Samuel Deurveilher

Indirect projections from the suprachiasmatic nucleus to major arousal-promoting cell groups in rat: Implications for the circadian control of behavioural state

The circadian clock housed in the suprachiasmatic nucleus (SCN) controls various circadian rhythms including daily sleep-wake cycles. Using dual tract-tracing, we recently showed that the medial preoptic area (MPA), subparaventricular zone (SPVZ) and dorsomedial hypothalamic nucleus (DMH) are well positioned to relay SCN output to two key sleep-promoting nuclei, namely, the ventrolateral and median preoptic nuclei. The present study examined the possibility that these three nuclei may link the SCN with wake-regulatory neuronal groups. Biotinylated dextran-amine with or without cholera toxin B subunit was injected into selected main targets of SCN efferents; the retrograde labeling in the SCN was previously analyzed. Here, anterograde labeling was analyzed in immunohistochemically identified cholinergic, orexin/hypocretin-containing and aminergic cell groups. Tracer injections into the MPA, SPVZ and DMH resulted in moderate to dense anterograde labeling of varicose fibers in the orexin field and the tuberomammillary nucleus. The locus coeruleus, particularly the dendritic field, contained moderate anterograde labeling from the MPA and DMH. The ventral tegmental area, dorsal raphe nucleus, and laterodorsal tegmental nucleus all showed moderate anterograde labeling from the DMH. The substantia innominata showed moderate anterograde labeling from the MPA. These results suggest that the MPA, SPVZ and DMH are possible relay nuclei for indirect SCN projections not only to sleep-promoting preoptic nuclei as previously shown, but also to wake-regulatory cell groups throughout the brain. In the absence of major direct SCN projections to most of these sleep/wake-regulatory regions, indirect neuronal pathways probably play an important role in the circadian control of sleep-wake cycles and other physiological functions.

Indirect projections from the suprachiasmatic nucleus to the ventrolateral preoptic nucleus: a dual tract-tracing study in rat

The suprachiasmatic nucleus (SCN) contains a master clock for most circadian rhythms in mammals, including daily sleep–wake cycles. The ventrolateral preoptic nucleus (VLPO) plays a key role in sleep generation and, as such, might be an important target of the SCN circadian signal. However, direct SCN projections to the VLPO are limited, suggesting that most of the SCN output to the VLPO might be conveyed indirectly. We examined this possibility by microinjecting selected known major targets of SCN efferents with biotinylated dextran‐amine and/or cholera toxin B subunit, followed by analyses of retrograde labelling in the SCN and anterograde labelling in the VLPO. Retrograde labelling results confirmed that the medial preoptic area, subparaventricular zone, dorsomedial hypothalamic nucleus and posterior hypothalamic area all received projections from the SCN; these projections arose predominantly from the shell, as opposed to the core, of the SCN. Anterograde labelling results indicated that these same nuclei also projected to the VLPO, mainly its medial and ventral aspects. Comparison of the results of injections of similar sizes across different target groups indicated that the rostral part of the medial preoptic area and the caudal part of the dorsomedial hypothalamic nucleus were particularly noteworthy for the abundance of both SCN source neurons and efferent fibres and terminals in the VLPO. These results suggest that the SCN might provide indirect input to the VLPO via the medial preoptic area and the dorsomedial hypothalamic nucleus, and that these indirect neuronal pathways might play a major role in circadian control of sleep–wake cycles.

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. J. Comp. Neurol. 498:667–689, 2006.

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.

Stimulant doses of caffeine induce c-FOS activation in orexin/hypocretin-containing neurons in rat.

Although caffeine is a commonly used CNS stimulant, neuronal mechanisms underlying its stimulatory effect are not fully understood. Orexin (hypocretin)-containing neurons play a critical role in arousal and might be activated by acute administration of caffeine. We examined this possibility by using dual-immunostaining for orexin B and c-Fos protein as a marker for neuronal activation. Rats were administered intraperitoneally with 10, 30 or 75 mg/kg caffeine, or saline. As previously reported, caffeine increased locomotion at 10 and 30 mg/kg, but not at 75 mg/kg. The numbers of orexin-immunoreactive and non-orexin-immunoreactive neurons expressing c-Fos were analysed using three counting boxes within the orexin field in the posterior hypothalamus. Compared with saline, all doses of caffeine increased the number of cells immunoreactive for both orexin and c-Fos. The average magnitude of this increase across doses in orexin neurons differed amongst regions; c-Fos expression increased by 343% in the perifornical area and by 158% in the more medial, dorsomedial nucleus. In the lateral hypothalamic area, c-Fos expression increased by 226% at 10 and 30 mg/kg but no change was seen at 75 mg/kg. In contrast, caffeine significantly increased the number of non-orexin-immunoreactive neurons expressing c-Fos only in the dorsomedial nucleus. These results indicate that systemically administered caffeine preferentially activates orexin neurons over non-orexin neurons in the same field, and that this activation is most pronounced in the perifornical region where orexin neurons are most concentrated. The activation of orexin neurons might play a role in the behavioural activation by caffeine.

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.

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.

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.

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.