Self-sustained Circadian Oscillations Research Articles

Autonomous and Self-sustained Circadian Oscillators Displayed in Human Primary Parathyroid Cell Culture Drive Parathormone Secretion

Abstract Background Most cells in our body possess circadian oscillators controlling diurnal rhythmicity of body metabolism. The role of molecular clocks in regulating the function and disfunction of human parathyroid gland (PG), responsible for calcium homeostasis, has not been unraveled. Hyperparathyroidism is a common endocrine pathology, characterized by an unadapted PTH secretion to calcium levels. Aims Here we aimed at characterizing molecular makeup of circadian clocks in human primary parathyroid cell culture (HPPCC), and at establishing differential transcriptional patterns of normal and pathological PGs. Methods RNA extracted from normal, adenomatous, and hyperplastic PG tissues was subjected to RNA sequencing (RNAseq) analysis. The rim of normal PG tissue was dissected from parathyroid adenoma specimens based on the higher autofluorescence intensity assessed by the near infra-red imaging. HPPCCs were established from adenomatous and hyperplastic PG biopsies. Efficient circadian clock disruption was achieved in HPPCCs by small interfering RNA-mediated knockdown of CLOCK. The functionality of circadian clock machinery was assessed by continuous recording of circadian bioluminescence introduced via lentivectors and paralleled with around-the-clock measurement of PTH secretion using perfusion system. Results The RNAseq analyses revealed transcriptional signatures that distinguished between pathological and normal PGs, and among adenomatous and hyperplastic PGs. We report, for the first time, robust anti-phasic circadian rhythmicity of Per2-luciferase and Bmal1-luciferase reporters in HPPCCs synchronized in vitro. Strikingly, we observed circadian rhythmicity of PTH secretion by parathyroid adenoma cells. Moreover, clock disruption in PG adenoma cells has an impact on transcription of functional genes. Conclusion Our data indicate presence of cell-autonomous molecular clock in human PG cells paralleled with circadian rhythmic PTH secretion and provide large-scale transcriptional pattern of parathyroid adenoma and hyperplastic tissues.

Evolution of self-sustained circadian rhythms is facilitated by seasonal change of daylight.

Self-sustained oscillation is a fundamental property of circadian rhythms and has been repeatedly tested since the early days of circadian research, resulting in the discovery of almost all organisms possessing self-sustained circadian oscillations. However, the evolutionary advantage of self-sustainability has been only speculatively discussed. In this theoretical study, we sought the environmental constraints and selection pressure that drive the acquisition or degeneration of self-sustainability through the process of evolution. We considered the dynamics of a gene regulatory network having a light input pathway under 12 h light and 12 h dark cycles or multiple day length conditions and then optimized the network structure using an evolutionary algorithm. By designing the fitness function in the evolutionary algorithm, we investigated the environmental conditions that led to the evolution of the self-sustained oscillators. Then, we found that self-sustained oscillation is rarer than damped oscillation and hourglass-type behaviour. Moreover, networks with self-sustainability have a markedly high fitness score when we assume that a network has to generate a constantly periodic expression profile regardless of day length. This study is, to our knowledge, the first to show that seasonality facilitated the evolution of the self-sustained circadian clock, which was consistent with empirical records.

Mathematical Modeling in Circadian Rhythmicity.

Circadian clocks are autonomous systems able to oscillate in a self-sustained manner in the absence of external cues, although such Zeitgebers are typically present. At the cellular level, the molecular clockwork consists of a complex network of interlocked feedback loops. This chapter discusses self-sustained circadian oscillators in the context of nonlinear dynamics theory. We suggest basic steps that can help in constructing a mathematical model and introduce how self-sustained generations can be modeled using ordinary differential equations. Moreover, we discuss how coupled oscillators synchronize among themselves or entrain to periodic signals. The development of mathematical models over the last years has helped to understand such complex network systems and to highlight the basic building blocks in which oscillating systems are built upon. We argue that, through theoretical predictions, the use of simple models can guide experimental research and is thus suitable to model biological systems qualitatively.

A Comparative Study of Circadian Rhythmicity and Photoperiodism in Closely Related Species of Blow Flies: External Coincidence, Maternal Induction, and Diapause at Northern Latitudes.

This review compares adult locomotor activity rhythms and photoperiodic induction of diapause in 3 common species of blow fly, Calliphora vicina, Lucilia sericata, and Protophormia terraenovae. Activity rhythms were broadly similar in all 3 species, although P. terraenovae is much less sensitive to constant light inducing arrhythmicity. Photoperiodic induction of diapause, on the other hand, varies more widely between species. C. vicina and L. sericata overwinter in a larval diapause induced by autumnal short days (long nights) acting both maternally and directly upon the larvae. P. terraenovae, on the other hand, shows an adult (reproductive) diapause induced by short daylength and low temperature experienced by the larvae. In the Nanda-Hamner protocol, C. vicina shows 3 clear peaks of high diapause incidence in cycle lengths close to 24, 48, and 72 h, without dampening and therefore suggesting a photoperiodic mechanism based on a self-sustained circadian oscillator acting in a clock of the external coincidence type. Entrainment of the locomotor activity rhythm to extended Nanda-Hamner photocycles, as well as to LD cycles close to the limits of the primary range of entrainment, demonstrates that overt circadian rhythmicity may act as ‘hands’ of the otherwise covert photoperiodic system, as suggested by Bünning, nearly 8 decades ago. In 24 h LD cycles, both locomotor activity rhythms and the photoperiodic oscillator are set to constant phase (CT 12) at light-off, so that the photoperiodic clock measures changes in nightlength by the coincidence (or not) of dawn light with a ‘photoinducible phase’ late in the subjective night (at about CT 21.5 h) as photoperiod changes with the seasons. Apparent differences between quantitative and qualitative photoperiodic responses are discussed.

BMAL1 dephosphorylation determines the pace of the circadian clock

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Circadian Vision in Zebrafish: From Molecule to Cell and from Neural Network to Behavior.

Most visual system functions, such as opsin gene expression, retinal neural transmission, light perception, and visual sensitivity, display robust day-night rhythms. The rhythms persist in constant lighting conditions, suggesting the involvement of endogenous circadian clocks. While the circadian pacemakers that control the rhythms of animal behaviors are mostly found in the forebrain and midbrain, self-sustained circadian oscillators are also present in the neural retina, where they play important roles in the regulation of circadian vision. This review highlights some of the correlative studies of the circadian control of visual system functions in zebrafish. Because zebrafish maintain a high evolutionary proximity to mammals, the findings from zebrafish research may provide insights for a better understanding of the mechanisms of circadian vision in other vertebrate species including humans.

Circadian clocks: an overview on its adaptive significance

ABSTRACT The metabolic, physiological, and behavioral processes in most of the living organisms exhibit an approximate 24-h rhythmic, self-sustained circadian (L. circa = about, dies = day) oscillations. These oscillations, apart from passively persisting in synchrony with external cues, exist in their absence as well, conferring adaptive advantage by following environmental cycle (extrinsic adaptation) and physiological harmony (intrinsic advantage), and tackling the adverse seasonal changes. Interestingly, these circadian perturbations generated at cellular level as rhythms are pretty much hierarchically similar across unicellular organisms to complex mammals. The biological clocks (master clock along with peripheral clocks) are able to influence biological fitness, life-history traits, physiology and behavior of organism. Given that the circadian clocks influence various biological processes, its evolutionary significance remained the subject of several laboratory studies with the view that imposing artificial selection for various clock properties such as phase, period, etc., might lead to the co-evolution of associated other clock-controlled evolutionary traits. In this review, we have presented key hypotheses pertaining to circadian rhythms, where by its presence to the owner can confer adaptive advantage intrinsically and extrinsically and enable endogenous clock to perform better in synchrony with the environment.

Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis

Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model.

Keeping Oxidative Metabolism on Time: Mitochondria as an Autonomous Redox Pacemaker Animated by H2O2 and Peroxiredoxin

In this issue of Molecular Cell, Kil etal. (2015) provide evidence for self-sustained circadian oscillations of the hyperoxidation of the mitochondrial Peroxiredoxin, PrxIII, and cytosolic release of mitochondrial H2O2, which might constitute one biochemical output coupling metabolic changes and transcriptional-based core clocks.

Chronobiology and obesity.

Chronobiology is a word derived from three Greek stems: kronos for time, bios for life and logos for study. From microarrays studies, now it is accepted that 10-30% of the human genome is under the control of circadian molecular clocks. This implies that most behavioral, physiological and biochemical variables display circadian rhythms in their expression. In its simplest form, circadian clocks are composed of a set of proteins that generate self-sustained circadian oscillations. The molecular clock comprises two transcription factors, CLOCK and BMAL1, whereas PERs and CRYs are responsible for the negative limb. One of the most important questions related to the circadian system and obesity, was to elucidate if adipose tissue displayed circadian rhythmicity or whether it had an internal peripheral clock. Our group of research has provided an overall view of the internal temporal order of circadian rhythms in human adipose tissue. A new concept related to illness is Chronodisruption (CD). It is defined as a relevant disturbance of the internal temporal order of physiological and behavioral circadian rhythms. In our modern society, CD may be common in several conditions such as jet lag, shift work, light at night, or social jet lag. In addition clock gene polymorphisms and aging may have also chronodisruptive effects. Our group has also demonstrated that Obesity and CD are also highly interconnected. With the help of chronobiology we can reach a new view of obesity considering not only "what" are the factors involved in obesity, but also "when" these factors are produced.

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep-wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.

Spatiotemporal dynamics of circadian clock in lettuce

Abstract Plant circadian clock controls many physiological events by synchronization with environmental changes. The clock can be controlled artificially by imposing various light conditions in a closed system for plant cultivation in plant factory. The plant circadian rhythm is formed by enormous self-sustained cellular circadian oscillators, so that the synchronization of circadian oscillators is of great importance to the formation of individual circadian rhythms. Therefore, the study of synchronization phenomena is important for precise control of the circadian rhythm. In this study, we have investigated the spatiotemporal dynamics of circadian oscillators in lettuce (Lactuca sativa L.). Bioluminescence of transgenic lettuce carrying a CCA1::LUC construct as a reporter of a circadian gene expression was measured in the leaf and root. We observed phase wave propagation in the leaf with a phase delay in the region of the primary vein, with the primary vein region showing lower amplitude than the other regions. Wave propagation occurred from the edge of the leaf inward. In addition, a striped wave traveled from the base to the tip along the roots, which were grown under continuous dark or light conditions. The features observed in the lettuce plants could be explained by phase oscillator models established in the study of Arabidopsis thaliana. The results of this study show the possibility of applying circadian clock control in a model plant for plant production.

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesisFollowing on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells.MethodsWe established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence–fluorescence time-lapse microscopy.ResultsHuman islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48 h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other.Conclusions/interpretationWe provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell cultures.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2779-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties

In the mammalian circadian system, cell-autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 (Luc) knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 (Luc) SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2 (Luc) fibroblasts. SCN mPer2 (Luc) cells generated self-sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co-cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 (Luc) fibroblasts decayed after 3-4 cycles in serum-shocked cultures but robustly persisted for 6-7 cycles in the presence of SCN2.2 cells. In the co-culture model, the circadian behavior of mPer2 (Luc) fibroblasts was dependent on the integrity of the molecular clockworks in co-cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1(ldc) /Per2 (ldc) ). Because immortalized mPer2 (Luc) SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real-time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night1–3. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and while rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes4, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism, and insulin signaling is delayed in circadian mutant mice, and both Clock5,6 and Bmal17 mutants exhibit impaired glucose tolerance, reduced insulin secretion, and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival, and synaptic vesicle assembly. Remarkably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective β-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the β-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger onset of diabetes mellitus.

Dependence of the period on the rate of protein degradation in minimal models for circadian oscillations

Circadian rhythms, which occur spontaneously with a period of about 24 h in a variety of organisms, allow their adaptation to the periodic variations of the environment. These rhythms are generated by a genetic regulatory network involving a negative feedback loop on transcription. Mathematical models based on the negative autoregulation of gene expression by the protein product of a clock gene account for the occurrence of self-sustained circadian oscillations. These models differ by their degree of complexity and, hence, by the number of variables considered. Some of these models can be considered as minimal because they contain a reduced number of biochemical processes and variables capable of producing sustained oscillations. In three of these minimal models, the period of the oscillations significantly changes with the rate of degradation of the clock protein. However, depending on the model considered, the period increases, decreases or passes through a maximum as a function of the protein degradation rate. We clarify the bases for these markedly different results by bringing to light the roles of (i) protein phosphorylation, which is required for protein degradation, and (ii) the velocity and degree of saturation of mRNA and protein degradation. Changes in the parameter values of the more complex of the minimal models can produce the period profiles observed in the other two models. The analysis allows us to reconcile the contradictory predictions for the dependence of the period on the clock protein degradation rate in three minimal models used to describe circadian rhythms.

Biological Rhythms: From molecular clocks to human health

Event Abstract Back to Event Biological Rhythms: From molecular clocks to human health Paul Pévet1* 1 Institute of cellular and Integrative Neurosciences LC2/UMR 7168, CNRS and University L. Pasteur, Department of Neurobiology of Rhythms, France Disorders of rhythmicity are characteristic of, and may underlie, a variety of troubles. Sleep and circadian rhythms are often disrupted in neurological disorders and increasing evidence indicates that alterations in the sleep/wake cycle accompany (or may be responsible for) many types of neurological disorders. To develop strategies to treat, prevent or delay such disturbances is a new challenge for medicine. The diurnal organisation of living organisms depends on a circadian network comprising circadian clocks, synchronizing inputs, various clock outputs as well as multiple peripheral self-sustained oscillators. In mammals, the focal point of this system is a master circadian clock within the suprachiasmatic nuclei (SCN). Self-sustained circadian oscillators are also present in numerous tissues. Peripheral oscillators (PO) share similar molecular mechanisms to generate rhythms like the central circadian clock, but they are distinct at the functional level. Even though PO in tissue culture or SCN-lesioned rodents can behave independently of SCN outputs, in intact animals all the identified synchronisers of PO (glucocorticoids, feeding behaviour) depend on the SCN for their temporal expression. The circadian clock thus has not only the capacity to build a circadian message, but can also distribute this signal to other structures. It is thus the complex interaction of neural, hormonal and behavioural outputs from the SCN that drive the circadian expression of events. One important question is thus the identification of the outputs pathways used by the circadian clock. To date, it is known that the SCN conveys its “timing” signal by using different tools: neural connections, hormonal cues (e.g. corticosterone, melatonin) and rhythmic behaviour cues. It is in this context of a complex and partially redundant system that we will analyse the role of the hormonal cues. Melatonin (MEL), as well as corticosterone (CORT), is efferent hormonal outputs of the circadian clock. The clock may use MEL or CORT signals to convey the circadian message to any system that can “read” it, i.e. to any structure/organ possessing MEL or CORT receptors. Potential sites for MEL binding are very numerous since more than 130 structures within the brain and periphery have been identified. Sites for the action of CORT are even more numerous. In the context of the multi-oscillatory nature of the circadian system, two modes of action have to be considered: 1) the hormone signal directly drives a rhythm; or 2) the hormone signal entrains PO. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Plenary lectures Citation: Pévet P (2009). Biological Rhythms: From molecular clocks to human health. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.122 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Nov 2009; Published Online: 23 Nov 2009. * Correspondence: Paul Pévet, Institute of cellular and Integrative Neurosciences LC2/UMR 7168, CNRS and University L. Pasteur, Department of Neurobiology of Rhythms, Strasbourg, France, pevet@inci-cnrs.unistra.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Paul Pévet Google Paul Pévet Google Scholar Paul Pévet PubMed Paul Pévet Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Organization of endogenous clocks in insects

Insect and mammalian circadian clocks show striking similarities. They utilize homologous clock genes, generating self-sustained circadian oscillations in distinct master clocks of the brain, which then control rhythmic behaviour. The molecular mechanisms of rhythm generation were first uncovered in the fruit fly Drosophila melanogaster, whereas cockroaches were among the first animals where the brain master clock was localized. Despite many similarities, there exist obvious differences in the organization and functioning of insect master clocks. These similarities and differences are reviewed on a molecular and anatomical level.

Organization of endogenous clocks in insects

Insect and mammalian circadian clocks show striking similarities. They utilize homologous clock genes, generating self-sustained circadian oscillations in distinct master clocks of the brain, which then control rhythmic behaviour. The molecular mechanisms of rhythm generation were first uncovered in the fruit fly Drosophila melanogaster, whereas cockroaches were among the first animals where the brain master clock was localized. Despite many similarities, there exist obvious differences in the organization and functioning of insect master clocks. These similarities and differences are reviewed on a molecular and anatomical level.

Independent Circadian Oscillations ofPeriod1in Specific Brain AreasIn VivoandIn Vitro

Behavioral and physiological circadian rhythms in mammals are controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN). Recently, circadian oscillations of hormone secretion, clock gene expression, and electrical activity have been demonstrated in explants of other brain regions. This suggests that some extra-SCN brain regions contain a functional, SCN-independent circadian clock, but in vivo evidence for intrinsic pacemaking is still lacking. We developed a novel method to image bioluminescence in vivo from the main olfactory bulbs (OB) of intact and SCN-lesioned (SCNX) Period1::luciferase rats for 2 d in constant darkness. The OBs expressed circadian rhythms in situ with a reliable twofold increase from day to night, similar to the phase and amplitude of ex vivo rhythms. In vivo cycling persisted for at least 1 month in the absence of the SCN. To assess indirectly in vivo rhythmicity of other brain areas, we measured the phase-dependence of their in vitro rhythms on the time of surgery. Surgery reliably reset the phase of the pineal gland and vascular organ of the lamina terminalis (VOLT) harvested from SCNX rats but had little effect on the phase of the OB. We deduce that the SCN and OB contain self-sustained circadian oscillators, whereas the pineal gland and VOLT are weak oscillators that require input from the SCN to show coordinated circadian rhythms. We conclude that the mammalian brain comprises a diverse set of SCN-dependent and SCN-independent circadian oscillators.