Non-rapid Eye Movement Sleep Delta Power Research Articles

Regulation of sleep amount by CRTC1 via transcription ofCrhin mice

The cAMP-response element binding protein (CREB) is required for regulation of daily sleep amount, whereas gain-of-function of CREB-regulated transcription coactivator 1 (CRTC1) causes severe insomnia in mice. However, the physiological functions of CRTCs and their downstream target genes in the regulation of sleep amount remain unclear. Here, we use adult brain chimeric (ABC)-expression/knockout platform for somatic genetics analysis of sleep in adult male mice. ABC-expression of constitutively active mutant CRTC1/2CAin the mouse brain neurons significantly reduces the amount of non-rapid eye movement sleep (NREMS) and/or REMS. Consistent with that SIK3 phosphorylates and inhibits CRTCs, ABC-expression of CRTC1/2/3CArescues the hypersomnia phenotype ofSleepy(Sik3Slp) mice. While ABC-Crtc2KOorCrtc3KOcauses no sleep phenotype, ABC-Crtc1KOor ABC-expression of dominant-negative CRTC (dnCRTC) results in modest reduction of NREMS amount accompanied with elevated NREMS delta power. Moreover, ABC-expression of CRTC1CAor dnCRTC in the excitatory neurons causes bidirectional changes of NREMS/REMS amount and/or NREMS delta power, whereas expression of CRTC1CAin the inhibitory neurons decreases REMS amount and increases NREMS delta power. The ability of CRTC1CAto regulate sleep requires its transactivation domain and CREB-binding domain and is dependent on CREB. Furthermore, we showed that inducible ABC-expression of corticotropin releasing hormone (Crh) and brain-derived neurotrophic factor (Bdnf)–two target genes of CRTCs–significantly reduces daily sleep amount. Notably, ABC-CrhKO, but notBdnfKO, rescues the insomnia phenotype of ABC-CRTC1CAmice. Taken together, these results indicate that CREB-CRTC1 complex regulates daily sleep amount by modulating the transcription ofCrhin the mouse brain neurons.Significance StatementCRTCs function as coactivators for CREB, a transcription factor required for the regulation of daily sleep amount in mice. Here, we found that CRTC1/2/3 function similarly to suppress NREMS and REMS in a CREB-dependent manner. Consistent with that CRTCs are substrates of SIK3 kinase, expression of constitutive active mutant CRTCsCArescue the hypersomnia phenotype ofSleepy(Sik3Slp) mice. Furthermore, we showed that CRTC1 reduces NREMS amount by upregulating the expression of neuropeptide CRH. These results elucidate the mechanism by which CRTCs regulates sleep amount and suggest a potential transcriptional mechanism in the stress-induced sleep/wake regulation.

Effect of N-Acetylcysteine on Sleep: Impacts of Sex and Time of Day.

Non-rapid eye movement sleep (NREMS) is accompanied by a decrease in cerebral metabolism, which reduces the consumption of glucose as a fuel source and decreases the overall accumulation of oxidative stress in neural and peripheral tissues. Enabling this metabolic shift towards a reductive redox environment may be a central function of sleep. Therefore, biochemical manipulations that potentiate cellular antioxidant pathways may facilitate this function of sleep. N-acetylcysteine increases cellular antioxidant capacity by serving as a precursor to glutathione. In mice, we observed that intraperitoneal administration of N-acetylcysteine at a time of day when sleep drive is naturally high accelerated the onset of sleep and reduced NREMS delta power. Additionally, N-acetylcysteine administration suppressed slow and beta electroencephalographic (EEG) activities during quiet wake, further demonstrating the fatigue-inducing properties of antioxidants and the impact of redox balance on cortical circuit properties related to sleep drive. These results implicate redox reactions in the homeostatic dynamics of cortical network events across sleep/wake cycles, illustrating the value of timing antioxidant administration relative to sleep/wake cycles. A systematic review of the relevant literature, summarized herein, indicates that this "chronotherapeutic hypothesis" is unaddressed within the clinical literature on antioxidant therapy for brain disorders such as schizophrenia. We, therefore, advocate for studies that systematically address the relationship between the time of day at which an antioxidant therapy is administered relative to sleep/wake cycles and the therapeutic benefit of that antioxidant treatment in brain disorders.

A signalling pathway for transcriptional regulation of sleep amount in mice.

In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation1-3. However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4. Chimeric knockout of LKB1 kinase-an activator of AMPK-related protein kinase SIK35-7-in adult mouse brain markedly reduces the amountand delta power-a measure of sleep depth-of non-rapid eye movement sleep (NREMS). Downstream of the LKB1-SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increasedsleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research.

Regulation of sleep quantity and intensity by long and short isoforms of SLEEPY kinase.

In Sleepy (Sik3Slp) or Sik3S551A mice, deletion or mutation of inhibitory phosphorylation site serine551 from salt-inducible kinase 3 (SIK3) markedly increases daily non-rapid eye movement sleep (NREMS) amount, accompanied with constitutively elevated NREMS delta power density-a measure of sleep intensity. Multiple SLP/SIK3 isoforms are expressed in mouse brain neurons, however, their respective roles in sleep regulation remain to be elucidated. Here, we identified a new and most abundant short isoform of SLP/SIK3 and examined sleep phenotypes resulted from isoform-specific expression of SLP-short (S) and long (L) isoforms. Adeno-associated virus (AAV)-mediated adult brain chimeric (ABC)-expression of SLP-S in neurons, but not in astrocytes, significantly and constitutively elevates NREMS delta power, whereas slightly increases NREMS amount. The ability of SLP-S to regulate sleep quantity/intensity is abrogated by kinase-inactivating mutations, suggesting that the sleep-promoting activity of SLP-S is dependent on its kinase activity. In Sik3S551A-L knock-in mice, isoform-specific expression of SIK3S551A-L (or SLP-L) significantly increases NREMS amount with a modest effect on NREMS delta power. ABC-expression of SLP-S complements the sleep phenotypes of heterozygous Sik3S551A-L mice by further increasing NREMS amount and NREMS delta power to levels of Sik3Slp or Sik3S551A mice. Taken together, these results indicate that both SLP-L and SLP-S isoforms contribute critically to the increases of sleep quantity and intensity in Sik3Slp or Sik3S551A mice.

Mice Lacking Cerebellar Cortex and Related Structures Show a Decrease in Slow-Wave Activity With Normal Non-REM Sleep Amount and Sleep Homeostasis.

In addition to the well-known motor control, the cerebellum has recently been implicated in memory, cognition, addiction, and social behavior. Given that the cerebellum contains more neurons than the cerebral cortex and has tight connections to the thalamus and brainstem nuclei, it is possible that the cerebellum also regulates sleep/wakefulness. However, the role of the cerebellum in sleep was unclear, since cerebellar lesion studies inevitably involved massive inflammation in the adjacent brainstem, and sleep changes in lesion studies were not consistent with each other. Here, we examine the role of the cerebellum in sleep and wakefulness using mesencephalon- and rhombomere 1-specific Ptf1a conditional knockout (Ptf1a cKO) mice, which lack the cerebellar cortex and its related structures, and exhibit ataxic gait. Ptf1a cKO mice had similar wake and non-rapid eye movement sleep (NREMS) time as control mice and showed reduced slow wave activity during wakefulness, NREMS and REMS. Ptf1a cKO mice showed a decrease in REMS time during the light phase and had increased NREMS delta power in response to 6 h of sleep deprivation, as did control mice. Ptf1a cKO mice also had similar numbers of sleep spindles and fear memories as control mice. Thus, the cerebellum does not appear to play a major role in sleep-wake control, but may be involved in the generation of slow waves.

0033 Dysregulated sleep and NREM sleep electroencephalogram delta power induced by intermittent hypoxia exposure are attenuated in NLRP3 knockout mice

Abstract Introduction The nucleotide-binding domain leucine rich family pyrin containing 3 (NLRP3) inflammasome protein complex activates caspase-1 to convert the pro-forms of IL-18 and IL-1 beta (IL-1β) into their active forms and is involved in homeostatic sleep and sleep responses to pathogenic stimuli. Intermittent hypoxia (IH) is a hallmark of sleep disordered breathing (SDB) and both IL-18 receptors are associated with SDB in humans. Thus, we hypothesized that NLRP3 inflammasomes are involved in SDB-related sleep disturbances. Methods Sleep architecture was assessed by polysomnography in NLRP3 knockout (KO) and wild-type (WT) mice (N = 8 per group). A gas exchange mixer delivered house air serving as a control or chronic IH that involved 90 second episodic oxygen reductions that consisted of ambient oxygen (21%) with brief hypoxic conditions (~10%) that lasted for 3 seconds for the first 10 h of the light period for 5 consecutive days. Gene expression and protein levels in the brain and lungs were assayed using real-time polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Results In WT mice, significant increased non-rapid-eye movement (NREM) sleep amounts and NREM sleep electroencephalogram delta power (0.5-4 Hz) were found after 1 day of IH compared to control conditions (p < 0.001). After 5 days of IH, WT mice showed a significant attenuation in NREM sleep amounts and NREM sleep delta power (p < 0.001) and increased wake bout frequency (p = 0.006) when compared to control conditions. However, the IH-induced NREM sleep and NREM sleep delta power enhancements and reductions were attenuated (21-35%) in NLRP3 KO mice compared to WT mice. In WT mice, NLRP3, IL-1β, IL-18 and caspase-1 gene expression, IL-1β and IL-18 protein levels, and caspase-1 activity were significantly increased in the somatosensory cortices, NTS, and lungs after both 1 and 5 days of IH when compared to control conditions (p < 0.05 for all), although NLRP3 KO mice did not exhibit significant differences in molecules downstream of NLRP3 inflammasome activation. Conclusion Our findings indicate that altered NLRP3 inflammasome activation contributes to dysregulated sleep occurring from IH and likely is involved in sleep disturbances in SDB. Support (If Any) VA Career Development Aware IBX002823, VA Merit BX004500, NIH/NIMH 1R21MH125242, NIH/NHLBI R03 HL154284, and NIH/NHLBI R35 HL135818. JTM received partial salary compensation and funding from Merck Investigator Sponsored Programs but has no conflict of interest with this work.

The Role of Reproductive Hormones in Sex Differences in Sleep Homeostasis and Arousal Response in Mice.

There are various sex differences in sleep/wake behaviors in mice. However, it is unclear whether there are sex differences in sleep homeostasis and arousal responses and whether gonadal hormones are involved in these sex differences. Here, we examined sleep/wake behaviors under baseline condition, after sleep deprivation by gentle handling, and arousal responses to repeated cage changes in male and female C57BL/6 mice that are hormonally intact, gonadectomized, or gonadectomized with hormone supplementation. Compared to males, females had longer wake time, shorter non-rapid eye movement sleep (NREMS) time, and longer rapid eye movement sleep (REMS) episodes. After sleep deprivation, males showed an increase in NREMS delta power, NREMS time, and REMS time, but females showed a smaller increase. Females and males showed similar arousal responses. Gonadectomy had only a modest effect on homeostatic sleep regulation in males but enhanced it in females. Gonadectomy weakened arousal response in males and females. With hormone replacement, baseline sleep in gonadectomized females was similar to that of intact females, and baseline sleep in gonadectomized males was close to that of intact males. Gonadal hormone supplementation restored arousal response in males but not in females. These results indicate that male and female mice differ in their baseline sleep–wake behavior, homeostatic sleep regulation, and arousal responses to external stimuli, which are differentially affected by reproductive hormones.

Homeostatic sleep and body temperature responses to acute sleep deprivation are preserved following chronic sleep restriction in rats.

Chronic sleep insufficiency is common in our society and has negative cognitive and health impacts. It can also alter sleep regulation, yet whether it affects subsequent homeostatic responses to acute sleep loss is unclear. We assessed sleep and thermoregulatory responses to acute sleep deprivation before and after a '3/1' chronic sleep restriction protocol in adult male Wistar rats. The 3/1 protocol consisted of continuous cycles of wheel rotations (3h on/1h off) for 4days. Sleep latency in a 2-h multiple sleep latency test starting 26h post-3/1 was unchanged, whereas non-rapid eye movement sleep (NREMS) and associated electroencephalogram delta power (a measure of sleep need) over a 24-h period beginning 54h post-3/1 were reduced, compared to respective pre-3/1 baseline levels. However, in response to acute sleep deprivation (6h by 'gentle handling') starting 78h post-3/1, the compensatory rebounds in NREMS and rapid eye movement sleep (REMS) amounts and NREMS delta power were unaltered. Body temperature increased progressively across the 3/1 protocol and returned to baseline levels on the second day post-3/1. The acute sleep deprivation also increased body temperature, followed by a decline below baseline levels, with no difference between before and after 3/1 sleep restriction. Non-sleep-restricted control rats showed responses to acute sleep deprivation similar to those observed in the sleep-restricted animals. These results suggest that the process of sleep homeostasis is altered on the third recovery day after a 4-day 3/1 sleep restriction protocol, whereas subsequent homeostatic sleep and temperature responses to brief sleep deprivation are not affected.

Multimodal neuroimaging correlates of NREM and REM sleep: EEG spectral power in aging

AbstractBackgroundSleep disturbances have been associated with cognitive decline and Alzheimer’s disease (AD), with growing efforts to understand how these changes may relate to AD pathophysiology. However, we lack a comprehensive overview of the associations between objectively‐measured sleep oscillations and brain changes in aging so that efficient treatment strategies could be developed. Using an unbiased, fast, and powerful quantitative (spectral) analysis of sleep EEG rhythms, we assessed the relationships between both non‐rapid eye movement (NREM) and REM sleep oscillations, and amyloid burden, gray matter (GM) volume and perfusion in cognitively unimpaired older adults (CUOA).Method125 CUOA from the Age‐Well randomized controlled trial underwent a neuropsychological assessment, an ambulatory polysomnography recording and a structural MRI. A Florbetapir‐PET scan was also performed with early and late acquisitions to measure brain perfusion and amyloid burden respectively. Normalized EEG spectral power values were obtained using the ASEEGA® algorithm for delta (0.1‐4 Hz), theta (4‐8 Hz), alpha (8‐12 Hz) and beta (16‐50 Hz) EEG frequency bands, over the Cz‐Pz derivation (Berthomier et al., 2007). Voxel‐wise multiple regressions were then conducted between EEG spectral power values and neuroimaging data (i.e., GM volume, perfusion and amyloid burden), separately for NREM and REM sleep, controlling for age, gender, education, the apnea‐hypopnea index, sleep medication use, and the ApoE4 status.ResultLower NREM sleep delta power, reflecting a decrease in slow wave activity, was related to decreased GM volume and perfusion in fronto‐cingulate areas, but not to amyloid deposition. By contrast, decreased REM sleep theta power was associated with i) greater amyloid deposition in frontal, cingulate, parietal and temporal areas; ii) lower GM volume in frontal, temporal, occipital and insular cortices, and iii) increased perfusion in frontal and parietal regions.ConclusionBoth NREM and REM sleep alterations were associated with structural and functional changes in regions sensitive to aging and AD pathophysiology. Importantly, REM sleep changes were associated with greater amyloid deposition. This may be due to early changes in cholinergic structures, acetylcholine playing a neuroprotective role against amyloid. Thus older individuals with REM sleep alterations may be more at risk of belonging to the Alzheimer’s continuum.

An EEG Investigation of Sleep Homeostasis in Healthy and CLN5 Batten Disease Affected Sheep

Sheep have large brains with human-like anatomy, making them a useful species for studying brain function. Sleep homeostasis has not been studied in sheep. Here, we establish correlates of sleep homeostasis in sheep through a sleep deprivation experiment. We then use these correlates to elucidate the nature of sleep deficits in a naturally occurring ovine model of neuronal ceroid lipofuscinosis (NCL, Batten disease) caused by a mutation in CLN5 In humans, mutations in this gene lead to cortical atrophy and blindness, as well as sleep abnormalities. We recorded electroencephalograms (EEGs) from unaffected and early stage CLN5(-/-) (homozygous, affected) sheep over 3 consecutive days, the second day being the sleep deprivation day. In unaffected sheep, sleep deprivation led to increased EEG delta (0.5-4 Hz) power during non-rapid eye movement (NREM) sleep, increased time spent in the NREM sleep state, and increased NREM sleep bout length. CLN5(-/-) sheep showed comparable increases in time spent in NREM sleep and NREM sleep bout duration, verifying the presence of increased sleep pressure in both groups. Importantly, CLN5(-/-) sheep did not show the increase in NREM sleep delta power seen in unaffected sheep. This divergent delta power response is consistent with the known cortical degeneration in CLN5(-/-) sheep. We conclude that, whereas sleep homeostasis is present in CLN5(-/-) sheep, underlying CLN5(-/-) disease processes prevent its full expression, even at early stages. Such deficits may contribute to early abnormalities seen in sheep and patients and warrant further study. Sleep abnormalities pervade most neurological diseases, including the neuronal ceroid lipofuscinoses (NCLs). Here, we show that, in an ovine model of a variant late-infantile NCL, there is abnormal expression of sleep homeostasis. Whereas some sleep pressure correlates respond to sleep deprivation, the strongest electroencephalogram (EEG) correlate of sleep pressure, non-REM delta power, failed to increase. This highlights the relevance of sleep deficits in this disease, in which the drive for sleep exists but the underlying disease prevents its full expression. Sleep abnormalities could contribute to early disease symptoms such as behavioral disorder and cognitive decline. Our study also shows sleep homeostatic EEG correlates in sheep, opening up new opportunities for studying sleep in a large social mammal with complex human-like brain neuroanatomy.

Effects of Macrophage Depletion on Sleep in Mice.

The reciprocal interaction between the immune system and sleep regulation has been widely acknowledged but the cellular mechanisms that underpin this interaction are not completely understood. In the present study, we investigated the role of macrophages in sleep loss- and cold exposure-induced sleep and body temperature responses. Macrophage apoptosis was induced in mice by systemic injection of clodronate-containing liposomes (CCL). We report that CCL treatment induced an immediate and transient increase in non-rapid-eye movement sleep (NREMS) and fever accompanied by decrease in rapid-eye movement sleep, motor activity and NREMS delta power. Chronically macrophage-depleted mice had attenuated NREMS rebound after sleep deprivation compared to normal mice. Cold-induced increase in wakefulness and decrease in NREMS, rapid-eye movement sleep and body temperature were significantly enhanced in macrophage-depleted mice indicating increased cold sensitivity. These findings provide further evidence for the reciprocal interaction among the immune system, sleep and metabolism, and identify macrophages as one of the key cellular elements in this interplay.

Increases in mature brain-derived neurotrophic factor protein in the frontal cortex and basal forebrain during chronic sleep restriction in rats: Possible role in initiating allostatic adaptation

Chronic sleep restriction (CSR) has various negative consequences on cognitive performance and health. Using a rat model of CSR that uses alternating cycles of 3h of sleep deprivation (using slowly rotating activity wheels) and 1h of sleep opportunity continuously for 4days (‘3/1’ protocol), we previously observed not only homeostatic but also allostatic (adaptive) sleep responses to CSR. In particular, non-rapid eye movement sleep (NREMS) electroencephalogram (EEG) delta power, an index of sleep intensity, increased initially and then declined gradually during CSR, with no rebound during a 2-day recovery period. To study underlying mechanisms of these allostatic responses, we examined the levels of brain-derived neurotrophic factor (BDNF), which is known to regulate NREMS EEG delta activity, during the same CSR protocol. Mature BDNF protein levels were measured in the frontal cortex and basal forebrain, two brain regions involved in sleep and EEG regulation, and the hippocampus, using Western blot analysis. Adult male Wistar rats were housed in motorized activity wheels, and underwent the 3/1 CSR protocol for 27h, for 99h, or for 99h followed by 24h of recovery. Additional rats were housed in either locked wheels (locked wheel controls [LWCs]) or unlocked wheels that rats could rotate freely (wheel-running controls [WRCs]). BDNF levels did not differ between WRC and LWC groups. BDNF levels were increased, compared to the control levels, in all three brain regions after 27h, and were increased less strongly after 99h, of CSR. After 24h of recovery, BDNF levels were at the control levels. This time course of BDNF levels parallels the previously reported changes in NREMS delta power during the same CSR protocol. Changes in BDNF protein levels in the cortex and basal forebrain may be part of the molecular mechanisms underlying allostatic sleep responses to CSR.

Time-of-day modulation of homeostatic and allostatic sleep responses to chronic sleep restriction in rats

To study sleep responses to chronic sleep restriction (CSR) and time-of-day influences on these responses, we developed a rat model of CSR that takes into account the polyphasic sleep patterns in rats. Adult male rats underwent cycles of 3 h of sleep deprivation (SD) and 1 h of sleep opportunity (SO) continuously for 4 days, beginning at the onset of the 12-h light phase ("3/1" protocol). Electroencephalogram (EEG) and electromyogram (EMG) recordings were made before, during, and after CSR. During CSR, total sleep time was reduced by ∼60% from baseline levels. Both rapid eye movement sleep (REMS) and non-rapid eye movement sleep (NREMS) during SO periods increased initially relative to baseline and remained elevated for the rest of the CSR period. In contrast, NREMS EEG delta power (a measure of sleep intensity) increased initially, but then declined gradually, in parallel with increases in high-frequency power in the NREMS EEG. The amplitude of daily rhythms in NREMS and REMS amounts was maintained during SO periods, whereas that of NREMS delta power was reduced. Compensatory responses during the 2-day post-CSR recovery period were either modest or negative and gated by time of day. NREMS, REMS, and EEG delta power lost during CSR were not recovered by the end of the second recovery day. Thus the "3/1" CSR protocol triggered both homeostatic responses (increased sleep amounts and intensity during SOs) and allostatic responses (gradual decline in sleep intensity during SOs and muted or negative post-CSR sleep recovery), and both responses were modulated by time of day.

Female Reproductive Hormones Alter Sleep Architecture in Ovariectomized Rats

Treating ovariectomized rats with physiological levels of estradiol and/or progesterone affects aspects of both baseline (24 h) sleep and recovery (18 h) sleep after 6 h of sleep deprivation. We have extended the analysis of these effects by examining several additional parameters of sleep architecture using the same data set as in our previous study (Deurveilher et al. SLEEP 2009;32(7):865-877). Sleep in ovariectomized rats implanted with oil, 17 β-estradiol and/or progesterone capsules was recorded using EEG and EMG before, during, and after 6 h of sleep deprivation during the light phase of a 12/12 h light/dark cycle. During the baseline dark, but not light, phase, treatments with estradiol alone or combined with progesterone decreased the mean duration of non-rapid eye movement sleep (NREMS) episodes and the number of REMS episodes, while also increasing brief awakenings, consistent with the previously reported lower baseline NREMS and REMS amounts. Following sleep deprivation, the hormonal treatments caused a larger percentage increase from baseline in the mean durations of NREMS and REMS episodes, and a larger percentage decrease in brief awakenings, consistent with the previously reported larger increase in recovery REMS amount. There were no hormonal effects on NREMS and REMS EEG power values, other than on recovery NREMS delta power, as previously reported. Physiological levels of estradiol and/or progesterone in female rats modulate sleep architecture differently at baseline and after acute sleep loss, fragmenting baseline sleep while consolidating recovery sleep. These hormones also play a role in the diurnal pattern of NREMS maintenance.

ATP and the purine type 2 X7 receptor affect sleep

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.

Sleep Deprivation Effects on Circadian Clock Gene Expression in the Cerebral Cortex Parallel Electroencephalographic Differences among Mouse Strains

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Alterations in EEG activity and sleep after influenza viral infection in GHRH receptor-deficient mice

Viral infections induce excess non-rapid eye movement sleep (NREMS) in mice. Growth hormone-releasing hormone receptor (GHRH receptor) was previously identified as a candidate gene responsible for NREMS responses to influenza challenge in mice. The dwarf lit/lit mouse with a nonfunctional GHRH receptor was used to assess the role of the GHRH receptor in viral-induced NREMS. After influenza A virus infection the duration and intensity [electroencephalogram (EEG) delta power] of NREMS increased in heterozygous mice with the normal phenotype, whereas NREMS and EEG delta power decreased in homozygous lit/lit mice. Lit/lit mice developed a pathological state with EEG slow waves and enhanced muscle tone. Other influenza-induced responses (decreases in rapid eye movement sleep, changes in the EEG high-frequency bands during the various stages of vigilance, hypothermia, and decreased motor activity) did not differ between the heterozygous and lit/lit mice. GH replacement failed to normalize the NREMS responses in the lit/lit mice after influenza inoculation. Decreases in NREMS paralleled hypothermia in the lit/lit mice. Lung virus levels were similar in the two mouse strains. Lit/lit mice had a higher death rate after influenza challenge than the heterozygotes. In conclusion, GHRH signaling is involved in the NREMS response to influenza infection.