TRPV1 participates in the activation of clock molecular machinery in the brown adipose tissue in response to light-dark cycle.

Transient receptor potential (TRPs) channels are involved in thermogenesis, and temperature and energy balance control. Mice lacking TrpV1 become more obese and develop insulin resistance when fed with high fat diet; however, a relationship between metabolic disorders, TRP channels, and clock genes is still unknown. Based on this, we hypothesized that TRPV1 channels would be involved in the synchronization of clock genes in the peripheral tissues. To address this question, we used wild type (WT) and TrpV1 knockout (KO) mice kept in constant darkness (DD) or in light-dark cycle (LD). In WT mouse brown adipose tissue (BAT), TrpV1 oscillated with higher expression at scotophase, Per1 and Per2 showed the same profile, and Bmal1 transcript only oscillated in DD. Interestingly, the oscillatory profile of these clock genes was abolished in TrpV1 KO mice. WT mouse Ucp1 was upregulated in LD as compared to DD, showing no temporal variation; mice lacking TrpV1 showed Ucp1 oscillation with a peak at the photophase. Remarkably, TrpV1 KO mice displayed less total activity than WT only when submitted to LD. We provide evidence that TRPV1 is an important modulator of BAT clock gene oscillations. Therefore, temperature and/or light-dependent regulation of TRPV1 activity might provide novel pharmacological approaches to treat metabolic disorders.

Effects of circadian disruption on methamphetamine consumption in methamphetamine-exposed rats.

RATIONALE: A substantial number of clinical studies indicate associations between sleep abnormalities and drug abuse; however, the role played by the circadian system in the development of addiction is largely unknown. OBJECTIVE: The aim of this study was to examine the effects of experimentally induced chronic jet lag on methamphetamine consumption in a rat model of methamphetamine drinking. METHODS: Male Sprague-Dawley rats (n = 32) were housed in running wheel cages in a 12:12 h light:dark cycle. One group of rats (n = 16) was given 2 weeks of forced methamphetamine consumption (0.01 % in drinking water; meth pre-exposed) while a second group (n = 16, not pre-exposed) received water only. This was followed by a 2-week abstinence period during which half of the animals from each group were exposed to four consecutive 6-h advancing phase shifts of the light:dark cycle, while the other half remained on the original light:dark cycle. Methamphetamine consumption was assessed in all rats following the deprivation period using a two-bottle choice paradigm. RESULTS: Methamphetamine consumption was initially lower in methamphetamine pre-exposed versus not pre-exposed rats. However, during the second week following abstinence, consumption was significantly higher in phase-shifted rats of the methamphetamine pre-exposed group compared to all other groups. CONCLUSIONS: These data reveal an effect of circadian rhythm disturbance on methamphetamine consumption and suggest that dysregulation of the circadian system be considered in the etiology of relapse and addiction.

Cold and hunger induce diurnality in a nocturnal mammal.

The mammalian circadian system synchronizes daily timing of activity and rest with the environmental light-dark cycle. Although the underlying molecular oscillatory mechanism is well studied, factors that influence phenotypic plasticity in daily activity patterns (temporal niche switching, chronotype) are presently unknown. Molecular evidence suggests that metabolism may influence the circadian molecular clock, but evidence at the level of the organism is lacking. Here we show that a metabolic challenge by cold and hunger induces diurnality in otherwise nocturnal mice. Lowering ambient temperature changes the phase of circadian light-dark entrainment in mice by increasing daytime and decreasing nighttime activity. This effect is further enhanced by simulated food shortage, which identifies metabolic balance as the underlying common factor influencing circadian organization. Clock gene expression analysis shows that the underlying neuronal mechanism is downstream from or parallel to the main circadian pacemaker (the hypothalamic suprachiasmatic nucleus) and that the behavioral phenotype is accompanied by phase adjustment of peripheral tissues. These findings indicate that nocturnal mammals can display considerable plasticity in circadian organization and may adopt a diurnal phenotype when energetically challenged. Our previously defined circadian thermoenergetics hypothesis proposes that such circadian plasticity, which naturally occurs in nocturnal mammals, reflects adaptive maintenance of energy balance. Quantification of energy expenditure shows that diurnality under natural conditions reduces thermoregulatory costs in small burrowing mammals like mice. Metabolic feedback on circadian organization thus provides functional benefits by reducing energy expenditure. Our findings may help to clarify relationships between sleep-wake patterns and metabolic phenotypes in humans.

Neurobiology: the eye within the brain.

All vertebrates except mammals have photoreceptors within their brains; however, the light-sensitive cells have never been unambiguously identified. A new paper provides direct evidence of photosensitivity in cerebrospinal fluid (CSF)-contacting neurons in quail brain that mediate the seasonal reproductive response.

The nocturnal bottleneck and the evolution of activity patterns in mammals.

In 1942, Walls described the concept of a 'nocturnal bottleneck' in placental mammals, where these species could survive only by avoiding daytime activity during times in which dinosaurs were the dominant taxon. Walls based this concept of a longer episode of nocturnality in early eutherian mammals by comparing the visual systems of reptiles, birds and all three extant taxa of the mammalian lineage, namely the monotremes, marsupials (now included in the metatherians) and placentals (included in the eutherians). This review describes the status of what has become known as the nocturnal bottleneck hypothesis, giving an overview of the chronobiological patterns of activity. We review the ecological plausibility that the activity patterns of (early) eutherian mammals were restricted to the night, based on arguments relating to endothermia, energy balance, foraging and predation, taking into account recent palaeontological information. We also assess genes, relating to light detection (visual and non-visual systems) and the photolyase DNA protection system that were lost in the eutherian mammalian lineage. Our conclusion presently is that arguments in favour of the nocturnal bottleneck hypothesis in eutherians prevail.

Effects of ovarian hormones on internal circadian organization in rats.

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is the central pacemaker driving rhythms in endocrine physiology. Gonadal steroid hormones affect behavioral rhythms and clock gene expression. However, the impact of fluctuating ovarian steroid levels during the estrous cycle on internal circadian organization remains to be determined. Further, it is not known if steroid hormone depletion, as in menopause, affects the timing system. To determine the influence of estrous cycle stage and steroid depletion on circadian organization, we measured clock gene expression in the SCN and peripheral tissues from cycling and ovariectomized (OVX) period1-luciferase (per1-luc) transgenic rats. The estrous cycle had modest effects on mean phase and phase distribution of per1-luc expression in the SCN. Surprisingly, peak per1-luc expression in the SCN was widely distributed mainly at night, regardless of cycle stage, an effect eliminated by OVX. Treatment of SCN tissue explants with ovarian steroids did not significantly affect per1-luc expression, suggesting that brain regions outside the SCN mediate the phasic effects of steroids. Our data demonstrate that estrous cycle stage has tissue-dependent effects on the phase of per1-luc expression, phase synchrony among oscillators, and the phase relationship between some peripheral clocks and the light-dark cycle. They also reveal that steroid hormone depletion following OVX alters the timing system, suggesting that the decline in hormone levels, common during the transition to menopause, may be associated with irregular internal circadian organization. This effect on the timing system could contribute to the behavioral and physiological changes associated with this transition.

Methamphetamine and dopamine receptor D1 regulate entrainment of murine circadian oscillators.

We investigated the effect of methamphetamine (MA) injections on the circadian organization of behavior and individual tissues in the mouse. Scheduled, daily injections of MA resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. Daily MA also shifted the peak time of PER2 expression in the liver, pituitary, and salivary glands. It has been suggested that reward pathways, and dopamine signaling in particular, may underlie the effects of MA on the circadian system. To test this hypothesis, we examined the effect of the D1 receptor antagonist SCH23390 (SCH) on circadian rhythms. The MA-induced shift in the phase of pituitary and salivary glands was attenuated by pretreatment with the D1 antagonist SCH23390 (SCH). Interestingly, daily SCH, administered alone, also affected some circadian oscillators. The livers and lungs (but not pituitaries or salivary glands) of mice treated with daily injections of SCH displayed disrupted rhythms of PER2 expression, suggesting that D1 receptor signaling is important for entrainment of these organs. From these results, we conclude that MA has widespread effects within the circadian system, and that these effects are mediated, at least in part, by the dopaminergic system. This study also identifies a role for dopamine signaling in normal entrainment of circadian oscillators.

Central control of peripheral circadian oscillators.

The suprachiasmatic nucleus of the hypothalamus and at least two other unidentified central pacemakers regulate the temporal structure of a circadian network that involves almost every organ in the body. Phase control is central to the efficient function of this system. Individual circadian oscillators in tissues and organs in the periphery bear adaptive phase relationships to the external light cycle, the central pacemakers and to each other. The known signals that regulate and maintain these phase relationships come from the autonomic nervous system, the pineal and adrenal glands, behavioral cycles of feeding and activity and the rhythm of body temperature. It is likely that there are many unknown signals as well. Disrupting the network can produce severe pathology.

Excess androgen during puberty disrupts circadian organization in female rats.

Circadian clocks have been described in each tissue of the hypothalamo-pituitary-ovarian axis. Although a role for the clock in the timing of ovulation is indicated, the impact of diseases that disrupt fertility on clock function or the clocks' role in the etiology of these pathologies has yet to be fully appreciated. Polycystic ovary syndrome (PCOS) is a particularly devastating endocrinopathy, affecting approximately 10% of women at childbearing age. Common features of PCOS are a polycystic ovary, amenorrhea, and excess serum androgen. Approximately 40% of these women have metabolic syndrome, including hyperinsulinemia, dyslipidemia, and hyperleptinemia. It has been suggested that excess androgen is a critical factor in the etiology of PCOS. We have examined the effects of androgen excess during puberty on the phase of circadian clocks in tissues of the metabolic and hypothalamo-pituitary-ovarian axes. Female period1-luciferase (per1-luc) rats were exposed to androgen (5α-dihydrotestosterone [DHT]) or placebo for 4-6 weeks (short term) or 9-15 weeks (long term). As expected, DHT-treated animals gained more weight than controls and had disrupted estrous cycles. At the end of treatment, tissues, including the liver, lung, kidney, white adipose, cornea, pituitary, oviduct, and ovarian follicles, were cultured, and per1-luc expression in each was recorded. Analysis of per1-luc expression revealed that DHT exposure increased phase distribution of multiple oscillators, including ovarian follicles, liver, and adipose, and altered phase synchrony between animals. These data suggest that excess androgen during puberty, a common feature of PCOS, negatively affects internal circadian organization in both the reproductive and metabolic axes.

Aging differentially affects the re-entrainment response of central and peripheral circadian oscillators.

Aging produces a decline in the amplitude and precision of 24 h behavioral, endocrine, and metabolic rhythms, which are regulated in mammals by a central circadian pacemaker within the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. Disruption of the circadian system, as experienced during transmeridian travel, can lead to adverse health consequences, particularly in the elderly. To test the hypothesis that age-related changes in the response to simulated jet lag will reflect altered circadian function, we examined re-entrainment of central and peripheral oscillators from young and old PER2::luciferase mice. As in previous studies, locomotor activity rhythms in older mice required more days to re-entrain following a shift than younger mice. At the tissue level, effects of age on baseline entrainment were evident, with older mice displaying earlier phases for the majority of peripheral oscillators studied and later phases for cells within most SCN subregions. Following a 6 h advance of the light:dark cycle, old mice displayed slower rates of re-entrainment for peripheral tissues but a larger, more rapid SCN response compared to younger mice. Thus, aging alters the circadian timing system in a manner that differentially affects the re-entrainment responses of central and peripheral circadian clocks. This pattern of results suggests that a major consequence of aging is a decrease in pacemaker amplitude, which would slow re-entrainment of peripheral oscillators and reduce SCN resistance to external perturbation.

Glucocorticoids as entraining signals for peripheral circadian oscillators.

Mammalian circadian organization is governed by pacemaker neurons in the brain that communicate with oscillators in peripheral tissues. Adrenal glucocorticoids are important time-giving signals to peripheral circadian oscillators. We investigated the rhythm of Per1-luc expression in pineal, pituitary, salivary glands, liver, lung, kidney, cornea as well as suprachiasmatic nucleus from adrenalectomized and sham-operated rats kept under light-dark cycles, or exposed to single 6-h phase delays or advances of their light cycles. Adrenalectomy shifted the phases of Per1-luc in liver, kidney, and cornea and caused phase desynchrony and significant dampening in the rhythmicity of cornea. Treatment with hydrocortisone shifted the phases of Per1-luc in most of the tissues examined, even those that were not affected by adrenalectomy. The rhythm in cornea recovered in animals given hydrocortisone in vivo or when corneas were treated with dexamethasone in vitro. Adrenalectomy increased the rate of reentrainment after phase shifts in liver, kidney, cornea, pineal, lung, and suprachiasmatic nucleus but not in pituitary and salivary glands. Our data show that glucocorticoids act as strong entraining signals for peripheral circadian oscillators and may feed back on central oscillators as well.

Photic resetting of the circadian clock is correlated with photic habitat in Anolis lizards.

Circadian rhythms are regulated by an internal clock, which is itself synchronized to environmental cues such as light and temperature. It is widely assumed that the circadian system is adapted to local cues, which vary enormously across habitats, yet the comparative data necessary for testing this idea are lacking. We examined photic and thermal resetting of the circadian clock in five species of Anolis lizards whose microhabitats differ in the amounts of sun and shade. The primary circadian oscillator in Anolis is the pineal gland, which produces the hormone melatonin. A flow-through culture system was employed to measure rhythmic melatonin output from individually cultured pineal glands. All species showed temperature-compensated circadian rhythms of pineal melatonin. Light caused significant phase delays of the melatonin rhythm, and this effect varied among species. Controlling for phylogenetic differences, the results indicate that the pineal glands of shade-dwelling species are more sensitive to photic resetting than species living in more brightly illuminated habitats. The differences were not due to variation in free-running period, but may be due to variation in oscillator phase and/or robustness. Surprisingly, thermal resetting was not statistically significant. Overall, the results suggest that the Anolis circadian system is adapted to photic habitat.

Photic induction of locomotor activity is correlated with photic habitat in Anolis lizards.

A variety of ecologically important behaviors, including circadian rhythms and seasonal reproduction, are influenced by non-visual responses to light, yet very little is known about the relationship between photic habitat and non-visual photoreception. Puerto Rican Anolis lizards have diverged into multiple photic niches, making them a good model for non-visual photosensory ecology. We investigated the photic induction of locomotor activity, a non-visual response to light, in four species of Anolis comprising two pairs of closely related, ecomorphologically similar species whose microhabitats differ in solar irradiance. We developed a device for continuous, automated detection and recording of anole locomotor activity, and used it to characterize activity under 12:12 h light-dark cycles. Next, we administered a series of 2-h light pulses during the dark period of the light-dark cycle and measured the increase in locomotor activity relative to baseline dark activity. Five different irradiances (ranging from very dim to daytime levels) were given to each individual lizard on separate nights. As expected, light caused an irradiance-dependent increase in locomotor activity in all four species. The responses at the highest irradiances were significantly greater in species occupying relatively more shaded habitats, suggesting that non-visual photoreception may be adapted to habitat light in Anolis lizards.

The effect of light on melatonin secretion in the cultured pineal glands of Anolis lizards.

Melatonin, a hormone produced by the pineal gland, is important for regulating circadian rhythms in many animals. Light at night causes an acute suppression of melatonin in nearly all vertebrate species. A previous study found that light failed to suppress melatonin in the lizard Anolis carolinensis. This is a surprising result given that the Anolis pineal gland is intrinsically photosensitive, is a key pacemaker controlling locomotor activity, and can be directly entrained to a light-dark cycle. To find out if the lack of photic suppression is widespread in the Anolis genus, we investigated the acute effects of light on melatonin secretion in five different species of Anolis using flow-through tissue culture. We administered a two-hour pulse of bright light to isolated pineal glands during the night. The results show photic suppression of melatonin in all five Anolis species, but the suppression is weak relative to that seen in other vertebrates. Moreover, Anolis species differ in the magnitude of the effect. These findings are discussed in the context of vertebrate pineal evolution and the ecology of Anolis lizards. Given their extensive phylogenetic and ecological divergence, Anolis lizards provide a promising system for investigating the ecology and evolution of circadian organization.

Resveratrol dietary supplementation shortens the free-running circadian period and decreases body temperature in a prosimian primate.

Resveratrol (RSV) is a dietary polyphenolic compound with several positive effects on metabolic functions and longevity. We tested the effect of RSV on the circadian clock in a nonhuman primate, the gray mouse lemur. The impact of a 2-week dietary supplementation of RSV on the rhythms of locomotor activity and body temperature in constant dark conditions (DD) was investigated in young (n = 7) and old (n = 6) animals. RSV supplementation followed 2 weeks in DD under normal diet (CTL). In both young and old animals receiving RSV, we observed a shortening of the free-running period compared to those under CTL (-15 minutes in young animals and -45 minutes in old animals), accompanied by a lower mean body temperature in both age groups and decreased locomotor activity in young animals. Thus, RSV is a food component capable of influencing a primate's circadian clock. This property may be of interest clinically in the context of the treatment of circadian disruption and in the context of the effects of RSV ingestion on health.

Circadian clocks in mammalian reproductive physiology: effects of the "other" biological clock on fertility.

As a discipline, chronobiology has come of age in the last 25 years. There has been an exponential increase in our understanding of the molecular mechanism underlying circadian rhythms of gene expression, physiology, and behavior. While the mammalian clock mechanism has not yet been fully described, most of the primary gears have probably been identified; however, there remains a large submerged portion of this physiological iceberg. What is the extent of "clock-controlled gene" expression in the myriad cell types in mammals? What are the cell specific physiological processes that depend either directly or indirectly on the clock? These questions remain largely unanswered, but recent advances suggest a substantial link between basic clock function and physiology in several systems. In the reproductive system, there has been a recent surge in research on molecular clock function in neuroendocrine and endocrine tissues. This makes sense a priori, given the established link between the circadian clock, behavior (including reproductive behavior), and endocrine physiology. By understanding the role of the clock in basic mammalian reproductive physiology, we can begin to explore its role in the onset and progression of diseases that negatively affect fertility. Advances in this area will certainly yield novel insights into the etiology of these disorders and may provide new and exciting avenues for clinical research in reproduction and fertility.

Circadian organization is governed by extra-SCN pacemakers.

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.

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.

Circadian clocks in the ovary.

Clock gene expression has been observed in tissues of the hypothalamic-pituitary-gonadal (HPG) axis. Whereas the contribution of hypothalamic oscillators to the timing of reproductive biology is well known, the role of peripheral oscillators like those in the ovary is less clear. Circadian clocks in the ovary might play a role in the timing of ovulation. Disruption of the clock in ovarian cells or desynchrony between ovarian clocks and circadian oscillators elsewhere in the body may contribute to the onset and progression of various reproductive pathologies. In this paper, we review evidence for clock function in the ovary across a number of species and offer a novel perspective into the role of this clock in normal ovarian physiology and in diseases that negatively affect fertility.

Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.

The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.