Protocol to study circadian food-anticipatory poking in mice using the feeding experimentation device version 3.

Food-anticipatory nose poking is a unique food-seeking behavior driven by the food-entrainable oscillator. Here, we present a protocol to record a novel food-seeking nose poking behavior in mice under temporally restricted feeding followed by food deprivation using the open-source feeding experimentation device version 3 (FED3). We describe steps for setting up the FED3 and cage, training, and habituation. We then detail procedures for setting up the schedule for time-restricted feeding and food deprivation and for generating ethograms from FED3 data. For complete details on the use and execution of this protocol, please refer to Ehichioya et al.1.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

Dietary restriction modulates ultradian rhythms and autocorrelation properties in mice behavior.

Animal behavior emerges from integration of many processes with different spatial and temporal scales. Dynamical behavioral patterns, including daily and ultradian rhythms and the dynamical microstructure of behavior (i.e., autocorrelations properties), can be differentially affected by external cues. Identifying these patterns is important for understanding how organisms adapt to their environment, yet unbiased methods to quantify dynamical changes over multiple temporal scales are lacking. Herein, we combine a wavelet approach with Detrended Fluctuation Analysis to identify behavioral patterns and evaluate changes over 42-days in mice subjected to different dietary restriction paradigms. We show that feeding restriction alters dynamical patterns: not only are daily rhythms modulated but also the presence, phase and/or strength of ~12h-rhythms, as well as the nature of autocorrelation properties of feed-intake and wheel running behaviors. These results highlight the underlying complexity of behavioral architecture and offer insights into the multi-scale impact of feeding habits on physiology.

Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

Neuronal deletion of the circadian clock gene Bmal1 induces cell-autonomous dopaminergic neurodegeneration.

Circadian rhythm dysfunction is a hallmark of Parkinson disease (PD), and diminished expression of the core clock gene Bmal1 has been described in patients with PD. BMAL1 is required for core circadian clock function but also serves nonrhythmic functions. Germline Bmal1 deletion can cause brain oxidative stress and synapse loss in mice, and it can exacerbate dopaminergic neurodegeneration in response to the toxin MPTP. Here we examined the effect of cell type-specific Bmal1 deletion on dopaminergic neuron viability in vivo. We observed that global, postnatal deletion of Bmal1 caused spontaneous loss of tyrosine hydroxylase+ (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc). This was not replicated by light-induced disruption of behavioral circadian rhythms and was not induced by astrocyte- or microglia-specific Bmal1 deletion. However, either pan-neuronal or TH neuron-specific Bmal1 deletion caused cell-autonomous loss of TH+ neurons in the SNpc. Bmal1 deletion did not change the percentage of TH neuron loss after α-synuclein fibril injection, though Bmal1-KO mice had fewer TH neurons at baseline. Transcriptomics analysis revealed dysregulation of pathways involved in oxidative phosphorylation and Parkinson disease. These findings demonstrate a cell-autonomous role for BMAL1 in regulating dopaminergic neuronal survival and may have important implications for neuroprotection in PD.

Gut microbes and the liver circadian clock partition glucose and lipid metabolism.

Circadian rhythms govern glucose homeostasis, and their dysregulation leads to complex metabolic diseases. Gut microbes exhibit diurnal rhythms that influence host circadian networks and metabolic processes, yet underlying mechanisms remain elusive. Here, we showed hierarchical, bidirectional communication among the liver circadian clock, gut microbes, and glucose homeostasis in mice. To assess this relationship, we utilized mice with liver-specific deletion of the core circadian clock gene Bmal1 via Albumin-cre maintained in either conventional or germ-free housing conditions. The liver clock, but not the forebrain clock, required gut microbes to drive glucose clearance and gluconeogenesis. Liver clock dysfunctionality expanded proportions and abundances of oscillating microbial features by 2-fold relative to that in controls. The liver clock was the primary driver of differential and rhythmic hepatic expression of glucose and fatty acid metabolic pathways. Absent the liver clock, gut microbes provided secondary cues that dampened these rhythms, resulting in reduced lipid fuel utilization relative to carbohydrates. All together, the liver clock transduced signals from gut microbes that were necessary for regulating glucose and lipid metabolism and meeting energy demands over 24 hours.

Metabolic and chemical architecture of the mammalian circadian clock.

Circadian rhythms are endogenous periodic biological processes that occur on a daily timescale. These rhythms are generated by a transcriptional/translational feedback loop that consists of the CLOCK-BMAL1 heterodimeric transcriptional activator complex and the PER1/2-CRY1/2-CK1δ/ε repressive complex. The output pathways of this molecular feedback loop generate circadian rhythmicity in various biological processes. Among these, metabolism is a primary regulatory target of the circadian clock which can also feedback to modulate clock function. This intertwined relationship between circadian rhythms and metabolism makes circadian clock components promising therapeutic targets. Despite this, pharmacological therapeutics that target the circadian clock are relatively rare. In this review, we hope to stimulate interest in chemical chronobiology by providing a comprehensive background on the molecular mechanism of mammalian circadian rhythms and their connection to metabolism, highlighting important studies in the chemical approach to circadian research, and offering our perspectives on future developments in the field.

The importance of sleep regularity: a consensus statement of the National Sleep Foundation sleep timing and variability panel.

OBJECTIVE: To develop and present consensus findings of the National Sleep Foundation sleep timing and variability panel regarding the impact of sleep timing variability on health and performance. METHODS: The National Sleep Foundation assembled a panel of sleep and circadian experts to evaluate the scientific evidence and conduct a formal consensus and voting procedure. A systematic literature review was conducted using the NIH National Library of Medicine PubMed database, and panelists voted on the appropriateness of 3 questions using a modified Delphi RAND/UCLA Appropriateness Method with 2 rounds of voting. RESULTS: The literature search and panel review identified 63 full text publications to inform consensus voting. Panelists achieved consensus on each question: (1) is daily regularity in sleep timing important for (a) health or (b) performance? and (2) when sleep is of insufficient duration during the week (or work days), is catch-up sleep on weekends (or non-work days) important for health? Based on the evidence currently available, panelists agreed to an affirmative response to all 3 questions. CONCLUSIONS: Consistency of sleep onset and offset timing is important for health, safety, and performance. Nonetheless, when insufficient sleep is obtained during the week/work days, weekend/non-work day catch-up sleep may be beneficial.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment in the mouse.

Nocturnal aversive stimuli presented to mice during eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus (SCN), the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our results support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders may represent the output of a fear-entrained clock. One-Sentence Summary: Cyclic fearful stimuli can entrain circadian rhythms in mice, and the molecular clock within the central circadian pacemaker is necessary but not sufficient for fear-entrainment.

Cry1 expression during postnatal development is critical for the establishment of normal circadian period.

The mammalian circadian system generates an approximate 24-h rhythm through a complex autoregulatory feedback loop. Four genes, Period1 (Per1), Period2 (Per2), Cryptochrome1 (Cry1), and Cryptochrome2 (Cry2), regulate the negative feedback within this loop. Although these proteins have distinct roles within the core circadian mechanism, their individual functions are poorly understood. Here, we used a tetracycline trans-activator system (tTA) to examine the role of transcriptional oscillations in Cry1 and Cry2 in the persistence of circadian activity rhythms. We demonstrate that rhythmic Cry1 expression is an important regulator of circadian period. We then define a critical period from birth to postnatal day 45 (PN45) where the level of Cry1 expression is critical for setting the endogenous free running period in the adult animal. Moreover, we show that, although rhythmic Cry1 expression is important, in animals with disrupted circadian rhythms overexpression of Cry1 is sufficient to restore normal behavioral periodicity. These findings provide new insights into the roles of the Cryptochrome proteins in circadian rhythmicity and further our understanding of the mammalian circadian clock.

Disease associations of excessive daytime sleepiness in multiple sclerosis: A prospective study.

Background: Excessive daytime sleepiness (EDS) in multiple sclerosis (MS) can be a significant source of disability. Despite this, its prevalence as a patient-reported outcome in this condition has not been well established, and its causes are not well understood. Methods: We prospectively assessed EDS as part of an observational study for patients referred for diagnostic neuro-ophthalmological testing. EDS was evaluated by the Epworth Sleepiness Scale (ESS), and visual data were also collected as part of a research protocol. Analysis with patient data was performed following the exclusion of patients with known primary sleep disorders. Results: A total of 69 patients with MS were included in the analysis. The mean ESS was 6.5 with a SD of 4.3. ESS ≥ 10 was present in 23% of the cohort even in the presence of minimal mean neurological disability (Patient Determined Disease Steps (PDDS) = 1.5). The ESS score was not associated with age, sex, disease-related disability, retinal nerve fiber layer (RNFL), or optic neuritis (ON), but displayed an association with visual dysfunction. Conclusions: There is an increased prevalence of EDS in MS. The increased values of the ESS are not explained by other sleep disorders, suggesting separate mechanisms. Further study of the underlying mechanisms is warranted.

Coupling-dependent metabolic ultradian rhythms in confluent cells.

Ultradian rhythms in metabolism and physiology have been described previously in mammals. However, the underlying mechanisms for these rhythms are still elusive. Here, we report the discovery of temperature-sensitive ultradian rhythms in mammalian fibroblasts that are independent of both the cell cycle and the circadian clock. The period in each culture is stable over time but varies in different cultures (ranging from 3 to 24 h). We show that transient, single-cell metabolic pulses are synchronized into stable ultradian rhythms across contacting cells in culture by gap junction-mediated coupling. Coordinated rhythms are also apparent for other metabolic and physiological measures, including plasma membrane potential (Δψp), intracellular glutamine, α-ketoglutarate, intracellular adenosine triphosphate (ATP), cytosolic pH, and intracellular calcium. Moreover, these ultradian rhythms require extracellular glutamine, several different ion channels, and the suppression of mitochondrial ATP synthase by α-ketoglutarate, which provides a key feedback mechanism. We hypothesize that cellular coupling and metabolic feedback can be used by cells to balance energy demands for survival.

A missense mutation in Kcnc3 causes hippocampal learning deficits in mice.

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named Clueless, with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 (Kcnc3) gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3G434V CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for Kcnc3 in learning and memory.

Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.

Caloric restriction (CR) prolongs life span, yet the mechanisms by which it does so remain poorly understood. Under CR, mice self-impose chronic cycles of 2-hour feeding and 22-hour fasting, raising the question of if it is calories, fasting, or time of day that is the cause of this increased life span. We show here that 30% CR was sufficient to extend the life span by 10%; however, a daily fasting interval and circadian alignment of feeding acted together to extend life span by 35% in male C57BL/6J mice. These effects were independent of body weight. Aging induced widespread increases in gene expression associated with inflammation and decreases in the expression of genes encoding components of metabolic pathways in liver from ad libitum-fed mice. CR at night ameliorated these aging-related changes. Our results show that circadian interventions promote longevity and provide a perspective to further explore mechanisms of aging.

Time to target the circadian clock for drug discovery.

The circadian clock is an intracellular timekeeping device that drives daily rhythms in diverse and extensive processes throughout the body. The clock mechanism comprises a core transcription/translation negative feedback loop that is modulated by a complex set of additional interlocking feedback loops. Pharmacological manipulation of the clock may be valuable for treating many maladies including jet lag, shift work and related sleep disorders, various metabolic diseases, and cancer. We review recent identification of small-molecule clock modulators and discuss the biochemical features of the core clock that may be amenable to future drug discovery.

Characterization of single nucleotide polymorphisms for a forward genetics approach using genetic crosses in C57BL/6 and BALB/c substrains of mice.

Forward genetics is a powerful approach based on chromosomal mapping of phenotypes and has successfully led to the discovery of many mouse mutations in genes responsible for various phenotypes. Although crossing between genetically remote strains can produce F2 and backcross mice for chromosomal mapping, the phenotypes are often affected by background effects from the partner strains in genetic crosses. Genetic crosses between substrains might be useful in genetic mapping to avoid genetic background effects. In this study, we investigated single nucleotide polymorphisms (SNPs) available for genetic mapping using substrains of C57BL/6 and BALB/c mice. In C57BL/6 mice, 114 SNP markers were developed and assigned to locations on all chromosomes for full utilization for genetic mapping using genetic crosses between the C57BL/6J and C57BL/6N substrains. Moreover, genetic differences were identified in the 114 SNP markers among the seven C57BL/6 substrains from five production breeders. In addition, 106 SNPs were detected on all chromosomes of BALB/cAJcl and BALB/cByJJcl substrains. These SNPs could be used for genotyping in BALB/cJ, BALB/cAJcl, BALB/cAnNCrlCrlj, and BALB/cCrSlc mice, and they are particularly useful for genetic mapping using crosses between BALB/cByJJcl and other BALB/c substrains. The SNPs characterized in this study can be utilized for genetic mapping to identify the causative mutations of the phenotypes induced by N-ethyl-N-nitrosourea mutagenesis and the SNPs responsible for phenotypic differences between the substrains of C57BL/6 and BALB/c mice.

Circadian rhythms in infectious diseases and symbiosis.

Timing is everything. Many organisms across the tree of life have evolved timekeeping mechanisms that regulate numerous of their cellular functions to optimize timing by anticipating changes in the environment. The specific environmental changes that are sensed depends on the organism. For animals, plants, and free-living microbes, environmental cues include light/dark cycles, daily temperature fluctuations, among others. In contrast, for a microbe that is never free-living, its rhythmic environment is its host's rhythmic biology. Here, we describe recent research on the interactions between hosts and microbes, from the perspective both of symbiosis as well as infections. In addition to describing the biology of the microbes, we focus specifically on how circadian clocks modulate these host-microbe interactions.

Introduction to the Clock System.

Circadian (24-h) rhythms dictate almost everything we do, setting our clocks for specific times of sleeping and eating, as well as optimal times for many other basic functions. The physiological systems that coordinate circadian rhythms are intricate, but at their core, they all can be distilled down to cell-autonomous rhythms that are then synchronized within and among tissues. At first glance, these cell-autonomous rhythms may seem rather straight-forward, but years of research in the field has shown that they are strikingly complex, responding to many different external signals, often with remarkable tissue-specificity. To understand the cellular clock system, it is important to be familiar with the major players, which consist of pairs of proteins in a triad of transcriptional/translational feedback loops. In this chapter, we will go through each of the core protein pairs one-by-one, summarizing the literature as to their regulation and their broader impacts on circadian gene expression. We will conclude by briefly examining the human genetics literature, as well as providing perspectives on the future of the study of the molecular clock.

Synchronization between peripheral circadian clock and feeding-fasting cycles in microfluidic device sustains oscillatory pattern of transcriptome.

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment.