The sleep-wake distribution contributes to clock gene expression: a descriptive and a mechanistic study in mice

The sleep-wake distribution (i.e. the duration and timing of sleep across the 24-h day) is orchestrated by the interaction of two processes: the sleep homeostat, which keeps track of time spent awake and asleep, and the circadian clock, which gates the timing of sleep. The mechanisms underlying the clock are well understood: overt rhythmic behavior in mammals is coordinated by the suprachiasmatic nucleus and at the molecular level by intertwining transcriptional-translational feedback loops of so-called clock genes, ensuring a period of ca. 24 hours. The substrate underlying the sleep homeostat is unknown, but clock genes appear implicated because: i) mutations and deletions of clock genes affect the sleep homeostat); ii) sleep deprivation (SD) impairs the binding of clock gene proteins to their target clock genes; iii) SD changes clock gene expression. In this thesis, a descriptive and a mechanistic study are presented to inspect more closely the sleep-wake distributions’ contribution to clock gene expression. The descriptive study made use of a mouse model where bioluminescence is measured as a proxy for period-2 (PER2) protein levels, combined with electroencephalogram (EEG) recordings to determine sleep-wake state. Under undisturbed conditions, PER2 bioluminescence changed as a function of sleep and wake. Twelve 2Hr SDs scheduled across two days reduced the amplitude of PER2 bioluminescence in 3 out of 4 mice. Thus, sleep-wake state contributes to PER2 bioluminescence. However, the reliability of PER2 bioluminescence as a proxy for PER2 protein levels remains to be verified. In the second study, the contribution of Cold Inducible RNA Binding Protein (CIRBP) to SD-incurred changes in clock gene expression was investigated, based on the observations that i) daily changes in cortical Cirbp appear mainly sleep-wake driven, possibly through cortical temperature; ii) CIRBP is necessary for high amplitude clock gene expression in vitro. First, we established that the sleep-wake distribution drives the changes in cortical temperature of the mouse. Second, we found that the SD induced changes in cortical Rev-erbα was attenuated in the absence of CIRBP, whereas the expression of Clock and Per2 was amplified. Third, and based on the premise that clock genes contribute to sleep regulation, we observed that Cirbp KO mice loose REM sleep after SD compared to their WT littermates. Altogether, this thesis i) supports the importance of considering the sleep-wake distribution when using clock gene expression as a state variable of the clock; ii) demonstrates that CIRBP modulates the SD incurred changes in cortical clock gene expression and contributes to REM sleep recovery

Cold-inducible RNA-binding protein (CIRBP) adjusts clock-gene expression and REM-sleep recovery following sleep deprivation.

Sleep depriving mice affects clock-gene expression, suggesting that these genes contribute to sleep homeostasis. The mechanisms linking extended wakefulness to clock-gene expression are, however, not well understood. We propose CIRBP to play a role because its rhythmic expression is i) sleep-wake driven and ii) necessary for high-amplitude clock-gene expression in vitro. We therefore expect Cirbp knock-out (KO) mice to exhibit attenuated sleep-deprivation-induced changes in clock-gene expression, and consequently to differ in their sleep homeostatic regulation. Lack of CIRBP indeed blunted the sleep-deprivation incurred changes in cortical expression of Nr1d1, whereas it amplified the changes in Per2 and Clock. Concerning sleep homeostasis, KO mice accrued only half the extra REM sleep wild-type (WT) littermates obtained during recovery. Unexpectedly, KO mice were more active during lights-off which was accompanied with faster theta oscillations compared to WT mice. Thus, CIRBP adjusts cortical clock-gene expression after sleep deprivation and expedites REM-sleep recovery

Galanin neurons unite sleep homeostasis and α2-adrenergic sedation

Our urge to sleep increases with time spent awake, until sleep becomes inescapable. The sleep following sleep deprivation is longer and deeper, with an increased power of delta (0.5 - 4 Hz) oscillations, a phenomenon termed sleep homeostasis [1-4]. Although widely-expressed genes regulate sleep homeostasis [1, 4-10], and the process is tracked by somnogens and phosphorylation [1, 3, 7, 11-14], at the circuit level sleep homeostasis has remained mysterious. Previously we found that sedation induced with 2 adrenergic agonists (e.g. dexmedetomidine) and sleep homeostasis both depend on the preoptic (PO) hypothalamus [15, 16]. Dexmedetomidine, increasingly used for long-term sedation in intensive care units [17], induces a NREM-like sleep but with undesirable hypothermia [18, 19]. Within the PO, various neuronal subtypes (e.g. GABA/galanin and glutamate/NOS1) induce NREM sleep [20-22] and concomitant body cooling [21, 22]. This could be because NREM sleep’s restorative effects depend on lower body temperature [23, 24]. Here, we show that mice with lesioned PO galanin neurons have reduced sleep homeostasis: in the recovery sleep following sleep deprivation, there is a diminished increase in delta power, and the mice catch up little on lost sleep. Furthermore, dexmedetomidine cannot induce high-power delta oscillations or sustained hypothermia. Some hours after dexmedetomidine administration to wild-type mice there is a rebound in delta power when they enter normal NREM sleep, reminiscent of emergence from torpor. This delta rebound is reduced in mice lacking PO galanin neurons. Thus, sleep homeostasis and dexmedetomidine-induced sedation require PO galanin neurons and likely share common mechanisms

The Temperature Dependence of Sleep

Mammals have evolved a range of behavioural and neurological mechanisms that coordinate cycles of thermoregulation and sleep. Whether diurnal or nocturnal, sleep onset and a reduction in core temperature occur together. Non-rapid eye movement (NREM) sleep episodes are also accompanied by core and brain cooling. Thermoregulatory behaviours, like nest building and curling up, accompany this circadian temperature decline in preparation for sleeping. This could be a matter of simply comfort as animals seek warmth to compensate for lower temperatures. However, in both humans and other mammals, direct skin warming can shorten sleep-latency and promote NREM sleep. We discuss the evidence that body cooling and sleep are more fundamentally connected and that thermoregulatory behaviours, prior to sleep, form warm microclimates that accelerate NREM directly through neuronal circuits. Paradoxically, this warmth might also induce vasodilation and body cooling. In this way, warmth seeking and nesting behaviour might enhance the circadian cycle by activating specific circuits that link NREM initiation to body cooling. We suggest that these circuits explain why NREM onset is most likely when core temperature is at its steepest rate of decline and why transitions to NREM are accompanied by a decrease in brain temperature. This connection may have implications for energy homeostasis and the function of sleep

Exploration of neuronal ensembles responsible for sleep and body temperature regulation

Sleep is a behaviour we experience every day but the fundamental function(s) and the neuronal circuitry underlying it remain mystery. Previous study from our laboratory suggests the lateral preoptic (LPO) area of the hypothalamus plays an important role in recovery sleep (RS) after sleep deprivation (SD) as well as 2-adrenergic agonist (dexmedetomidine)-induced sedation and hypothermia. Preliminary data of whole-brain mapping of the neuronal activity of 5-hr SD mice and 2- hr RS (followed 5-hr SD) mice by cFos expression confirmed that the LPO shows higher neuronal activity in RS mice compared to SD mice. Cell-type specific ablation of galaninergic neurons in the LPO abolished sleep homeostasis in mice, in terms of the amount of RS as well as the increased slow wave activity (SWA) of RS after SD which is a hallmark of sleep homeostasis. In addition, mice with ablation of LPOGal neurons have a permanent elevation in their body temperature compared to control mice. LPOGal neurons are also involved in mediating dexmedetomidine (DEX)-induced sedation and hypothermia. Mice without LPOGal neurons have reduced effects: administration of DEX cannot induce high-power oscillations or sustained hypothermia. Together, LPOGal neurons unite sleep homeostasis and 2-adrenergic sedation. Preliminary whole-brain cFos mapping also revealed a few other potential brain regions that might be involved in sleep/wake regulation, including the ventral tegmental area (VTA). Chemogenetic activation and inactivation increase the neuronal activity of VTAVglut2 and VTAVgat neurons, respectively, and both increase wakefulness. VTAVglut2/Nos1 neurons promote wakefulness by sending excitatory projections to both the lateral hypothalamus (LH) and nucleus accumbens (NAc), whereas the wake-inhibiting effect of VTAVgat neurons is achieved by sending inhibitory projections to local VTAVglut2 and VTADA neurons as well as to the orexin neurons in the LH, implying the significance of the VTA in sleep/wake regulation.Open Acces