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.

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.

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.

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.

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.

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.

The methamphetamine-sensitive circadian oscillator does not employ canonical clock genes.

The "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus controls most behavioral, physiological, and molecular circadian rhythms in mammals. However, there are other, still unidentified, circadian oscillators that are able to carry out some SCN functions. Here we show that one of these, the methamphetamine-sensitive circadian oscillator (MASCO), which generates behavioral rhythms in the absence of the SCN, is based on an entirely different molecular mechanism. We tested mice lacking, or with mutations of, genes that form the canonical circadian machinery. In all cases, animals that were arrhythmic as a consequence of genetic defect expressed circadian locomotor rhythms when treated with methamphetamine. These results strongly support the hypothesis that the mechanism generating MASCO does not involve the molecular feedback loops that underlie canonical circadian rhythmicity. The properties of MASCO may provide insight into the evolution of circadian mechanisms. Importantly, MASCO may play a role in addiction to psychostimulants.

Retinal pathways influence temporal niche.

In mammals, light input from the retina entrains central circadian oscillators located in the suprachiasmatic nuclei (SCN). The phase of circadian activity rhythms with respect to the external light:dark cycle is reversed in diurnal and nocturnal species, although the phase of SCN rhythms relative to the light cycle remains unchanged. Neural mechanisms downstream from the SCN are therefore believed to determine diurnality or nocturnality. Here, we report a switch from nocturnal to diurnal entrainment of circadian activity rhythms in double-knockout mice lacking the inner-retinal photopigment melanopsin (OPN4) and RPE65, a key protein used in retinal chromophore recycling. These mice retained only a small amount of rod function. The change in entrainment phase of Rpe65(-/-);Opn4(-/-) mice was accompanied by a reversal of the rhythm of clock gene expression in the SCN and a reversal in acute masking effects of both light and darkness on activity, suggesting that the nocturnal to diurnal switch is due to a change in the neural response to light upstream from the SCN. A switch from nocturnal to diurnal activity rhythms was also found in wild-type mice transferred from standard intensity light:dark cycles to light:dark cycles in which the intensity of the light phase was reduced to scotopic levels. These results reveal a novel mechanism by which changes in retinal input can mediate acute temporal-niche switching.

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.

Ontogeny of circadian organization in the rat.

The mammalian circadian system is orchestrated by a master pacemaker in the brain, but many peripheral tissues also contain independent or quasi-independent circadian oscillators. The adaptive significance of clocks in these structures must lie, in large part, in the phase relationships between the constituent oscillators and their micro- and macroenvironments. To examine the relationship between postnatal development, which is dependent on endogenous programs and maternal/environmental influences, and the phase of circadian oscillators, the authors assessed the circadian phase of pineal, liver, lung, adrenal, and thyroid tissues cultured from Period 1-luciferase (Per1-luc ) rat pups of various postnatal ages. The liver, thyroid, and pineal were rhythmic at birth, but the phases of their Per1-luc expression rhythms shifted remarkably during development. To determine if the timing of the phase shift in each tissue could be the result of changing environmental conditions, the behavior of pups and their mothers was monitored. The circadian phase of the liver shifted from the day to night around postnatal day (P) 22 as the pups nursed less during the light and instead ate solid food during the dark. Furthermore, the phase of Per1-luc expression in liver cultures from nursing neonates could be shifted experimentally from the day to the night by allowing pups access to the dam only during the dark. Peak Per1-luc expression also shifted from midday to early night in thyroid cultures at about P20, concurrent with the shift in eating times. The phase of Per1-luc expression in the pineal gland shifted from day to night coincident with its sympathetic innervation at around P5. Per1-luc expression was rhythmic in adrenal cultures and peaked around the time of lights-off throughout development; however, the amplitude of the rhythm increased at P25. Lung cultures were completely arrhythmic until P12 when the pups began to leave the nest. Taken together, the data suggest that the molecular machinery that generates circadian oscillations matures at different rates in different tissues and that the phase of at least some peripheral organs is malleable and may shift as the organ's function changes during development.

Deletion of TASK1 and TASK3 channels disrupts intrinsic excitability but does not abolish glucose or pH responses of orexin/hypocretin neurons.

The firing of hypothalamic hypocretin/orexin neurons is vital for normal sleep-wake transitions, but its molecular determinants are not well understood. It was recently proposed that TASK (TWIK-related acid-sensitive potassium) channels [TASK1 (K(2P)3.1) and/or TASK3 (K(2P)9.1)] regulate neuronal firing and may contribute to the specialized responses of orexin neurons to glucose and pH. Here we tested these theories by performing patch-clamp recordings from orexin neurons directly identified by targeted green fluorescent protein labelling in brain slices from TASK1/3 double-knockout mice. The deletion of TASK1/3 channels significantly reduced the ability of orexin cells to generate high-frequency firing. Consistent with reduced excitability, individual action potentials from knockout cells had lower rates of rise, higher thresholds and more depolarized after-hyperpolarizations. However, orexin neurons from TASK1/3 knockout mice retained typical responses to glucose and pH, and the knockout animals showed normal food-anticipatory locomotor activity. Our results support a novel role for TASK genes in enhancing neuronal excitability and promoting high-frequency firing, but suggest that TASK1/3 subunits are not essential for orexin cell responses to glucose and pH.

Lithium and genetic inhibition of GSK3beta enhance the effect of methamphetamine on circadian rhythms in the mouse.

Lithium, a drug commonly used to treat mood disorders, and the psychostimulant methamphetamine are both capable of altering circadian rhythmicity. Although the actions of lithium on the circadian system are thought to occur through inhibition of glycogen synthase kinase-3beta (GSK3beta), the mechanism by which methamphetamine alters circadian rhythms is unknown. We tested the effects of concurrent methamphetamine and lithium treatment on the circadian wheel-running behavior of mice. Methamphetamine alone lengthened both the active duration and the free-running period of locomotor activity in animals housed in constant conditions. Administering lithium enhanced the period-lengthening effects of methamphetamine in animals housed in constant darkness. This effect was even more pronounced when animals were housed in constant light. Lithium increased both methamphetamine intake and serum levels of methamphetamine, possibly contributing to the effects on circadian behavior. We also tested the effect of methamphetamine in mutant mice possessing only one allele for Gsk3beta. These animals, when treated with methamphetamine, responded like wild-type mice treated with a combination of methamphetamine and lithium, displaying long, free-running rhythms. These data, together with many others in the literature, point to a complicated interaction between the circadian system and the development and possible treatment of psychopathologies such as bipolar disorder and drug addiction.

Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons.

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.

Interactions between dopamine and melatonin organize circadian rhythmicity in the retina of the green iguana.

Circadian physiology in the vertebrate retina is regulated by several neurotransmitters. In the lateral eyes of the green iguana the circadian rhythm of melatonin content peaks during the night while the rhythm of dopamine peaks during the day. In the present work, the authors explore the interaction of these 2 neurotransmitters during the circadian cycle. They depleted retinal dopamine with intravitreal injections of 6-hydroxydopamine (6-OHDA) and measured ocular melatonin content in vivo throughout 1 circadian cycle. The circadian rhythm of ocular melatonin not only persisted but increased 10-fold in amplitude. This increase was substantially reduced by the intraocular administration of dopamine. 6-OHDA-treated retinas, unlike those from untreated animals, did not express a circadian rhythm of melatonin synthesis in vitro. To deplete retinal melatonin, the authors pinealectomized iguanas and blocked retinal melatonin synthesis by depleting serotonin with intraocular injections of 5,6-dihydroxytryptamine. In animals so treated, they found that the circadian rhythm of retinal dopamine content was abolished, the levels of dopamine were lowered, and the levels of dopamine metabolites were greatly increased. The data suggest that in iguanas, the amplitude of the circadian rhythm of melatonin synthesis in the eye is suppressed by dopamine while the rhythm of dopamine depends, at least in part, on the presence of melatonin.

The methamphetamine-sensitive circadian oscillator (MASCO) in mice.

The suprachiasmatic nucleus (SCN) orchestrates synchrony among many peripheral oscillators and is required for circadian rhythms of locomotor activity and many physiological processes. However, the unique effects of methamphetamine (MAP) on circadian behavior suggest the presence of an SCN-independent, methamphetamine-sensitive circadian oscillator (MASCO). Substantial data collected using rat models show that chronic methamphetamine dramatically lengthens circadian period of locomotor activity rhythms and induces rhythms in animals lacking an SCN. However, the anatomical substrate and the molecular components of the MASCO are unknown. The response to MAP is less well studied in mice, a model that would provide the genetic tools to probe the molecular components of this extra-SCN oscillator. The authors tested the effects of chronic MAP on 2 strains of intact and SCN-lesioned mice in constant dark and constant light. Furthermore, they applied various MAP availability schedules to SCN-lesioned mice to confirm the circadian nature of the underlying oscillator. The results indicate that this oscillator has circadian properties. In intact mice, the MASCO interacts with the SCN in a manner that is strain, sex, and dose dependent. In SCN-lesioned mice, it induces robust free-running locomotor rhythmicity, which persists for up to 14 cycles after methamphetamine is withdrawn. In the future, localization of the MASCO and characterization of its underlying molecular mechanism, as well as its interactions with other oscillators in the body, will be essential to a complete understanding of the organization of the mammalian circadian system.

Birds of a feather clock together--sometimes: social synchronization of circadian rhythms.

Biological systems use internal circadian clocks to efficiently organize physiological and behavioral activity within the 24-hour time domain. In the absence of time cues, circadian periods vary slightly from 24 hours, but in nature, ambient light serves as the most salient synchronizer for these rhythms, fine-tuning them to exactly 24 hours each day. For some species, social cues can serve to synchronize circadian rhythms in the absence of other time cues or to amplify ambiguous light cues. This has been demonstrated to various degrees in fruit flies, degus, birds, fish, bats, beavers and humans; however, studies in rats and hamsters have shown that social cues are less salient time cues for these species. Social influences on circadian timing might function to tightly organize the social group, thereby decreasing the chances of predation and increasing the likelihood of mating.

Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization.

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles but, in the absence of external rhythmic input, may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. The authors asked whether individual central and peripheral circadian oscillators were rhythmic in LL-treated arrhythmic rats and, if rhythmic, what were the phase relationships between them. The authors prepared SCN, pineal gland, pituitary, and cornea cultures from transgenic Period1-luciferaserats whose body temperature and locomotor activity were arrhythmic and from several groups of rhythmic rats held in LD, DD, and short-term LL. The authors measured mPer1gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this, the authors cultured tissues at 2 different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary, and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats, oscillators in the pineal, pituitary, and SCN had been arrhythmic or severely damped in vivo, while the cornea oscillator was free running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LD-entrained rats was strongly influenced when the procedure was carried out at any time except the 2nd half of the day.

Circadian rhythms in isolated brain regions.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus has been referred to as the master circadian pacemaker that drives daily rhythms in behavior and physiology. There is, however, evidence for extra-SCN circadian oscillators. Neural tissues cultured from rats carrying the Per-luciferase transgene were used to monitor the intrinsic Per1 expression patterns in different brain areas and their response to changes in the light cycle. Although many Per-expressing brain areas were arrhythmic in culture, 14 of the 27 areas examined were rhythmic. The pineal and pituitary glands both expressed rhythms that persisted for >3 d in vitro, with peak expression during the subjective night. Nuclei in the olfactory bulb and the ventral hypothalamus expressed rhythmicity with peak expression at night, whereas other brain areas were either weakly rhythmic and peaked at night, or arrhythmic. After a 6 hr advance or delay in the light cycle, the pineal, paraventricular nucleus of the hypothalamus, and arcuate nucleus each adjusted the phase of their rhythmicity with different kinetics. Together, these results indicate that the brain contains multiple, damped circadian oscillators outside the SCN. The phasing of these oscillators to one another may play a critical role in coordinating brain activity and its adjustment to changes in the light cycle.