No loss of orexin/hypocretin, melanin-concentrating hormone or locus coeruleus noradrenergic neurons in a rat model of chronic sleep restriction.

Chronic sleep restriction (CSR) is common in modern society, adversely affecting cognitive performance and health. Yet how it impacts neurons regulating sleep remains unclear. Several studies using mice reported substantial losses of wake-active orexin/hypocretin and locus coeruleus (LC) noradrenergic neurons, but not rapid eye movement sleep-active melanin-concentrating hormone (MCH) neurons, following CSR. Here, we used immunohistochemistry and stereology to examine orexin, MCH and LC noradrenergic neurons in a rat model of CSR that uses programmed wheel rotation (3 h on/1 h off; '3/1' protocol). Adult male Wistar rats underwent one or four cycles of the 4-day 3/1 CSR protocol, with 2-day recovery between cycles in home cages. Time-matched control rats were housed in locked wheels/home cages. We found no significant differences in the numbers of orexin, MCH and LC noradrenergic neurons following either one- or four-cycle CSR protocol compared to respective controls. Similarly, the four-cycle CSR protocol had no effect on the densities of orexin axon terminals in the LC, noradrenergic dendrites in the LC and noradrenergic axon terminals in the frontal cortex. Body weights, however, decreased after one cycle of CSR and then increased with diminishing slope over the next three cycles. Thus, we found no evidence for loss of orexin or LC noradrenergic neurons following one and four cycles of the 4-day 3/1 CSR protocol in rats. Differences in CSR protocols and/or possible species differences in neuronal vulnerability to sleep loss may account for the discrepancy between the current results in rats and previous findings in mice.

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 (3 h on/1 h off) for 4 days. Sleep latency in a 2-h multiple sleep latency test starting 26 h 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 54 h post-3/1 were reduced, compared to respective pre-3/1 baseline levels. However, in response to acute sleep deprivation (6 h by 'gentle handling') starting 78 h 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.

Sex differences in age-related changes in the sleep-wake cycle.

Age-related changes in sleep and circadian regulation occur as early as the middle years of life. Research also suggests that sleep and circadian rhythms are regulated differently between women and men. However, does sleep and circadian rhythms regulation age similarly in men and women? In this review, we present the mechanisms underlying age-related differences in sleep and the current state of knowledge on how they interact with sex. We also address how testosterone, estrogens, and progesterone fluctuations across adulthood interact with sleep and circadian regulation. Finally, we will propose research avenues to unravel the mechanisms underlying sex differences in age-related effects on sleep.

Ovarian hormones promote recovery from sleep deprivation by increasing sleep intensity in middle-aged ovariectomized rats.

Sleep disturbances are commonly associated with menopause. Hormone replacement therapy is often used to treat various menopausal symptoms, but its efficacy for improving sleep is a matter of debate. We addressed this question by using a rodent model of ovarian hormone loss and replacement in midlife. Middle-aged female rats were ovariectomized and implanted with capsules containing estradiol with or without progesterone, or oil. After two weeks, sleep/wake states were recorded polygraphically during a 24-h baseline period, followed by 6h of sleep deprivation in the second half of the light phase, and a 24-h recovery period. During the baseline dark phase, hormone treatments increased wakefulness, and decreased non-rapid eye movement sleep (NREMS) by shortening NREMS episodes; however, NREMS EEG delta power or energy (cumulative power) was unaffected by combined hormones. Following sleep deprivation, all the groups showed NREMS and rapid eye movement sleep (REMS) rebounds, with similar relative increases from respective baseline levels. The increases in NREMS EEG delta power/energy during recovery were enhanced by combined hormones. These results from middle-aged ovariectomized rats indicate that replacement with estrogen with or without progesterone reduces baseline NREMS without affecting sleep intensity, particularly during the dark (active) phase, whereas following sleep deprivation the same hormone treatments do not affect the ability to increase NREMS or REMS, but treatment with both hormones, in particular, enhances the intensity of recovery sleep. These results support the usefulness of ovariectomized middle-aged rats as a model system to study the biological effects of hormone replacement on sleep regulation.

Microglia dynamics in sleep/wake states and in response to sleep loss.

Sleep has an essential role for optimal brain function, but the cellular substrates for sleep regulation are not fully understood. Microglia, the immune cells of the brain, have gained increasingly more attention over the last two decades for their important roles in various brain functions that extend beyond their well-known immune function, including brain development, neuronal protection, and synaptic plasticity. Here we review recent advances in understanding: i) morphological and phenotypic dynamics of microglia including process motility/growth and gene/protein expression, and ii) microglia-neuron interactions including phagocytosis and contact at synapses which alters neuronal circuit activity, both under physiological state in the adult brain. We discuss how the microglia-neuron interactions particularly at synapses could influence microglia and neuronal activities across circadian cycles and sleep/wake states. We also review recent findings on how microglia respond to sleep loss. We conclude by pointing out key questions and proposing suggestions for future research to better understand the role of microglia in sleep regulation, sleep homeostasis, and the function of sleep.

Altered circadian activity and sleep/wake rhythms in the stable tubule only polypeptide (STOP) null mouse model of schizophrenia.

Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.

Homeostatic state of microglia in a rat model of chronic sleep restriction.

Chronic sleep restriction (CSR) negatively impacts brain functions. Whether microglia, the brain's resident immune cells, play any role is unknown. We studied microglia responses to CSR using a rat model featuring slowly rotating wheels (3 h on/1 h off), which was previously shown to induce both homeostatic and adaptive responses in sleep and attention. Adult male rats were sleep restricted for 27 or 99 h. Control rats were housed in locked wheels. After 27 and/or 99 h of CSR, the number of cells immunoreactive for the microglia marker ionized calcium-binding adaptor molecule-1 (Iba1) and the density of Iba1 immunoreactivity were increased in 4/10 brain regions involved in sleep/wake regulation and cognition, including the prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus. CSR neither induced mitosis in microglia (assessed with bromodeoxyuridine) nor impaired blood-brain barrier permeability (assessed with Evans Blue). Microglia appeared ramified in all treatment groups and, when examined quantitatively in the prelimbic cortex, their morphology was not affected by CSR. After 27 h, but not 99 h, of CSR, mRNA levels of the anti-inflammatory cytokine interleukin-10 were increased in the frontal cortex. Pro-inflammatory cytokine mRNA levels (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) were unchanged. Furthermore, cortical microglia were not immunoreactive for several pro- and anti-inflammatory markers tested, but were immunoreactive for the purinergic P2Y12 receptor. These results suggest that microglia respond to CSR while remaining in a physiological state and may contribute to the previously reported homeostatic and adaptive responses to CSR.

Role of polysialylated neural cell adhesion molecule in rapid eye movement sleep regulation in rats.

Recent evidence suggests that synaptic plasticity occurs during homeostatic processes, including sleep-wakefulness regulation, although the underlying mechanisms are not well understood. Polysialylated neural cell adhesion molecule (PSA NCAM) is a transmembrane protein that has been implicated in various forms of plasticity. To investigate whether PSA NCAM is involved in the neuronal plasticity associated with spontaneous sleep-wakefulness regulation and sleep homeostasis, four studies were conducted using rats. First, we showed that PSA NCAM immunoreactivity is present in close proximity to key neurons in several nuclei of the sleep-wakefulness system, including the tuberomammillary hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. Second, using western blot analysis and densitometric image analysis of immunoreactivity, we found that 6 h of sleep deprivation changed neither the levels nor the general location of PSA NCAM in the sleep-wakefulness system. Finally, we injected endoneuraminidase (Endo N) intracerebroventricularly to examine the effects of polysialic acid removal on sleep-wakefulness states and electroencephalogram (EEG) slow waves at both baseline and during recovery from 6 h of sleep deprivation. Endo N-treated rats showed a small but significant decrease in baseline rapid eye movement (REM) sleep selectively in the late light phase, and a facilitated REM sleep rebound after sleep deprivation, as compared with saline-injected controls. Non-REM sleep and wakefulness were unaffected by Endo N. These results suggest that PSA NCAM is not particularly involved in the regulation of wakefulness or non-REM sleep, but plays a role in the diurnal pattern of REM sleep as well as in some aspects of REM sleep homeostasis.

Estradiol and progesterone modulate spontaneous sleep patterns and recovery from sleep deprivation in ovariectomized rats.

STUDY OBJECTIVES: Women undergo hormonal changes both naturally during their lives and as a result of sex hormone treatments. The objective of this study was to gain more knowledge about how these hormones affect sleep and responses to sleep loss. DESIGN: Rats were ovariectomized and implanted subcutaneously with Silastic capsules containing oil vehicle, 17 beta-estradiol and/or progesterone. After 2 weeks, sleep/wake states were recorded during a 24-h baseline period, 6 h of total sleep deprivation induced by gentle handling during the light phase, and an 18-h recovery period. MEASUREMENTS AND RESULTS: At baseline and particularly in the dark phase, ovariectomized rats treated with estradiol or estradiol plus progesterone spent more time awake at the expense of non-rapid eye movement sleep (NREMS) and/or REMS, whereas those given progesterone alone spent less time in REMS than ovariectomized rats receiving no hormones. Following sleep deprivation, all rats showed rebound increases in NREMS and REMS, but the relative increase in REMS was larger in females receiving hormones, especially high estradiol. In contrast, the normal increase in NREMS EEG delta power (an index of NREMS intensity) during recovery was attenuated by all hormone treatments. CONCLUSIONS: Estradiol promotes arousal in the active phase in sleep-satiated rats, but after sleep loss, both estradiol and progesterone selectively facilitate REMS rebound while reducing NREMS intensity. These results indicate that effects of ovarian hormones on recovery sleep differ from those on spontaneous sleep. The hormonal modulation of recovery sleep architecture may affect recovery of sleep related functions after sleep loss.

Transforming growth factor-alpha and glial fibrillary acidic protein in the hamster circadian system: daily profile and cellular localization.

Transforming growth factor-alpha (TGF-alpha) has been identified as a potential output signal of the principal circadian pacemaker housed in the mammalian suprachiasmatic nucleus (SCN). The goal of the present study was to characterize the temporal pattern and cellular localization of TGF-alpha immunoreactivity (IR), and to examine its localization relative to astrocytic and neuronal markers in the hamster circadian system. In contrast to previous reports of circadian rhythms in TGF-alpha mRNA levels in the hamster SCN, we did not detect any statistically significant changes in the levels of TGF-alpha protein IR in the hamster SCN across a 14:10 light-dark cycle using densitometric analyses. TGF-alpha was found to be colocalized with glial fibrillary acidic protein (GFAP), but not with the general neuronal marker NeuN, or calbindin-D28K which is present in a subgroup of SCN neurons. GFAP IR showed a small but significant daily variation in the SCN, with higher levels early in the light phase compared to the middle of the dark phase. The thalamic intergeniculate leaflet (IGL), another component of the circadian regulatory system, did not show any TGF-alpha IR or any detectable daily variation in GFAP IR. These results suggest that daily variations of TGF-alpha mRNA levels in the hamster SCN are not accompanied by corresponding rhythms of TGF-alpha protein levels, and confirm that TGF-alpha is present primarily in astrocytes within the SCN.

Region-specific increases in FosB/ΔFosB immunoreactivity in the rat brain in response to chronic sleep restriction.

Using a rat model of chronic sleep restriction (CSR) featuring periodic sleep deprivation with slowly rotating wheels (3h on/1h off), we previously observed that 99h of this protocol induced both homeostatic and allostatic (adaptive) changes in physiological and behavioural measures. Notably, the initial changes in sleep intensity and attention performance gradually adapted during CSR despite accumulating sleep loss. To identify brain regions involved in these responses, we used FosB/ΔFosB immunohistochemistry as a marker of chronic neuronal activation. Adult male rats were housed in motorized activity wheels and underwent the 3/1 CSR protocol for 99h, or 99h followed by 6 or 12days of recovery. Control rats were housed in home cages, locked activity wheels, or unlocked activity wheels that the animals could turn freely. Immunohistochemistry was conducted using an antibody that recognized both FosB and ΔFosB, and 24 brain regions involved in sleep/wake, autonomic, and limbic functions were examined. The number of darkly-stained FosB/ΔFosB-immunoreactive cells was increased immediately following 99h of CSR in 8/24 brain regions, including the medial preoptic and perifornical lateral hypothalamic areas, dorsomedial and paraventricular hypothalamic nuclei, and paraventricular thalamic nucleus. FosB/ΔFosB labeling was at control levels in all 8 brain areas following 6 or 12 recovery days, suggesting that most of the immunoreactivity immediately after CSR reflected FosB, the more transient marker of chronic neuronal activation. This region-specific induction of FosB/ΔFosB following CSR may be involved in the mechanisms underlying the allostatic changes in behavioural and physiological responses to CSR.

Differential c-Fos immunoreactivity in arousal-promoting cell groups following systemic administration of caffeine in rats.

Despite the widespread use of caffeine, the neuronal mechanisms underlying its stimulatory effects are not completely understood. By using c-Fos immunohistochemistry as a marker of neuronal activation, we recently showed that stimulant doses of caffeine activate arousal-promoting hypothalamic orexin (hypocretin) neurons. In the present study, we investigated whether other key neurons of the arousal system are also activated by caffeine, via dual immunostaining for c-Fos and transmitter markers. Rats were administered three doses of caffeine or saline vehicle during the light phase. Caffeine at 10 and 30 mg/kg, i.p., increased motor activities, including locomotion, compared with after saline or a higher dose, 75 mg/kg. The three doses of caffeine induced distinct dose-related patterns of c-Fos immunoreactivity in several arousal-promoting areas, including orexin neurons and adjacent neurons containing neither orexin nor melanin-concentrating hormone; tuberomammillary histaminergic neurons; locus coeruleus noradrenergic neurons; noncholinergic basal forebrain neurons that do not contain parvalbumin; and nondopaminergic neurons in the ventral tegmental area. At any dose used, caffeine induced little or no c-Fos expression in cholinergic neurons of the basal forebrain and mesopontine tegmentum; dopaminergic neurons of the ventral tegmental area, central gray, and substantia nigra pars compacta; and serotonergic neurons in the dorsal raphe nucleus. Saline controls exhibited only few c-Fos-positive cells in most of the cell groups examined. These results indicate that motor-stimulatory doses of caffeine induce a remarkably restricted pattern of c-Fos expression in the arousal-promoting system and suggest that this specific neuronal activation may be involved in the behavioral arousal by caffeine.

Disruptions of Sleep/Wake Patterns in the Stable Tubule Only Polypeptide (STOP) Null Mouse Model of Schizophrenia.

Disruption of sleep/wake cycles is common in patients with schizophrenia and correlates with cognitive and affective abnormalities. Mice deficient in stable tubule only polypeptide (STOP) show cognitive, behavioral, and neurobiological deficits that resemble those seen in patients with schizophrenia, but little is known about their sleep phenotype. We characterized baseline sleep/wake patterns and recovery sleep following sleep deprivation in STOP null mice. Polysomnography was conducted in adult male STOP null and wild-type (WT) mice under a 12:12 hours light:dark cycle before, during, and after 6 hours of sleep deprivation during the light phase. At baseline, STOP null mice spent more time awake and less time in non-rapid eye movement sleep (NREMS) over a 24-hour period, with more frequent transitions between wake and NREMS, compared to WT mice, especially during the dark phase. The distributions of wake, NREMS and REMS across the light and the dark phases differed by genotype, and so did features of the electroencephalogram (EEG). Following sleep deprivation, both genotypes showed homeostatic increases in sleep duration, with no significant genotype differences in the initial compensatory increase in sleep intensity (EEG delta power). These results indicate that STOP null mice sleep less overall, and their sleep and wake periods are more fragmented than those of WT mice. These features in STOP null mice are consistent with the sleep patterns observed in patients with schizophrenia.

Psychomotor vigilance task performance during and following chronic sleep restriction in rats.

STUDY OBJECTIVES: Chronic sleep restriction (CSR) impairs sustained attention in humans, as commonly assessed with the psychomotor vigilance task (PVT). To further investigate the mechanisms underlying performance deficits during CSR, we examined the effect of CSR on performance on a rat version of PVT (rPVT). DESIGN: Adult male rats were trained on a rPVT that required them to press a bar when they detected irregularly presented, brief light stimuli, and were then tested during CSR. CSR consisted of 100 or 148 h of continuous cycles of 3-h sleep deprivation (using slowly rotating wheels) alternating with a 1-h sleep opportunity (3/1 protocol). MEASUREMENTS AND RESULTS: After 28 h of CSR, the latency of correct responses and the percentages of lapses and omissions increased, whereas the percentage of correct responses decreased. Over 52-148 h of CSR, all performance measures showed partial or nearly complete recovery, and were at baseline levels on the first or second day after CSR. There were large interindividual differences in the magnitude of performance impairment during CSR, suggesting differential vulnerability to the effects of sleep loss. Wheel-running controls showed no changes in performance. CONCLUSIONS: A 28-h period of the 3/1 chronic sleep restriction (CSR) protocol disrupted performance on a sustained attention task in rats, as sleep deprivation does in humans. Performance improved after longer periods of CSR, suggesting allostatic adaptation, contrary to some reports of progressive deterioration in psychomotor vigilance task performance during CSR in humans. However, as observed in humans, there were individual differences among rats in the vulnerability of their attention performance to CSR.

The cholinesterase inhibitor DFP facilitates the expression of paradoxical sleep (PS) propensity in rats subjected to short-term PS deprivation.

Short-term paradoxical sleep (PS) deprivation was used to examine the effects of chronic exposure to subtoxic doses of the cholinesterase inhibitor diisopropylfluorophosphate (DFP) on PS regulation. Rats were injected once daily with DFP (0.2 mg/kg per day; s.c.) for 11 consecutive days; control rats received a daily injection of oil vehicle. The experiment was conducted on the 10th and 11th days of treatment, when brain cholinesterase inhibition induced by DFP exposure was maximal. On the 10th day, an 8-h baseline recording was carried out. On the 11th day, a 6-h PS deprivation was carried out by manually awaking rats each time they showed polygraphic signs of PS; recordings were then continued for another 2 h to examine recovery sleep. During deprivation, though they slept less than controls, DFP-treated rats made more attempts to enter PS. After deprivation, their PS rebound had an overall amount comparable to that of the controls, but its time course was shortened: whereas PS elevation was manifested through the 2 h of recovery in the control group, it occurred only during the first hour in the DFP group. These results demonstrate that chronic, low-level DFP exposure facilitated the expression of the PS propensity that accumulated as a result of PS deprivation: it enhanced the tendency for PS during deprivation; it accelerated the rate of compensatory PS expression after deprivation. They support the hypothesis that DFP promotes PS initiation by increasing cholinergic transmission.

Indirect projections from the suprachiasmatic nucleus to the median preoptic nucleus in rat.

We recently showed, using dual tract-tracing, that the suprachiasmatic nucleus (SCN), the site of the principal circadian clock in mammals, may have indirect projections to the sleep-promoting ventrolateral preoptic nucleus (VLPO) via relays in the medial preoptic area (MPA), dorsomedial hypothalamic nucleus (DMH), and, to a lesser extent, the subparaventricular zone (SPVZ). Here, we found that the injection of the rostral MPA, the periventricular nucleus/medial SPVZ, and the caudal DMH with a mixture of anterograde and retrograde tracers resulted in dense anterograde labeling in the median preoptic nucleus (MnPO), another key sleep-promoting nucleus in the preoptic region. The retrograde labeling in the SCN was evident as previously reported. The injections in either the MPA or the DMH produced similar densities of varicose fibers between the MnPO and the VLPO, while the injections in the SPVZ yielded a greater density of varicose fibers in the MnPO than in the VLPO. These results suggest that the MPA and DMH are potential relay nuclei to mediate SCN output to the MnPO, as well as to the VLPO, for the circadian control of sleep-wake states.

Social and environmental contexts modulate sleep deprivation-induced c-Fos activation in rats.

People often sleep deprive themselves voluntarily for social and lifestyle reasons. Animals also appear to stay awake longer as a result of their natural curiosity to explore novel environments and interact socially with conspecifics. Although multiple arousal systems in the brain are known to act jointly to promote and maintain wakefulness, it remains unclear whether these systems are similarly engaged during voluntary vs. forced wakefulness. Using c-Fos immunohistochemistry, we compared neuronal responses in rats deprived of sleep for 2 h by gentle sensory stimulation, exploration under social isolation, or exploration with social interaction, and rats under undisturbed control conditions. In many arousal, limbic, and autonomic nuclei examined (e.g., anterior cingulate cortex and locus coeruleus), the two sleep deprivation procedures involving exploration were similarly effective, and both were more effective than sleep deprivation with sensory stimulation, in increasing the number of c-Fos immunoreactive neurons. However, some nuclei (e.g., paraventricular hypothalamic nucleus and select amygdala nuclei) were more responsive to exploration with social interaction, while others (e.g., histaminergic tuberomammillary nucleus) responded more strongly to exploration in social isolation. In the rostral basal forebrain, cholinergic and GABAergic neurons responded preferentially to exploration with social interaction, whereas resident neurons in general responded most strongly to exploration without social interaction. These results indicate that voluntary exploration with/without social interaction is more effective than forced sleep deprivation with gentle sensory stimulation for inducing c-Fos in arousal and limbic/autonomic brain regions, and suggest that these nuclei participate in different aspects of arousal during sustained voluntary wakefulness.

Estradiol treatment modulates spontaneous sleep and recovery after sleep deprivation in castrated male rats.

Exogenous estradiol (E) is used occasionally to treat the side effects associated with androgen-deprivation in men, but its effects on sleep patterns have received little attention. We examined whether E modulates sleep patterns and recovery from sleep loss in castrated male rats. Adult male rats were castrated and implanted subcutaneously with Silastic tubes containing either oil (Cast+Oil) or E (Cast+E). Sham-operated male rats (Intact) were implanted with oil-filled tubes. All rats were also implanted with EEG and EMG electrodes for sleep/wake recordings. After two weeks, polysomnographic recordings were made before, during, and following 6h of sleep deprivation (SD). At baseline, the Cast+Oil group showed sleep and EEG patterns similar to those in the Intact group. Compared to these groups, the Cast+E group spent more time awake during the dark (active) phase, and showed higher EEG theta power (a measure of cortical activation) during wake and rapid eye movement (REM) sleep in both the light and dark phases. Following SD, the Cast+E group showed a larger increase from baseline in REM sleep amount, compared to the Cast+Oil group. The Cast+Oil group showed prolonged rebound in non-REM sleep and EEG delta power, and reduced REM sleep rebound, compared to the other two groups. These results indicate that E treatment in castrated male rats promotes baseline wakefulness during the active phase, and facilitates recovery of REM sleep after acute sleep loss. The possible benefit of E treatment for improving sleep quality in androgen-deprived men remains to be investigated.

Female reproductive hormones alter sleep architecture in ovariectomized rats.

STUDY OBJECTIVES: 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). DESIGN: Sleep in ovariectomized rats implanted with oil, 17 ß-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. MEASUREMENTS AND RESULTS: 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. CONCLUSIONS: 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.

Behavioural and neuronal activation after microinjections of AMPA and NMDA into the perifornical lateral hypothalamus in rats.

The perifornical lateral hypothalamic area (PeFLH), which houses orexin/hypocretin (OX) neurons, is thought to play an important role in arousal, feeding, and locomotor activity. The present study examined behavioural effects of activating PeFLH neurons with microinjections of ionotropic glutamate receptor agonists. Three separate unilateral microinjections of either (1) AMPA (1 and 2mM in 0.1 µL artificial cerebrospinal fluid, ACSF) and ACSF, or (2) NMDA (1 and 10mM in 0.1 µL ACSF), and ACSF were made into the PeFLH of adult male rats. Following each injection, the rats were placed into an open field for behavioural scoring for 45 min. Rats were perfused after the third injection for immunohistochemistry for c-Fos and OX to assess the level of activation of OX neurons. Behavioural analyses showed that, as compared to ACSF conditions, AMPA injections produced a dose-dependent increase in locomotion and rearing that persisted throughout the 45 min recording period, and an increase in drinking. Injection of NMDA at 10mM, but not 1mM, induced a transient increase in locomotion and an increase in feeding. Histological analyses showed that while both agonists increased the number of neurons immunoreactive for c-Fos in the PeFLH, only AMPA increased the number of neurons immunoreactive for both c-Fos and OX. There were positive correlations between the number of c-Fos/OX-immunoreactive neurons and the amounts of locomotion, rearing, and drinking. These results support the role of ionotropic glutamate receptors on OX and other neurons in the PeFLH in the regulation of locomotor and ingestive behaviours.