baseline and experimental conditions. Nevertheless, the spectra were modified by the short LD-cycle. In NREMS, the values in the middle and high frequencies (greater than 6 Hz in the rest period; greater than 11 Hz in the activity period) were lower in the 1-h light intervals than in the 1-h dark intervals. In contrast, activity in some frequency bands during waking and REMS was higher in the light than in the dark intervals. It is concluded that the short LD-cycle modulates the vigilance states and induces state-specific changes in the EEG, whereas circadian aspects of sleep are little affected.     

Effects of circadian phase and duration of sleep deprivation on sleep and EEG power spectra in the cat

The electroencephalogram (EEG) of cats was recorded under baseline conditions (LD 12:12) and after 4 and 8 h of sleep deprivation (SD). The EEG was analyzed by visual scoring and by spectral analysis. Under baseline conditions the 24-h distribution of sleep was bimodal: the smallest amounts of sleep occurred at the light-dark and dark-light transitions. EEG slow-wave activity (power density in the delta frequency range: 0.5-4.0 Hz) in non-rapid-eye-movement sleep (NREMS) showed a small variation over the 24-h period. When recovery sleep, following 4 h and 8 h of SD, started at the beginning of the dark period, no significant rebound of NREMS and REMS occurred during the 24-h recovery period. When recovery sleep, after 4 h of SD, started at the fifth hour of the light period, the amount of NREMS was increased. In all experiments the EEG power density in NREMS was enhanced after SD in the entire frequency range studied (0.5-31.5 Hz), but more prominently in the delta and theta (4.5-7.0 Hz) frequency bands. The effects dissipated in the course of the recovery period. The magnitude and duration of the enhancements of EEG power densities were dependent on the duration of SD and on the circadian phase at which SD was scheduled. It is concluded that in the cat sleep is a function of both circadian and homeostatic processes and that especially the EEG power density in NREMS is highly responsive to sleep loss.     

Interferon type I receptor-deficient mice have altered disease symptoms in response to influenza virus

The role of type I interferons (IFNs) in mediation of acute viral symptoms (fever, somnolence, anorexia, etc.) is unknown. To determine the role of type I IFN in selected symptom development, body temperature and sleep responses to a marginally lethal dose of X-31 influenza virus were examined in mice with a targeted mutation of the IFN receptor type I (IFN-RI knockouts) and compared to wild-type 129 SvEv control mice. Mice were monitored for 48 h to determine baseline temperature and sleep profiles prior to infection, and then for 9 days following infection. Hypothermic responses to virus were perceptible beginning at 64 h post-infection (PI) and were more marked in KO mice until 108 h, when hypothermia became more exaggerated in wild-type controls. Temperatures of wild-type mice continued to decline through day 9 while temperatures in IFN-RI KO mice stabilized. Time spent in non-rapid eye movement sleep (NREMS) increased in KO mice when hypothermia was marked and then returned to baseline levels, while NREMS continued to increase in wild-type mice through day 9. Other sleep parameters [time spent in rapid eye movement sleep (REMS), relative NREMS EEG slow wave activity, NREMS EEG power density] were all reduced in wild-type mice compared to KOs from days 3 to 8 while REMS low frequency EEG power density increased in wild-type relative to KOs. In conclusion, our results indicate that the presence of functional type I IFN slightly ameliorates disease symptoms early in the X-31 infection while exacerbating disease symptoms later in the infection.     

Sleep and immunomodulatory responses to systemic lipopolysaccharide in mice selectively expressing interleukin‐1 receptor 1 on neurons or astrocytes

Sleep‐wake behavior is altered in response to immune challenge. Although the precise mechanisms that govern sickness‐induced changes in sleep are not fully understood, interleukin‐1β (IL‐1) is one mediator of these responses. To better understand mechanisms underlying sleep and inflammatory responses to immune challenge, we used two transgenic mouse strains that express IL‐1 receptor 1 (IL1R1) only in the central nervous system and selectively on neurons or astrocytes. Electroencephalographic recordings from transgenic and wild‐type mice reveal that systemic challenge with lipopolysaccharide (LPS) fragments sleep, suppresses rapid eye movement sleep (REMS), increases non‐REMS (NREMS), diminishes NREM delta power, and induces fever in all genotypes. However, the magnitude of REMS suppression is greater in mice expressing IL1R1 on astrocytes compared with mice in which IL1R1 is selectively expressed on neurons. Furthermore, there is a delayed increase in NREM delta power when IL1R1 is expressed on astrocytes. LPS‐induced sleep fragmentation is reduced in mice expressing IL1R1 on neurons. Although LPS increases IL‐1 and IL‐6 in brain of all genotypes, this response is attenuated when IL1R1 is expressed selectively on neurons or on astrocytes. Collectively, these data suggest that in these transgenic mice under the conditions of this study it is neuronal IL1R1 that plays a greater role in LPS‐induced suppression of REMS and NREM delta power, whereas astroglial IL1R1 is more important for sleep fragmentation after this immune challenge. Thus, aspects of central responses to LPS are modulated by IL1R1 in a cell type‐specific manner. GLIA 2016;64:780–791     

Deficiency of corticotropin-releasing hormone type-2 receptor alters sleep responses to bacterial lipopolysaccharide in mice

In response to infectious stimuli, enhanced non-rapid eye movement sleep (NREMS) occurs, which is driven by pro-inflammatory cytokines. Those cytokines further elicit the release of corticotropin-releasing hormone (CRH), resulting in the activation of the hypothalamic–pituitary–adrenocortical axis. Signals of CRH are mediated by two receptor types, namely CRH-R1 and -R2. The role of CRH-R1 in wake-promoting effects of CRH has been rather clarified, whereas the involvement of CRH-R2 in sleep-wake regulation is poorly understood. To investigate whether CRH-R2 interferes with sleep responses to immune challenge, this study examined effects of bacterial lipopolysaccharide (LPS) on sleep in CRH-R2 deficient (KO) mice. CRH-R2 KO mice and control littermates (CL) were implanted with electrodes for recording electroencephalogram (EEG) and electromyogram. After recovery, LPS was applied by intraperitoneal injection at doses of 0.1, 1.0, or 10 μg at dark onset. In response to LPS injection NREMS of both genotypes was enhanced in a dose-dependent manner. However, CRH-R2 KO mice showed a larger increase, in particular after 10 μg of LPS compared to CL mice. During postinjection, reduced delta power for NREMS was detected in both genotypes after each dose, but the highest dose evoked a marked elevation of EEG activity in a limited frequency band (4 Hz). However, the EEG power of lower frequencies (1–2 Hz) increased more in CRH-R2 KO than in CL mice. The results indicated that CRH-R2 KO mice show greater NREMS responses to LPS, providing evidence that CRH-R2 participates in sleep-wake regulation via an interaction with the activated immune system.     

Spontaneous sleep in mice with targeted disruptions of neuronal or inducible nitric oxide synthase genes

Nitric oxide (NO) affects almost every physiological process, including the regulation of sleep. There is strong evidence that NO plays an important role in rapid eye movement sleep (REMS) regulation. To further investigate the role of NO in sleep, we characterized spontaneous sleep in mice with targeted disruptions (knockout; KO) in the neuronal nitric oxide synthase (nNOS) or inducible (i)NOS genes. REMS in nNOS KO mice was substantially lower than that of their control mice. In contrast, the iNOS KO mice had significantly more REMS than their controls. Inducible NOS KO mice also had less non-REMS (NREMS) during the dark period. Results suggest that nNOS and iNOS play opposite roles in REMS regulation.     

The effects of intracerebroventricular application of 8-Br-cGMP and LY-83,583, a guanylyl cyclase inhibitor, on sleep-wake activity in rats

Cyclic GMP is the second messenger that mediates most of the neuronal effects of nitric oxide (NO). Several lines of evidence suggest that NO-ergic mechanisms play an integral role in the regulation of vigilance. In the present study, we tested the effects of the activation of cGMP-receptive mechanisms and the inhibitor of guanylyl cyclase (GC), LY-83,583, on sleep in rats. Rats were injected intracerebroventricularly (icv) with 0.16, 4, 100, and 500 microg or 2.5 mg 8-Br-cGMP, a membrane-permeable analogue of cGMP, or 1 and 100 microg LY-83,583. Administration of 4 microg-2.5 mg 8-Br-cGMP increased wakefulness and suppressed rapid-eye-movement sleep (REMS) and non-REMS (NREMS) in rats when given before dark onset but not when given before the light period. The GC inhibitor LY-83,583 strongly promoted NREMS and suppressed REMS during the light period of the day. Furthermore, LY-83,583 induced striking increases in the delta-wave activity of the electroencephalogram (EEG) during NREMS, whereas EEG activity above the 4.5 Hz wave range was suppressed in all vigilance states. Our finding that cGMP has an arousal-promoting activity is in line with the hypothesis that NO/cGMP signaling pathway is involved in the regulation of vigilance.     

Altered rapid eye movement sleep timing in serotonin transporter knockout mice

The monoamine neurotransmitter serotonin has long been implicated in development and maintenance of sleep patterns, yet the role of the serotonin transporter (SERT) in these processes has not been evaluated in detail. We report that genetically engineered SERT knockout mice exhibit more REM sleep (REMS) than wild type littermates (11 vs 7% of recording time under baseline conditions) and display more frequent REMS bouts that last longer. This phenotype resembles the previously reported long-term effect of repeated treatment with SERT inhibitor compounds rather than the acute REMS suppressing effect of treatment with such compounds, and is thus likely to reflect neuroadaptations to the absence of SERT, rather than an acute effect of its absence in the adult. While electroencephalographic (EEG) spectra did not differ between SERT knockout and wild type mice during non-REM sleep (NREMS) or REMS, the dynamics of the EEG during the transition from NREMS to REMS differed between the genotypes. The surge in EEG power in both the 6-9 Hz and 10-16 Hz ranges that occurs just prior to the onset of REMS (pre-REMS power surge) is of greater magnitude in SERT knockout mice than in wild type littermate controls. This observation contrasts with the reduced magnitude pre-REMS power surge observed in rats subjected to REMS deprivation relative to yoked controls. These results indicate that the pre-REMS power surge is influenced by REMS history and by monoaminergic transmission. Genetic differences in serotonin systems and developmental exposure to SERT blockers are likely to exert effects on REMS.     

The effects of immunolesions of nerve growth factor-receptive neurons by 192 IgG-saporin on sleep

Low-affinity nerve growth factor (NGF) receptors are present on the cholinergic neurons of the basal forebrain. We studied the effects of 192 IgG-saporin, a specific immunotoxin for the NGF receptor-positive, cholinergic basal forebrain neurons, on sleep, the power spectrum of the electroencephalogram (EEG), and body temperature. After 3 d baseline recordings, 12 male rats were injected intracerebroventricularly with 4 μg 192 IgG-saporin. EEG, motor activity, and brain temperature were recorded for 23 h on the first, third, fifth, and seventh day after the treatment. 192 IgG-saporin did not affect the total daily amounts but altered the circadian distribution of sleep. On days 1 and 3 after the injection of the immunotoxin, the amount of non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) increased during the dark period, whereas during the light both NREMS and REMS decreased. On day 5, these changes were less pronounced and sleep completely returned to the baseline by day 7. The EEG was suppressed in each frequency band and each vigilance state, and, in contrast to sleep, these changes in EEG persisted for 7 days. Brain temperature was decreased from day 3. These results suggest that NGF receptor-positive, cholinergic basal forebrain neurons are not necessary for the maintenance of total sleep time but contribute to the generation of normal EEG and the maintenance of brain temperature.     

Sex with knockout models: behavioral studies of estrogen receptor α

Estrogens are an important class of steroid hormones, having multiple targets, in the body and brain, and exerting ubiquitous effects on behavior. At present, two estrogen receptors (ERα and β) have been cloned and sequenced in mammals. In the brain these receptors are regionally specific, but both have widespread distributions, which are largely non-overlapping. Given the newly emerging complexities of estrogen's mechanisms of action it is important to distinguish which pathways are involved in modifying which behaviors. We use a knockout mouse, lacking functional copies of the estrogen receptor α (ERα) gene, to study the mechanisms by which estrogens mediate behaviors. There are pronounced ramifications of ERα gene disruption on behavior. First, female ERα knockout (ERαKO) mice do not display normal feminine sexual behavior. Second, treatment of adult mice with androgens promotes masculine sexual behavior in both sexes. However, male-typical sexual behavior is severely compromised in male and female ERαKOs. Third, male ERαKOs do not exhibit the same social preferences for female mice as do wildtype (WT) littermates. Thus, the ERα is essential for normal expression of sexual behaviors. In addition, gonadectomized ERαKO and WT mice rapidly learn to escape from the Morris water maze. Exogenous estrogen treatment prevents WT females from learning this task, yet, has no effect in ERαKO mice, suggesting that estrogens effects on learning in adult females involves the ERα. Based on these data we hypothesize that ERα mediates many of the effects of estrogen on sexual behavior, learning, and memory.