Estrogen-mediated coupling via gap junctions in the suprachiasmatic nucleus.

The circadian clock orchestrates many physiological and behavioural rhythms in mammals with 24-h periodicity, through a hierarchical organisation, with the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus. The circuits of the SCN generate circadian rhythms with precision, relying on intrinsic coupling mechanisms, for example, neurotransmitters like arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), neuronal gamma-aminobutyric acid (GABA) signalling and astrocytes connected by gap junctions composed of connexins (Cx). In female rodents, the presence of estrogen receptors (ERs) in the dorsal SCN suggests an influence of estrogen (E2) on the circuit timekeeping that could regulate circadian rhythm and coupling. To investigate this, we used SCN explants together with hypothalamic neurons and astrocytes. First, we showed that E2 stabilised the circadian amplitude in the SCN when rAVPs (receptor-associated vasopressin peptides) were inhibited. However, the phase delay induced by VIPAC2 (VIP receptors) inhibition remained unaffected by E2. We then showed that E2 exerted its effects in the SCN via ERß (estrogen receptor beta), resulting in increased expression of Cx36 and Cx43. Notably, specific inhibition of both connexins resulted in a significant reduction in circadian amplitude within the SCN. Remarkably, E2 restored the period with inhibited Cx36 but not with Cx43 inhibition. This implies that the network between astrocytes and neurons, responsible for coupling in the SCN, can be reinforced through E2. In conclusion, these findings provide new insights into how E2 regulates circadian rhythms ex vivo in an ERß-dependent manner, underscoring its crucial role in fortifying the SCN's rhythm.

Circle(s) of Life: The Circadian Clock from Birth to Death.

Most lifeforms on earth use endogenous, so-called circadian clocks to adapt to 24-h cycles in environmental demands driven by the planet's rotation around its axis. Interactions with the environment change over the course of a lifetime, and so does regulation of the circadian clock system. In this review, we summarize how circadian clocks develop in humans and experimental rodents during embryonic development, how they mature after birth and what changes occur during puberty, adolescence and with increasing age. Special emphasis is laid on the circadian regulation of reproductive systems as major organizers of life segments and life span. We discuss differences in sexes and outline potential areas for future research. Finally, potential options for medical applications of lifespan chronobiology are discussed.

Genetic and environmental circadian disruption induce weight gain through changes in the gut microbiome.

OBJECTIVE: Internal clocks time behavior and physiology, including the gut microbiome, in a circadian (∼24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown. METHODS: Core and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the host's physiology. RESULTS: Both chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW-associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation. CONCLUSIONS: Collectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work.

Studying Circadian Clock Entrainment by Hormonal Signals.

In mammals, molecular circadian clocks not only exist in the suprachiasmatic nucleus (SCN) but in almost all organ systems. Intriguingly, tissue clocks can operate in both isolated tissues and cell lines with endocrine signals mediating the circadian expression of local transcriptomes. This can be demonstrated by treating tissue explants with endocrine cues in a phase- and dose-dependent manner. In this chapter we provide an overview of methods to study the effects of candidate hormonal time cues on tissue clock resetting. We propose an experimental procedure based on an in vitro setup consisting of several consecutive steps in which organotypic tissue cultures or cells can be used. Our approach targets the potential resetting mechanism at three levels: the hormone, the direct clock gene target, and the tissue clock response.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Restructuring of the male mice peripheral circadian network after bariatric surgery.

Bariatric surgery is still the most effective long-term weight-loss therapy. Recent data indicate that surgical outcomes may be affected by diurnal food intake patterns. In this study, we aimed to investigate how surgery-induced metabolic adaptations (i.e. weight loss) interact with circadian clock function. For that reason, vertical sleeve gastrectomy (VSG) was performed in obese mice and rhythms in behavior, tissue rhythmicity, and white adipose tissue transcriptome were evaluated. VSG under constant darkness conditions led to a maximum weight loss of 18% compared to a loss of 3% after sham surgery. Post-surgical weight development was characterized by two distinct intervals of catabolic and subsequent anabolic metabolic state. Locomotor activity was not affected. However, VSG significantly increased active phase meal frequency in the anabolic state. No significant effects on clock gene rhythmicity were detected in adrenal and white adipose tissue (WAT) explant cultures. Transcriptome rhythm analyses of subcutaneous WAT revealed a reduction of cycling genes after VSG (sham: 2493 vs VSG: 1013) independent of sustained rhythms in core clock gene expression. This may be a consequence of weight loss-induced morphological reconstruction of WAT that overwrites the direct influence of the local clock machinery on the transcriptome. However, VSG altered rhythmic transcriptional regulation of WAT lipid metabolism pathways. Thus, our data suggest a reorganization of diurnal metabolic rhythms after VSG downstream of the molecular clock machinery.

Rapid Jetlag Resetting of Behavioral, Physiological, and Molecular Rhythms in Proestrous Female Mice.

A gradual adaptation to a shifted light-dark (LD) cycle is a key element of the circadian clock system and believed to be controlled by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Endocrine factors have a strong influence on the regulation of the circadian clock network and alter acute photic responses of the SCN clock. In females, endocrine function depends on the stage of the ovarian cycle. So far, however, little is known about the effect of the estrous cycle on behavioral and molecular responses to shifts in the LD rhythm. Based on this, we investigated whether estrous state affects the kinetics of phase shift during jetlag in behavior, physiology, and molecular clock rhythms in the SCN and in peripheral tissues. Female mice exposed to an advanced LD phase at proestrous or metestrous showed different phase-shift kinetics, with proestrous females displaying accelerated adaptation in behavior and physiology. Constant darkness release experiments suggest that these fast phase shifts do not reflect resetting of the SCN pacemaker. Explant experiments on SCN, adrenal gland, and uterus confirmed this finding with proestrous females showing significantly faster clock phase shifts in peripheral tissues compared with the SCN. Together, these findings provide strong evidence for an accelerated adaptation of proestrous compared with metestrous females to new LD conditions that is accompanied by rapid behavioral, physiological, and molecular rhythm resetting. Not only do these findings open up a new avenue to understand the effect of estrous cycle on the clock network under changing environmental conditions but also imply a greater susceptibility in proestrous females.

The Concept of Coupling in the Mammalian Circadian Clock Network.

The circadian clock network regulates daily rhythms in mammalian physiology and behavior to optimally adapt the organism to the 24-h day/night cycle. A central pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), coordinates subordinate cellular oscillators in the brain, as well as in peripheral organs to align with each other and external time. Stability and coordination of this vast network of cellular oscillators is achieved through different levels of coupling. Although coupling at the molecular level and across the SCN is well established and believed to define its function as pacemaker structure, the notion of coupling in other tissues and across the whole system is less well understood. In this review, we describe the different levels of coupling in the mammalian circadian clock system - from molecules to the whole organism. We highlight recent advances in gaining knowledge of the complex organization and function of circadian network regulation and its significance for the generation of stable but plastic intrinsic 24-h rhythms.

The role of the circadian clock system in physiology.

Life on earth is shaped by the 24-h rotation of our planet around its axes. To adapt behavior and physiology to the concurring profound but highly predictable changes, endogenous circadian clocks have evolved that drive 24-h rhythms in invertebrate and vertebrate species. At the molecular level, circadian clocks comprised a set of clock genes organized in a system of interlocked transcriptional-translational feedback loops. A ubiquitous network of cellular central and peripheral tissue clocks coordinates physiological functions along the day through activation of tissue-specific transcriptional programs. Circadian rhythms impact on diverse physiological processes including the cardiovascular system, energy metabolism, immunity, hormone secretion, and reproduction. This review summarizes our current understanding of the mechanisms of circadian timekeeping in different species, its adaptation by external timing signals and the pathophysiological consequences of circadian disruption.

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.

Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light.

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting.

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors.

Cold and hunger induce diurnality in a nocturnal mammal.

The mammalian circadian system synchronizes daily timing of activity and rest with the environmental light-dark cycle. Although the underlying molecular oscillatory mechanism is well studied, factors that influence phenotypic plasticity in daily activity patterns (temporal niche switching, chronotype) are presently unknown. Molecular evidence suggests that metabolism may influence the circadian molecular clock, but evidence at the level of the organism is lacking. Here we show that a metabolic challenge by cold and hunger induces diurnality in otherwise nocturnal mice. Lowering ambient temperature changes the phase of circadian light-dark entrainment in mice by increasing daytime and decreasing nighttime activity. This effect is further enhanced by simulated food shortage, which identifies metabolic balance as the underlying common factor influencing circadian organization. Clock gene expression analysis shows that the underlying neuronal mechanism is downstream from or parallel to the main circadian pacemaker (the hypothalamic suprachiasmatic nucleus) and that the behavioral phenotype is accompanied by phase adjustment of peripheral tissues. These findings indicate that nocturnal mammals can display considerable plasticity in circadian organization and may adopt a diurnal phenotype when energetically challenged. Our previously defined circadian thermoenergetics hypothesis proposes that such circadian plasticity, which naturally occurs in nocturnal mammals, reflects adaptive maintenance of energy balance. Quantification of energy expenditure shows that diurnality under natural conditions reduces thermoregulatory costs in small burrowing mammals like mice. Metabolic feedback on circadian organization thus provides functional benefits by reducing energy expenditure. Our findings may help to clarify relationships between sleep-wake patterns and metabolic phenotypes in humans.

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli.

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1ε normally serves. Here, we reveal that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers.

Working for food shifts nocturnal mouse activity into the day.

Nocturnal rodents show diurnal food anticipatory activity when food access is restricted to a few hours in daytime. Timed food access also results in reduced food intake, but the role of food intake in circadian organization per se has not been described. By simulating natural food shortage in mice that work for food we show that reduced food intake alone shifts the activity phase from the night into the day and eventually causes nocturnal torpor (natural hypothermia). Release into continuous darkness with ad libitum food, elicits immediate reversal of activity to the previous nocturnal phase, indicating that the classical circadian pacemaker maintained its phase to the light-dark cycle. This flexibility in behavioral timing would allow mice to exploit the diurnal temporal niche while minimizing energy expenditure under poor feeding conditions in nature. This study reveals an intimate link between metabolism and mammalian circadian organization.

Age and oestrus cycle-related changes in glucocorticoid excretion and wheel-running activity in female mice carrying mutations in the circadian clock genes Per1 and Per2.

In mammals, numerous physiological and behavioural functions are controlled by an endogenous circadian clock located in the suprachiasmatic nuclei (SCN). Within the SCN neurons, clock genes such as Per1 and Per2 interact in a molecular clockwork regulating the expression of hundreds of output genes. Through the timed release of humoral and neuronal signals, the rhythmicity of numerous biological processes, including reproductive behaviour, the oestrus cycle and endocrine parameters is controlled by the SCN. Mutations in Per genes in mice affect a wide array of physiological functions. Interestingly, most of these studies use only male animals, thus neglecting potential gender-specificities in clock function. In an attempt to broaden this perspective we have investigated the impact of Per1 and Per2 mutations on both glucocorticoid (GC) metabolite excretion and locomotor activity in relation to age and oestrus cycle stage of female mice. We show that the Per2 mutation dampens daily GC rhythms in young adult females. While locomotor activity does not vary along the different oestrus stages in Per2 mutant females, oestrus effects on GC excretion and locomotor activity are largely comparable between Per1 mutants and wild-type animals. 20 month-old, acyclic Per1 and wild-type females show reduced GC levels when compared to young adults while aged Per2 mutants retain their normal GC rhythmicity. Correlating with this, onsets of locomotor activity do not change with age in Per2 mutant females. Together, our data highlight specific roles for Per1 and Per2 in both the regulation of locomotor activity and endocrine functions in the female organism.

Low reproductive success in Per1 and Per2 mutant mouse females due to accelerated ageing?

Recent studies on mice with mutations in the Clock gene have shown that this mutation disrupts oestrus cyclicity and interferes with successful pregnancy. In order to determine whether two other molecular components of the main clock, namely the period genes, Per1 and Per2, have an effect on the length of the oestrous cycle and the reproductive success, we used Per1- and Per2-deficient females. We show that although fecundity of young adult Per mutant females does not differ from that of wild-type females, middle-aged Per mutant mice are characterised by lower reproductive success than the control group. This may be a consequence of irregularity and acyclicity of the oestrous cycle of the middle-aged mutants. Besides, we demonstrate that Per mutant females have significantly more embryonal implantations in the uterus than successfully delivered offspring. The reproductive deficits of the middle-aged Per mutant females are comparable with those seen in aged wild-type mice. This suggests that Per1 and Per2 mutations cause an advanced ageing.

The cost of a specific immune response in young guinea pigs.

The specific immune system is a protective mechanism that detects infection and fights it by production of antibodies. Newborns are especially susceptible to infections because their immune system is not yet as fully developed as that of adults. This has been well established in altricial mammals. Fighting infection is associated with costs (metabolic rate, protein synthesis) potentially affecting other developmental processes. We investigated the specific immune response in a precocial mammal, by testing the response of 3 and 7 day old young guinea pigs (Cavia aperea f. porcellus) against a non-pathogenic antigen (KLH) and determined the effect of the immune response on growth and metabolic rate. Challenged young produced a substantial specific immune response (IgG). The efficiency of the immune response was almost identical in 3 and 7 day old young, but lower than in adult females. Antibody titres achieved by actively immunised young pups were as high as titres transferred transplacentally by mothers immunised on day 40 and 47 of pregnancy. In comparison to a control group, the immune response did not influence growth and metabolic rate measured on day 4 after each immune challenge and was not reflected by changes in hematocrit value. We discuss whether the weaker immune response of pups is caused by reduced allocation of limited resources in growing young or by the immature immune system of young animals.