Ruminiclostridium 5, Parabacteroides distasonis, and bile acid profile are modulated by prebiotic diet and associate with facilitated sleep/clock realignment after chronic disruption of rhythms.

Chronic disruption of rhythms (CDR) impacts sleep and can result in circadian misalignment of physiological systems which, in turn, is associated with increased disease risk. Exposure to repeated or severe stressors also disturbs sleep and diurnal rhythms. Prebiotic nutrients produce favorable changes in gut microbial ecology, the gut metabolome, and reduce several negative impacts of acute severe stressor exposure, including disturbed sleep, core body temperature rhythmicity, and gut microbial dysbiosis. In light of previous compelling evidence that prebiotic diet broadly reduces negative impacts of acute, severe stressors, we hypothesize that prebiotic diet will also effectively mitigate the negative impacts of chronic disruption of circadian rhythms on physiology and sleep/wake behavior. Male, Sprague Dawley rats were fed diets enriched in prebiotic substrates or calorically matched control chow. After 5 weeks on diet, rats were exposed to CDR (12 h light/dark reversal, weekly for 8 weeks) or remained on undisturbed normal light/dark cycles (NLD). Sleep EEG, core body temperature, and locomotor activity were recorded via biotelemetry in freely moving rats. Fecal samples were collected on experimental days -33, 0 (day of onset of CDR), and 42. Taxonomic identification and relative abundances of gut microbes were measured in fecal samples using 16S rRNA gene sequencing and shotgun metagenomics. Fecal primary, bacterially modified secondary, and conjugated bile acids were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prebiotic diet produced rapid and stable increases in the relative abundances of Parabacteroides distasonis and Ruminiclostridium 5. Shotgun metagenomics analyses confirmed reliable increases in relative abundances of Parabacteroides distasonis and Clostridium leptum, a member of the Ruminiclostridium genus. Prebiotic diet also modified fecal bile acid profiles; and based on correlational and step-wise regression analyses, Parabacteroides distasonis and Ruminiclostridium 5 were positively associated with each other and negatively associated with secondary and conjugated bile acids. Prebiotic diet, but not CDR, impacted beta diversity. Measures of alpha diversity evenness were decreased by CDR and prebiotic diet prevented that effect. Rats exposed to CDR while eating prebiotic, compared to control diet, more quickly realigned NREM sleep and core body temperature (ClockLab) diurnal rhythms to the altered light/dark cycle. Finally, both cholic acid and Ruminiclostridium 5 prior to CDR were associated with time to realign CBT rhythms to the new light/dark cycle after CDR; whereas both Ruminiclostridium 5 and taurocholic acid prior to CDR were associated with NREM sleep recovery after CDR. These results support our hypothesis and suggest that ingestion of prebiotic substrates is an effective strategy to increase the relative abundance of health promoting microbes, alter the fecal bile acid profile, and facilitate the recovery and realignment of sleep and diurnal rhythms after circadian disruption.

Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

Resource competition shapes biological rhythms and promotes temporal niche differentiation in a community simulation.

Competition for resources often contributes strongly to defining an organism's ecological niche. Endogenous biological rhythms are important adaptations to the temporal dimension of niches, but how other organisms influence such temporal niches has not been much studied, and the role of competition in particular has been even less examined. We investigated how interspecific competition and intraspecific competition for resources shape an organism's activity rhythms.To do this, we simulated communities of one or two species in an agent-based model. Individuals in the simulation move according to a circadian activity rhythm and compete for limited resources. Probability of reproduction is proportional to an individual's success in obtaining resources. Offspring may have variance in rhythm parameters, which allow for the population to evolve over time.We demonstrate that when organisms are arrhythmic, one species will always be competitively excluded from the environment, but the existence of activity rhythms allows niche differentiation and indefinite coexistence of the two species. Two species which are initially active at the same phase will differentiate their phase angle of entrainment over time to avoid each other. When only one species is present in an environment, competition within the species strongly selects for niche expansion through arrhythmicity, but the addition of an interspecific competitor facilitates evolution of increased rhythmic amplitude when combined with additional adaptations for temporal specialization. Finally, if individuals preferentially mate with others who are active at similar times of day, then disruptive selection by intraspecific competition can split one population into two reproductively isolated groups separated in activity time.These simulations suggest that biological rhythms are an effective method to temporally differentiate ecological niches and that competition is an important ecological pressure promoting the evolution of rhythms and sleep. This is the first study to use ecological modeling to examine biological rhythms.

Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight.

BACKGROUND: Space environment imposes a range of challenges to mammalian physiology and the gut microbiota, and interactions between the two are thought to be important in mammalian health in space. While previous findings have demonstrated a change in the gut microbial community structure during spaceflight, specific environmental factors that alter the gut microbiome and the functional relevance of the microbiome changes during spaceflight remain elusive. METHODS: We profiled the microbiome using 16S rRNA gene amplicon sequencing in fecal samples collected from mice after a 37-day spaceflight onboard the International Space Station. We developed an analytical tool, named STARMAPs (Similarity Test for Accordant and Reproducible Microbiome Abundance Patterns), to compare microbiome changes reported here to other relevant datasets. We also integrated the gut microbiome data with the publically available transcriptomic data in the liver of the same animals for a systems-level analysis. RESULTS: We report an elevated microbiome alpha diversity and an altered microbial community structure that were associated with spaceflight environment. Using STARMAPs, we found the observed microbiome changes shared similarity with data reported in mice flown in a previous space shuttle mission, suggesting reproducibility of the effects of spaceflight on the gut microbiome. However, such changes were not comparable with those induced by space-type radiation in Earth-based studies. We found spaceflight led to significantly altered taxon abundance in one order, one family, five genera, and six species of microbes. This was accompanied by a change in the inferred microbial gene abundance that suggests an altered capacity in energy metabolism. Finally, we identified host genes whose expression in the liver were concordantly altered with the inferred gut microbial gene content, particularly highlighting a relationship between host genes involved in protein metabolism and microbial genes involved in putrescine degradation. CONCLUSIONS: These observations shed light on the specific environmental factors that contributed to a robust effect on the gut microbiome during spaceflight with important implications for mammalian metabolism. Our findings represent a key step toward a better understanding the role of the gut microbiome in mammalian health during spaceflight and provide a basis for future efforts to develop microbiota-based countermeasures that mitigate risks to crew health during long-term human space expeditions.

Genetics of Circadian Rhythms.

In mammals, genetic influences of circadian rhythms occur at many levels. A set of core "clock genes" have been identified that form a feedback loop of gene transcription and translation. The core genetic clockwork generates circadian rhythms in cells throughout the body. Polymorphisms in both core clock genes and interacting genes contribute to individual differences in the expression and properties of circadian rhythms. The circadian clock profoundly influences the patterns of gene expression and cellular functions, providing a mechanistic basis for the impact of the genetic circadian system on normal physiological processes as well as the development of diseases.

The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Parkinson's Disease is Associated with Dysregulations of a Dopamine-Modulated Gene Network Relevant to Sleep and Affective Neurobehaviors in the Striatum.

In addition to the characteristic motor symptoms, Parkinson's disease (PD) often involves a constellation of sleep and mood symptoms. However, the mechanisms underlying these comorbidities are largely unknown. We have previously reconstructed gene networks in the striatum of a population of (C57BL/6J x A/J) F2 mice and associated the networks to sleep and affective phenotypes, providing a resource for integrated analyses to investigate perturbed sleep and affective functions at the gene network level. Combining this resource with PD-relevant transcriptomic datasets from humans and mice, we identified four networks that showed elevated gene expression in PD patients, including a circadian clock and mitotic network that was altered similarly in mouse models of PD. We then utilized multiple types of omics data from public databases and linked this gene network to postsynaptic dopamine signaling in the striatum, CDK1-modulated transcriptional regulation, and the genetic susceptibility of PD. These findings suggest that dopamine deficiency, a key aspect of PD pathology, perturbs a circadian/mitotic gene network in striatal neurons. Since the normal functions of this network were relevant to sleep and affective behaviors, these findings implicate that dysregulation of functional gene networks may be involved in the emergence of non-motor symptoms in PD. Our analyses present a framework for integrating multi-omics data from diverse sources in mice and humans to reveal insights into comorbid symptoms of complex diseases.

Light/Dark Shifting Promotes Alcohol-Induced Colon Carcinogenesis: Possible Role of Intestinal Inflammatory Milieu and Microbiota.

BACKGROUND: Colorectal cancer (CRC) is associated with the modern lifestyle. Chronic alcohol consumption-a frequent habit of majority of modern societies-increases the risk of CRC. Our group showed that chronic alcohol consumption increases polyposis in a mouse mode of CRC. Here we assess the effect of circadian disruption-another modern life style habit-in promoting alcohol-associated CRC. METHOD: TS4Cre × adenomatous polyposis coli (APC)lox468 mice underwent (a) an alcohol-containing diet while maintained on a normal 12 h light:12 h dark cycle; or (b) an alcohol-containing diet in conjunction with circadian disruption by once-weekly 12 h phase reversals of the light:dark (LD) cycle. Mice were sacrificed after eight weeks of full alcohol and/or LD shift to collect intestine samples. Tumor number, size, and histologic grades were compared between animal groups. Mast cell protease 2 (MCP2) and 6 (MCP6) histology score were analyzed and compared. Stool collected at baseline and after four weeks of experimental manipulations was used for microbiota analysis. RESULTS: The combination of alcohol and LD shifting accelerated intestinal polyposis, with a significant increase in polyp size, and caused advanced neoplasia. Consistent with a pathogenic role of stromal tryptase-positive mast cells in colon carcinogenesis, the ratio of mMCP6 (stromal)/mMCP2 (intraepithelial) mast cells increased upon LD shifting. Baseline microbiota was similar between groups, and experimental manipulations resulted in a significant difference in the microbiota composition between groups. CONCLUSIONS: Circadian disruption by Light:dark shifting exacerbates alcohol-induced polyposis and CRC. Effect of circadian disruption could, at least partly, be mediated by promoting a pro-tumorigenic inflammatory milieu via changes in microbiota.

Night workers with circadian misalignment are susceptible to alcohol-induced intestinal hyperpermeability with social drinking.

Alcohol-induced intestinal hyperpermeability (AIHP) is a known risk factor for alcoholic liver disease (ALD), but only 20-30% of heavy alcoholics develop AIHP and ALD. The hypothesis of this study is that circadian misalignment would promote AIHP. We studied two groups of healthy subjects on a stable work schedule for 3 mo [day workers (DW) and night workers (NW)]. Subjects underwent two circadian phase assessments with sugar challenge to access intestinal permeability between which they drank 0.5 g/kg alcohol daily for 7 days. Sleep architecture by actigraphy did not differ at baseline or after alcohol between either group. After alcohol, the dim light melatonin onset (DLMO) in the DW group did not change significantly, but in the NW group there was a significant 2-h phase delay. Both the NW and DW groups had no change in small bowel permeability with alcohol, but only in the NW group was there an increase in colonic and whole gut permeability. A lower area under the curve of melatonin inversely correlated with increased colonic permeability. Alcohol also altered peripheral clock gene amplitude of peripheral blood mononuclear cells in CLOCK, BMAL, PER1, CRY1, and CRY2 in both groups, and inflammatory markers lipopolysaccharide-binding protein, LPS, and IL-6 had an elevated mesor at baseline in NW vs. DW and became arrhythmic with alcohol consumption. Together, our data suggest that central circadian misalignment is a previously unappreciated risk factor for AIHP and that night workers may be at increased risk for developing liver injury with alcohol consumption.

Osteoarthritis-like pathologic changes in the knee joint induced by environmental disruption of circadian rhythms is potentiated by a high-fat diet.

A variety of environmental factors contribute to progressive development of osteoarthritis (OA). Environmental factors that upset circadian rhythms have been linked to various diseases. Our recent work establishes chronic environmental circadian disruption - analogous to rotating shiftwork-associated disruption of circadian rhythms in humans - as a novel risk factor for the development of OA. Evidence suggests shift workers are prone to obesity and also show altered eating habits (i.e., increased preference for high-fat containing food). In the present study, we investigated the impact of chronic circadian rhythm disruption in combination with a high-fat diet (HFD) on progression of OA in a mouse model. Our study demonstrates that when mice with chronically circadian rhythms were fed a HFD, there was a significant proteoglycan (PG) loss and fibrillation in knee joint as well as increased activation of the expression of the catabolic mediators involved in cartilage homeostasis. Our results, for the first time, provide the evidence that environmental disruption of circadian rhythms plus HFD potentiate OA-like pathological changes in the mouse joints. Thus, our findings may open new perspectives on the interactions of chronic circadian rhythms disruption with diet in the development of OA and may have potential clinical implications.

Environmental disruption of circadian rhythm predisposes mice to osteoarthritis-like changes in knee joint.

Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA-like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild-type (WT) mice were maintained on a constant 12 h:12 h LD cycle (12:12 LD) or exposed to weekly 12 h phase shifts. Alternatively, male circadian mutant mice (Clock(Δ19) or Csnk1e(tau) mutants) were compared with age-matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix-degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ-ERK-RUNX2/NFκB and ß-catenin signaling pathways, stimulation of MMP-13 and ADAMTS-5, as well as suppression of the anabolic mediators SOX9 and TIMP-3 in articular chondrocytes of phase-shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA-like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA-like pathological changes in the mouse knee joint.

Validation of video motion-detection scoring of forced swim test in mice.

BACKGROUND: The forced swim test (FST) is used to predict the effectiveness of novel antidepressant treatments. In this test, a mouse or rat is placed in a beaker of water for several minutes, and the amount of time spent passively floating is measured; antidepressants reduce the amount of such immobility. Though the FST is commonly used, manually scoring the test is time-consuming and involves considerable subjectivity. NEW METHOD: We developed a simple MATLAB-based motion-detection method to quantify mice's activity in videos of FST. FST trials are video-recorded from a side view. Each pixel of the video is compared between subsequent video frames; if the pixel's color difference surpasses a threshold, a motion count is recorded. RESULTS: Human-scored immobility time correlates well with total motion detected by the computer (r=-0.80) and immobility time determined by the computer (r=0.83). Our computer method successfully detects group differences in activity between genotypes and different days of testing. Furthermore, we observe heterosis for this behavior, in which (C57BL/6J×A/J) F1 hybrid mice are more active in the FST than the parental strains. COMPARISON WITH EXISTING METHODS: This computer-scoring method is much faster and more objective than human scoring. Other automatic scoring methods exist, but they require the purchase of expensive hardware and/or software. CONCLUSION: This computer-scoring method is an effective, fast, and low-cost method of quantifying the FST. It is validated by replicating statistical differences observed in traditional visual scoring. We also demonstrate a case of heterosis in the FST.

The circadian clock gene Csnk1e regulates rapid eye movement sleep amount, and nonrapid eye movement sleep architecture in mice.

STUDY OBJECTIVES: Efforts to identify the genetic basis of mammalian sleep have included quantitative trait locus (QTL) mapping and gene targeting of known core circadian clock genes. We combined three different genetic approaches to identify and test a positional candidate sleep gene - the circadian gene casein kinase 1 epsilon (Csnk1e), which is located in a QTL we identified for rapid eye movement (REM) sleep on chromosome 15. MEASUREMENTS AND RESULTS: Using electroencephalographic (EEG) and electromyographic (EMG) recordings, baseline sleep was examined in a 12-h light:12-h dark (LD 12:12) cycle in mice of seven genotypes, including Csnk1e(tau/tau) and Csnk1e(-/-) mutant mice, Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice, and their respective wild-type littermate control mice. Additionally, Csnk1e(tau/tau) and wild-type mice were examined in constant darkness (DD). Csnk1e(tau/tau) mutant mice and both Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice showed significantly higher proportion of sleep time spent in REM sleep during the dark period than wild-type controls - the original phenotype for which the QTL on chromosome 15 was identified. This phenotype persisted in Csnk1e(tau/tau) mice while under free-running DD conditions. Other sleep phenotypes observed in Csnk1e(tau/tau) mice and congenics included a decreased number of bouts of nonrapid eye movement (NREM) sleep and an increased average NREM sleep bout duration. CONCLUSIONS: These results demonstrate a role for Csnk1e in regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture, and support Csnk1e as a causal gene in the sleep QTL on chromosome 15.

Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation.

The circadian clock orchestrates temporal patterns of physiology and behavior relative to the environmental light:dark cycle by generating and organizing transcriptional and biochemical rhythms in cells and tissues throughout the body. Circadian clock genes have been shown to regulate the physiology and function of the gastrointestinal tract. Disruption of the intestinal epithelial barrier enables the translocation of proinflammatory bacterial products, such as endotoxin, across the intestinal wall and into systemic circulation; a process that has been linked to pathologic inflammatory states associated with metabolic, hepatic, cardiovascular and neurodegenerative diseases - many of which are commonly reported in shift workers. Here we report, for the first time, that circadian disorganization, using independent genetic and environmental strategies, increases permeability of the intestinal epithelial barrier (i.e., gut leakiness) in mice. Utilizing chronic alcohol consumption as a well-established model of induced intestinal hyperpermeability, we also found that both genetic and environmental circadian disruption promote alcohol-induced gut leakiness, endotoxemia and steatohepatitis, possibly through a mechanism involving the tight junction protein occludin. Circadian organization thus appears critical for the maintenance of intestinal barrier integrity, especially in the context of injurious agents, such as alcohol. Circadian disruption may therefore represent a previously unrecognized risk factor underlying the susceptibility to or development of alcoholic liver disease, as well as other conditions associated with intestinal hyperpermeability and an endotoxin-triggered inflammatory state.

Environmental perturbation of the circadian clock disrupts pregnancy in the mouse.

BACKGROUND: The circadian clock has been linked to reproduction at many levels in mammals. Epidemiological studies of female shift workers have reported increased rates of reproductive abnormalities and adverse pregnancy outcomes, although whether the cause is circadian disruption or another factor associated with shift work is unknown. Here we test whether environmental disruption of circadian rhythms, using repeated shifts of the light:dark (LD) cycle, adversely affects reproductive success in mice. METHODOLOGY/PRINCIPAL FINDINGS: Young adult female C57BL/6J (B6) mice were paired with B6 males until copulation was verified by visual identification of vaginal plug formation. Females were then randomly assigned to one of three groups: control, phase-delay or phase-advance. Controls remained on a constant 12-hr light:12-hr dark cycle, whereas phase-delayed and phase-advanced mice were subjected to 6-hr delays or advances in the LD cycle every 5-6 days, respectively. The number of copulations resulting in term pregnancies was determined. Control females had a full-term pregnancy success rate of 90% (11/12), which fell to 50% (9/18; p<0.1) in the phase-delay group and 22% (4/18; p<0.01) in the phase-advance group. CONCLUSIONS/SIGNIFICANCE: Repeated shifting of the LD cycle, which disrupts endogenous circadian timekeeping, dramatically reduces pregnancy success in mice. Advances of the LD cycle have a greater negative impact on pregnancy outcomes and, in non-pregnant female mice, require longer for circadian re-entrainment, suggesting that the magnitude or duration of circadian misalignment may be related to the severity of the adverse impact on pregnancy. These results explicitly link disruptions of circadian entrainment to adverse pregnancy outcomes in mammals, which may have important implications for the reproductive health of female shift workers, women with circadian rhythm sleep disorders and/or women with disturbed circadian rhythms for other reasons.

Rhythmic leptin is required for weight gain from circadian desynchronized feeding in the mouse.

The neuroendocrine and metabolic effects of leptin have been extensively researched since the discovery, and the later identification, of the leptin gene mutated within the ob/ob mouse. Leptin is required for optimal health in a number of physiological systems (e.g. fertility, bone density, body weight regulation). Despite the extensive leptin literature and many observations of leptin's cyclical pattern over the 24-hour day, few studies have specifically examined how the circadian rhythm of leptin may be essential to leptin signaling and health. Here we present data indicating that a rhythmic leptin profile (e.g. 1 peak every 24 hours) leads to excessive weight gain during desynchronized feeding whereas non-rhythmic leptin provided in a continuous manner does not lead to excessive body weight gain under similar feeding conditions. This study suggests that feeding time can interact with leptin's endogenous rhythm to influence metabolic signals, specifically leading to excessive body weight gains during 'wrongly' timed feeding.

Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway.

Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2(Luciferase) (Per2(Luc)) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.

The mouse Clock mutation reduces circadian pacemaker amplitude and enhances efficacy of resetting stimuli and phase-response curve amplitude.

The mouse Clock gene encodes a basic helix-loop-helix-PAS transcription factor, CLOCK, that acts in concert with BMAL1 to form the positive elements of the circadian clock mechanism in mammals. The original Clock mutant allele is a dominant negative (antimorphic) mutation that deletes exon 19 and causes an internal deletion of 51 aa in the C-terminal activation domain of the CLOCK protein. Here we report that heterozygous Clock/+ mice exhibit high-amplitude phase-resetting responses to 6-h light pulses (Type 0 resetting) as compared with wild-type mice that have low amplitude (Type 1) phase resetting. The magnitude and time course of acute light induction in the suprachiasmatic nuclei of the only known light-induced core clock genes, Per1 and Per2, are not affected by the Clock/+ mutation. However, the amplitude of the circadian rhythms of Per gene expression are significantly reduced in Clock homozygous and heterozygous mutants. Rhythms of PER2::LUCIFERASE expression in suprachiasmatic nuclei explant cultures also are reduced in amplitude in Clock heterozygotes. The phase-response curves to changes in culture medium are Type 0 in Clock heterozygotes, but Type 1 in wild types, similar to that seen for light in vivo. The increased efficacy of resetting stimuli and decreased PER expression amplitude can be explained in a unified manner by a model in which the Clock mutation reduces circadian pacemaker amplitude in the suprachiasmatic nuclei.