Ketema N. Paul

Dysregulation of Inflammatory Responses by Chronic Circadian Disruption

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

Reproductive hormone replacement alters sleep in mice

Several studies have reported that reproductive hormones can alter baseline sleep-wake states, however, no studies in mice have examined whether reproductive hormone replacement in adult females and males influences sleep. In this study, we determined whether androgen replacement in males and estrogen replacement in females alter sleep-wake amount and sleep rebound after extended wakefulness. The gonads from adult male and female C57BL/6J mice were removed and animals were implanted with continuous release hormone or placebo pellets. Male mice received testosterone and females received 17beta-estradiol. Recording electrodes were implanted to monitor sleep-wake states under baseline conditions and in response to 6h of sleep deprivation. During baseline recording estradiol-treated females exhibited a reduction in NREM sleep amount that was predominant during the dark phase. Testosterone-treated males conversely, exhibited an increase in NREM sleep amount. After sleep deprivation, hormone-treated males and females exhibited similar amounts of recovery sleep however males exhibited slightly more sleep than placebo-treated controls. The results of these experiments demonstrate that the androgens and estrogens are primarily responsible for sex differences in baseline sleep-wake amount but do not have substantial effects on homeostatic sleep rebound after extended wakefulness.

The role of retinal photoreceptors in the regulation of circadian rhythms

The circadian clock is an evolutionarily, highly conserved feature of most organisms. This internal timing mechanism coordinates biochemical, physiological and behavioral processes to maintain synchrony with the environmental cycles of light, temperature and nutrients. Several studies have shown that light is the most potent cue used by most organisms (humans included) to synchronize daily activities. In mammals, light perception occurs only in the retina; three different types of photoreceptors are present within this tissue: cones, rods and the newly discovered intrinsically photosensitive retinal ganglion cells (ipRGCs). Researchers believe that the classical photoreceptors (e.g., the rods and the cones) are responsible for the image-forming vision, whereas the ipRGCs play a key role in the non-image forming vision. This non-image-forming photoreceptive system communicates not only with the master circadian pacemaker located in the suprachiasmatic nuclei of the hypothalamus, but also with many other brain areas that are known to be involved in the regulation of several functions; thus, this non-image forming system may also affect several aspects of mammalian health independently from the circadian system.

Influence of Sex on Sleep Regulatory Mechanisms

The ability of biological sex and sex-driven characteristics to alter sleep states may contribute to gender disparities in sleep disorders. Sex influences sleep-wake amount, the daily timing of the sleep-wake cycle, and the ability to restore sleep after extended wakefulness. Several lines of evidence suggest that in mammals, reproductive hormones are responsible for the effects of sex on sleep and may have organizational and activational influences on sleep regulatory mechanisms. In humans, exogenously administered estrogens and progestins generally enhance sleep amount and continuity, whereas androgens appear to have a positive impact on rapid eye movement (REM) sleep but disrupt sleep consolidation. In rodent studies, however, female reproductive hormones appear to enhance wakefulness, and male gonadal hormones reinforce sleep. Rodent studies have also revealed that neonatal exposure to reproductive hormones organizes adult sleep-wake architecture. This paper reviews how sex and reproductive hormones interact with circadian and homeostatic sleep regulatory mechanisms in humans and animal models. We examine the organizational and activational nature of these interactions and also review how these interactions change with advancing age. Finally, we discuss the potential for genetic sex to influence sleep states. It is our hope that a better understanding of the mechanisms through which sex influences sleep-wake states will lead to improvements in the design of studies that examine gender disparities in sleep-wake disorders.

N-acetylserotonin promotes hippocampal neuroprogenitor cell proliferation in sleep-deprived mice

N-acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole, is regulated in a circadian rhythm. NAS swiftly activates TrkB in a circadian manner and exhibits antidepressant effect in a TrkB-dependent manner. Here we show that NAS regulates an early event of neurogenesis by increasing neuronal progenitor cell (NPC) proliferation. Subchronic and chronic NAS administration induces NPC proliferation in adult mice. Chronic NAS treatment triggers TrkB receptor activation and its downstream signaling in NPCs. Blockade of TrkB abolishes NAS-elicited neurogenesis in TrkBF616A knockin mice, suggesting that TrkB activation is essential for the effect of NAS-induced NPC proliferation. Moreover, NAS induces NPC proliferation in both active and sleeping phases of the mice. Strikingly, NAS significantly enhances NPC proliferation in sleep-deprived mice. Thus, our finding demonstrates a unique function of NAS in promoting robust NPC proliferation, which may contribute to hippocampal plasticity during sleeping period.

GABAA receptor activation suppresses Period 1 mRNA and Period 2 mRNA in the suprachiasmatic nucleus during the mid-subjective day.

The mammalian circadian clock can be entrained by photic and nonphotic environmental time cues. γ‐aminobutyric acid (GABA) is a nonphotic stimulus that induces phase advances in the circadian clock during the middle of the subjective day. Several nonphotic stimuli suppress Period 1‐ and Period 2 mRNA expression in the suprachiasmatic nucleus (SCN); however, the effect of GABA on Period mRNA is unknown. In the present study we demonstrate that microinjection of the GABAA receptor agonist muscimol into the SCN region suppresses the expression of Period 1 mRNA in the hamster. A significant suppression of Period 2 mRNA following microinjection of muscimol was not observed in free‐running conditions. However, Period 2 mRNA was significantly reduced following muscimol treatment when animals were maintained under a light cycle and transferred to constant darkness 42 h prior to treatment. An additional study investigated the maximum behavioural phase advance inducible by GABAA receptor activation.Together, these data indicate that, like other nonphotic stimuli, GABA suppresses Period 1‐ and Period 2 mRNA in the SCN.

Light and GABAA receptor activation alter Period mRNA levels in the SCN of diurnal Nile grass rats

We examined Period (Per) mRNA rhythms in the suprachiasmatic nucleus (SCN) of a diurnal rodent and assessed how phase‐shifting stimuli acutely affect SCN Per mRNA using semiquantitative in situ hybridization. First, Per1 and Per2 varied rhythmically in the SCN over the course of one circadian cycle in constant darkness: Per1 mRNA was highest in the early to mid‐subjective day, while Per2 mRNA levels peaked in the late subjective day. Second, acute light exposure in the early subjective night significantly increased both Per1 and Per2 mRNA. Third, Per2 but not Per1 levels decreased 1 and 2 h after injection of the γ‐aminobutyric acid (GABA)A receptor agonist muscimol into the SCN during the subjective day. Fourth, muscimol also reduced the light‐induced Per2 in the early subjective night, but Per1 induction by light was not significantly affected. Consistent with previous studies, these data demonstrate that diurnal and nocturnal animals show very similar daily patterns of Per mRNA and light‐induced Per increases in the SCN. As with light, muscimol alters circadian phase, and daytime phase alterations induced by muscimol are associated with significant decreases in Per2 mRNA. In diurnal animals, muscimol‐induced decreases in Per are associated with phase delays rather than advances. The direction of the daytime phase shift may be determined by the relative suppression of Per1 vs. Per2 in SCN cells. As in nocturnal animals, changes in Per1 and Per2 mRNA by photic and non‐photic stimuli appear to be associated with circadian phase alteration.

Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture, Reduces Diurnal Corticosterone Levels, and Enhances Spatial Memory

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Nav1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also proposed in studies examining the genetic basis of attempted suicide and bipolar disorder. In addition, mice with a Scn8a loss-of-function mutation (Scn8amed-Tg/+) show altered anxiety and depression-like phenotypes. Because psychiatric abnormalities are often associated with altered sleep and hormonal patterns, we evaluated heterozygous Scn8amed-jo/+ mutants for alterations in sleep-wake architecture, diurnal corticosterone levels, and behavior. Compared with their wild-type littermates, Scn8amed-jo/+ mutants experience more non-rapid eye movement (non-REM) sleep, a chronic impairment of REM sleep generation and quantity, and a lowered and flattened diurnal rhythm of corticosterone levels. No robust differences were observed between mutants and wild-type littermates in locomotor activity or in behavioral paradigms that evaluate anxiety or depression-like phenotypes; however, Scn8amed-jo/+ mutants did show enhanced spatial memory. This study extends the spectrum of phenotypes associated with mutations in Scn8a and suggests a novel role for altered sodium channel function in human sleep disorders.

Altered Sleep Regulation in a Mouse Model of SCN1A-Derived Genetic Epilepsy with Febrile Seizures Plus (GEFS+)

Mutations in the voltage‐gated sodium channel (VGSC) gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition to seizures, patients with SCN1A mutations often experience sleep abnormalities, suggesting that SCN1A may also play a role in the neuronal pathways involved in the regulation of sleep. However, to date, a role for SCN1A in the regulation of sleep architecture has not been directly examined. To fill this gap, we tested the hypothesis that SCN1A contributes to the regulation of sleep architecture, and by extension, that SCN1A dysfunction contributes to the sleep abnormalities observed in patients with SCN1A mutations.

Regulation of light's action in the mammalian circadian clock: role of the extrasynaptic GABAA receptor

GABA(A) receptor agonists act in the suprachiasmatic nucleus (SCN) to reset circadian rhythms during the day but inhibit the ability of light to reset rhythms during the night. In the present study, we examined whether these paradoxical differences in the effect of GABA(A) receptor stimulation on the circadian system are mediated by separate GABA(A) receptor subtypes. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a GABA(A) receptor agonist, preferentially activates GABA(A) receptors in extrasynaptic locations. THIP, muscimol (a GABA(A) agonist), or vehicle were microinjected into the SCN region of Syrian hamsters free-running in constant darkness during the mid-subjective day, early subjective night, or late subjective night. The subjective night injections were followed by a light pulse or sham control. Behavioral phase shifts of wheel running rhythms and both Period1 (Per1) and Per2 mRNA levels in the SCN were assessed. Animals that received THIP during the subjective day did not exhibit significant phase alterations. During the early and late subjective night, however, THIP abolished the phase-shifting effects of light and the ability of light to increase Per1 and Per2 mRNA levels. The ability of N-methyl-d-aspartic acid to phase-shift wheel running rhythms was also attenuated by THIP. Together these data demonstrate that THIP does not produce phase shifts during the subjective day, but does inhibit the ability of light to produce phase shifts. Thus, extrasynaptic GABA(A) receptors appear to play a role in regulating light input to the SCN, while a different population of GABA(A) receptors appears to be responsible for daytime effects of GABA.