The Suprachiasmatic Nucleus at 50: Looking Back, Then Looking Forward.

It has been 50 years since the suprachiasmatic nucleus (SCN) was first identified as the central circadian clock and 25 years since the last overview of developments in the field was published in the . Here, we explore new mechanisms and concepts that have emerged in the subsequent 25 years. Since 1997, methodological developments, such as luminescent and fluorescent reporter techniques, have revealed intricate relationships between cellular and network-level mechanisms.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ().

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage.

Deconstructing circadian disruption: Assessing the contribution of reduced peripheral oscillator amplitude on obesity and glucose intolerance in mice.

Disturbing the circadian regulation of physiology by disruption of the rhythmic environment is associated with adverse health outcomes but the underlying mechanisms are unknown. Here, the response of central and peripheral circadian clocks to an advance or delay of the light-dark cycle was determined in mice. This identified transient damping of peripheral clocks as a consequence of an advanced light-dark cycle.

Periodic Parasites and Daily Host Rhythms.

Biological rhythms appear to be an elegant solution to the challenge of coordinating activities with the consequences of the Earth's daily and seasonal rotation. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are well understood. In contrast, the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites remain mysterious.

Clocks and meals keep mice from being cool.

Daily torpor is used by small mammals to reduce daily energy expenditure in response to energetic challenges. Optimizing the timing of daily torpor allows mammals to maximize its energetic benefits and, accordingly, torpor typically occurs in the late night and early morning in most species. However, the regulatory mechanisms underlying such temporal regulation have not been elucidated.

Functionally Complete Excision of Conditional Alleles in the Mouse Suprachiasmatic Nucleus by Vgat-ires-Cre.

Mice with targeted gene disruption have provided important information about the molecular mechanisms of circadian clock function. A full understanding of the roles of circadian-relevant genes requires manipulation of their expression in a tissue-specific manner, ideally including manipulation with high efficiency within the suprachiasmatic nuclei (SCN). To date, conditional manipulation of genes within the SCN has been difficult.

Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes.

Circadian disruption as a result of shift work is associated with adverse metabolic consequences. Internal desynchrony between the phase of the suprachiasmatic nuclei (SCN) and peripheral clocks is widely believed to be a major factor contributing to these adverse consequences, but this hypothesis has never been tested directly. A GABAergic Cre driver combined with conditional casein kinase mutations ( ) was used to lengthen the endogenous circadian period in GABAergic neurons, including the SCN, but not in peripheral tissues, to create a Discordant mouse model.

The Circadian Clock Gene Coordinates Intestinal Regeneration.

Background & Aims: The gastrointestinal syndrome is an illness of the intestine caused by high levels of radiation. It is characterized by extensive loss of epithelial tissue integrity, which initiates a regenerative response by intestinal stem and precursor cells. The intestine has 24-hour rhythms in many physiological functions that are believed to be outputs of the circadian clock: a molecular system that produces 24-hour rhythms in transcription/translation.

Single-Cell Transcriptional Analysis Reveals Novel Neuronal Phenotypes and Interaction Networks Involved in the Central Circadian Clock.

Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN). Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity.

The hepatic circadian clock modulates xenobiotic metabolism in mice.

The circadian clock generates daily cycles of gene expression that regulate physiological processes. The liver plays an important role in xenobiotic metabolism and also has been shown to possess its own cell-based clock. The liver clock is synchronized by the master clock in the brain, and a portion of rhythmic gene expression can be driven by behavior of the organism as a whole even when the hepatic clock is suppressed.

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration.

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus.

Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β.

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators.

Integrative gene regulatory network analysis reveals light-induced regional gene expression phase shift programs in the mouse suprachiasmatic nucleus.

We use the multigenic pattern of gene expression across suprachiasmatic nuclei (SCN) regions and time to understand the dynamics within the SCN in response to a circadian phase-resetting light pulse. Global gene expression studies of the SCN indicate that circadian functions like phase resetting are complex multigenic processes. While the molecular dynamics of phase resetting are not well understood, it is clear they involve a "functional gene expression program", e.

Antibodies for assessing circadian clock proteins in the rodent suprachiasmatic nucleus.

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation.

Distinct patterns of Period gene expression in the suprachiasmatic nucleus underlie circadian clock photoentrainment by advances or delays.

The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities--advances vs. delays--engage the molecular clockwork remains incompletely understood.

The period of the circadian oscillator is primarily determined by the balance between casein kinase 1 and protein phosphatase 1.

Mounting evidence suggests that PERIOD (PER) proteins play a central role in setting the speed (period) and phase of the circadian clock. Pharmacological and genetic studies have shown that changes in PER phosphorylation kinetics are associated with changes in circadian rhythm period and phase, which can lead to sleep disorders such as Familial Advanced Sleep Phase Syndrome in humans. We and others have shown that casein kinase 1δ and ε (CK1δ/ε) are essential PER kinases, but it is clear that additional, unknown mechanisms are also crucial for regulating the kinetics of PER phosphorylation.

Photic resetting and entrainment in CLOCK-deficient mice.

Mice lacking the CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hour light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle. The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock(-/-) mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock(-/-) mice exhibit very large phase advances after 4-hour light pulses in the late subjective night but have relatively normal responses to light at other phases.

Disrupting the circadian clock: gene-specific effects on aging, cancer, and other phenotypes.

The circadian clock imparts 24-hour rhythmicity on gene expression and cellular physiology in virtually all cells. Disruption of the genes necessary for the circadian clock to function has diverse effects, including aging-related phenotypes. Some circadian clock genes have been described as tumor suppressors, while other genes have less clear functions in aging and cancer.

Altered body mass regulation in male mPeriod mutant mice on high-fat diet.

The circadian clock orchestrates most physiological processes in mammals. Disruption of circadian rhythms appears to contribute to the development of obesity and metabolic syndrome. The Period genes mPer1 and mPer2, but not mPer3, are essential for core clock function in mice.

Casein kinase 1 delta (CK1delta) regulates period length of the mouse suprachiasmatic circadian clock in vitro.

Background: Casein kinase 1 delta (CK1delta) plays a more prominent role in the regulation of circadian cycle length than its homologue casein kinase 1 epsilon (CK1epsilon) in peripheral tissues such as liver and embryonic fibroblasts. Mice lacking CK1delta die shortly after birth, so it has not been possible to assess the impact of loss of CK1delta on behavioral rhythms controlled by the master circadian oscillator in the suprachiasmatic nuclei (SCN).

Methodology/principal Findings: In the present study, mPER2::LUCIFERASE bioluminescence rhythms were monitored from SCN explants collected from neonatal mice.

The circadian clock protein Period 1 regulates expression of the renal epithelial sodium channel in mice.

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the alpha subunit of the epithelial sodium channel (alphaENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1.

Deletion of the secretory vesicle proteins IA-2 and IA-2beta disrupts circadian rhythms of cardiovascular and physical activity.

Targeted deletion of IA-2 and IA-2beta, major autoantigens in type 1 diabetes and transmembrane secretory vesicle proteins, results in impaired secretion of hormones and neurotransmitters. In the present study, we evaluated the effect of these deletions on daily rhythms in blood pressure, heart rate, core body temperature, and spontaneous physical and neuronal activity. We found that deletion of both IA-2 and IA-2beta profoundly disrupts the usual diurnal variation of each of these parameters, whereas the deletion of either IA-2 or IA-2beta alone did not produce a major change.

Casein kinase 1 delta regulates the pace of the mammalian circadian clock.

Both casein kinase 1 delta (CK1delta) and epsilon (CK1epsilon) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1delta and CK1epsilon in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1delta(Delta2)).