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The circadian clock times physiological and behavioural processes and resets on a daily basis to synchronize with the environment. The involvement of the circadian clock in photoperiodic time measurement synchronising annual rhythms is still under debate and different models have been proposed explaining their integration. Insects overcome unfavourable conditions in diapause, a form of dormancy. A latitudinal cline in diapause induction in the parasitoid wasp Nasonia vitripennis as well as a difference in circadian light sensitivity between north and south provide us with additional evidence that the circadian system of Nasonia is involved in photoperiodic time measurement and that circadian adaptation is a potential mechanism driving latitudinal adaptation in photoperiodism. We tested diapause induction in a range of T-cycles and photoperiods and found diapause induction in short photoperiods in all T-cycles in the northern line but in the southern line diapause only occurred in T-cycles close to 24 h. Due to a lower light sensitivity in the southern line, a wider distribution of phase angles of entrainment can be expected at a specific T-cycle duration, while the range of entrainment will decrease. Taking these oscillator properties into account, our data can be explained by an external coincidence model involving a single oscillator with a light-sensitive phase that drives annual timing of diapause in Nasonia vitripennis.

This work was supported in part by the Netherlands Forensic Institute, Netherlands Genomics Initiative/Netherlands Organization for Scientific Research within the framework of the Forensic Genomics Consortium Netherlands, the 6th Framework project EUCLOCK (018741), and UK Biotechnology and Biological Sciences Research Council Grant BB/I019405/1. Additional funding was received from the Cancer Research UK Cancer Therapeutics Unit award (Ref: C2739/A22897) and a Cancer Therapeutics Centre award (Ref: C309/A25144 to FR), the NWO-STW Perspective Program grant ‘OnTime’ (project 12185 to TW and RAH). Circadian rhythms influence physiology, metabolism, and molecular processes in the human body. Estimation of individual body time (circadian phase) is therefore highly relevant for individual optimization of behavior (sleep, meals, sports), diagnostic sampling, medical treatment, and for treatment of circadian rhythm disorders. Here, we provide a partial least squares regression (PLSR) machine learning approach that uses plasma-derived metabolomics data in one or more samples to estimate dim light melatonin onset (DLMO) as a proxy for circadian phase of the human body. For this purpose, our protocol was aimed to stay close to real-life conditions. We found that a metabolomics approach optimized for either women or men under entrained conditions performed equally well or better than existing approaches using more labor-intensive RNA sequencing-based methods. Although estimation of circadian body time using blood-targeted metabolomics requires further validation in shift work and other real-world conditions, it currently may offer a robust, feasible technique with relatively high accuracy to aid personalized optimization of behavior and clinical treatment after appropriate validation in patient populations. Publisher PDF

The vertebrate photoperiodic neuroendocrine system uses the photoperiod as a proxy to time the annual rhythms in reproduction. The thyrotropin receptor (TSHR) is a key protein in the mammalian seasonal reproduction pathway. Its abundance and function can tune sensitivity to the photoperiod. To investigate seasonal adaptation in mammals, the hinge region and the first part of the transmembrane domain of the Tshr gene were sequenced for 278 common vole (Microtus arvalis) specimens from 15 localities in Western Europe and 28 localities in Eastern Europe. Forty-nine single nucleotide polymorphisms (SNPs; twenty-two intronic and twenty-seven exonic) were found, with a weak or lack of correlation with pairwise geographical distance, latitude, longitude, and altitude. By applying a temperature threshold to the local photoperiod–temperature ellipsoid, we obtained a predicted critical photoperiod (pCPP) as a proxy for the spring onset of local primary food production (grass). The obtained pCPP explains the distribution of the genetic variation in Tshr in Western Europe through highly significant correlations with five intronic and seven exonic SNPs. The relationship between pCPP and SNPs was lacking in Eastern Europe. Thus, Tshr, which plays a pivotal role in the sensitivity of the mammalian photoperiodic neuroendocrine system, was targeted by natural selection in Western European vole populations, resulting in the optimized timing of seasonal reproduction.

Climate change will strongly affect the developmental timing of insects, as their development rate depends largely on ambient temperature. However, we know little about the genetic mechanisms underlying the temperature sensitivity of embryonic development in insects. We investigated embryonic development rate in the winter moth (Operophtera brumata), a species with egg dormancy which has been under selection due to climate change. We used RNA sequencing to investigate which genes are involved in the regulation of winter moth embryonic development rate in response to temperature. Over the course of development, we sampled eggs before and after an experimental change in ambient temperature, including two early development weeks when the temperature sensitivity of eggs is low and two late development weeks when temperature sensitivity is high. We found temperature-responsive genes that responded in a similar way across development, as well as genes with a temperature response specific to a particular development week. Moreover, we identified genes whose temperature effect size changed around the switch in temperature sensitivity of development rate. Interesting candidate genes for regulating the temperature sensitivity of egg development rate included genes involved in histone modification, hormonal signalling, nervous system development and circadian clock genes. The diverse sets of temperature-responsive genes we found here indicate that there are many potential targets of selection to change the temperature sensitivity of embryonic development rate. Identifying for which of these genes there is genetic variation in wild insect populations will give insight into their adaptive potential in the face of climate change.

Policymakers aim to move toward animal-free alternatives for scientific research and have introduced very strict regulations for animal research. We argue that, for neuroscience research, until viable and translational alternatives become available and the value of these alternatives has been proven, the use of animals should not be compromised., We would like to thank Loren Frank, UCSF, USA; Sheena Josselyn, Hospital for Sick Children, University of Toronto, Canada; Shantanu Jadhav, Brandeis University, USA; the European Animal Research Association (EARA); the Federation of European Neuroscience Societies Committee on Animals in Research (CARE); the Swiss Society for Neuroscience; the Society for Neuroscience Committee on Animals in Research (CAR); and Stichting Informatie Dierproeven (the Dutch foundation for public information on animal testing: SID) for input on and support for this article.

Many mammalian species use photoperiod as a predictive cue to time seasonal reproduction. In addition, metabolic effects on the reproductive axis may also influence seasonal timing, especially in female small, short-lived mammals. To get a better understanding of how annual cycling environmental cues impact reproductive function and plasticity in small, short-lived herbivores with different geographic origins, we investigated the mechanisms underlying integration of temperature in the photoperiodic-axis regulating female reproduction in a Northern vole species (tundra vole, Microtus oeconomus) and in a Southern vole species (common vole, Microtus arvalis). We show that photoperiod and temperature interact to determine appropriate physiological responses; there is species-dependent annual variation in the sensitivity to temperature for reproductive organ development. In common voles, temperature can overrule photoperiodical spring-programmed responses, with reproductive organ mass being higher at 10°C than at 21°C, whereas in autumn they are less sensitive to temperature. These findings are in line with our census data, showing an earlier onset of spring reproduction in cold springs, while reproductive offset in autumn is synchronized to photoperiod. The reproductive organs of tundra voles were relatively insensitive to temperature, whereas hypothalamic gene expression was generally upregulated at 10°C. Thus, both vole species use photoperiod, whereas only common voles use temperature as a cue to control spring reproduction, which indicates species-specific reproductive strategies. Due to global warming, spring reproduction in common voles will be delayed, perhaps resulting in shorter breeding seasons and thus declining populations, as observed throughout Europe.

While stress does not affect the phase or period of the central pacemaker in the suprachiasmatic nucleus, it can shift clocks in peripheral tissues. Our previous studies showed significant delays of the PER2 rhythms in lung and kidney following social defeat stress. The mechanism underlying these effects is not fully understood, but might involve glucocorticoids (GC) released during the stressor. In the present study, we performed social defeat stress in adrenalectomized (ADX) mice to see if the induction of endogenous GC is necessary for the stress-induced phase shifts of peripheral clocks. We used mice that carry a luciferase reporter gene fused to the circadian clock gene Period2 (PER2::LUC) to examine daily rhythms of PER2 expression in various peripheral tissues. Mice were exposed to 5 consecutive daily social defeat stress in the late dark phase (ZT21-22). Running wheel rotations were recorded during 7 baseline and 5 social defeat days, which showed that social defeat stress suppressed locomotor activity without affecting the phase of the rhythm. This suppression of activity was not prevented by ADX. One hour after the last stressor, tissue samples from the liver, kidney and lung were collected and cultured for ex vivo bioluminescence recordings. In the liver, PER2 rhythms were not affected by social defeat stress or ADX. In the kidney, social defeat stress caused a > 4 h phase delay of the PER2 rhythm, which was prevented by ADX, supporting the hypothesis of a crucial role of GC in this stress effect. In the lung, social defeat stress caused an 8 h phase delay, but, surprisingly, a similar phase delay was seen in ADX animals independent of defeat. The latter indicates complex effects of stress and stress hormones on the lung clock. In conclusion, the findings show that repeated social defeat stress in the dark phase can shift PER2 rhythms in some tissues (lung, kidney) and not others (liver). Moreover, the social defeat stress effect in some tissues appears to be mediated by glucocorticoids (kidney) whereas the mechanism in other tissues is more complex (lung).

Uncontrollable stress is linked to the development of many diseases, some of which are associated with disrupted daily rhythms in physiology and behavior. While available data indicate that the master circadian pacemaker in the suprachiasmatic nucleus (SCN) is unaffected by stress, accumulating evidence suggest that circadian oscillators in peripheral tissues and organs can be shifted by a variety of stressors and stress hormones. In the present study, we examined effects of acute and chronic social defeat stress in mice and addressed the question of whether effects of uncontrollable stress on peripheral clocks are tissue specific and depend on time of day of stress exposure. We used mice that carry a luciferase reporter gene fused to the circadian clock gene Period2 (PER2::LUC) to examine daily rhythms of PER2 expression in various peripheral tissues. Mice were exposed to social defeat stress in the early (ZT13-14) or late (ZT21-22) dark phase, either once (acute stress) or repeatedly on 10 consecutive days (chronic stress). One hour after the last stressor, tissue samples from liver, lung, kidney, and white adipose tissue (WAT) were collected. Social defeat stress caused a phase delay of several hours in the rhythm of PER2 expression in lung and kidney, but this delay was stronger after chronic than after acute stress. Moreover, shifts only occurred after stress in the late dark phase, not in the early dark phase. PER2 rhythms in liver and WAT were not significantly shifted by social defeat, suggesting a different response of various peripheral clocks to stress. This study indicates that uncontrollable social defeat stress is capable of shifting peripheral clocks in a time of day dependent and tissue specific manner. These shifts in peripheral clocks were smaller or absent after a single stress exposure and may therefore be the consequence of a cumulative chronic stress effect.

Seasonal timing of reproduction in voles is driven by photoperiod. We hypothesized that a negative energy balance can modify spring-programmed photoperiodic responses in the hypothalamus, controlling reproductive organ development. We manipulated energy balance by the ‘work-for-food’ protocol, in which voles were exposed to increasing levels of food scarcity at different ambient temperatures under long photoperiod. We found that in common voles (Microtus arvalis) and tundra voles (Microtus oeconomus), photoperiod-induced pars tuberalis thyroid-stimulating hormone β-subunit (Tshβ) expression is reduced to potentially inhibit gonadal development when food is scarce. Reduction in gonadal size is more pronounced in tundra voles, in which anterior hypothalamic Kiss1 is additionally downregulated, especially in males. Low temperature additionally leads to decreased hypothalamic Rfrp expression, which potentially may facilitate further suppression of gonadal growth. Shutting off the photoperiodic axis when food is scarce in spring may be an adaptive response to save energy, leading to delayed reproductive organ development until food resources are sufficient for reproduction, lactation and offspring survival. Defining the mechanisms through which metabolic cues modify photoperiodic responses will be important for a better understanding of how environmental cues impact reproduction.

In mammals, daily rhythms in physiology and behavior are under control of a circadian pacemaker situated in the suprachiasmatic nucleus (SCN). This master clock receives photic input from the retina and coordinates peripheral oscillators present in other tissues, maintaining all rhythms in the body synchronized to the environmental light-dark cycle. In line with its function as a master clock, the SCN appears to be well protected against unpredictable stressful stimuli. However, available data indicate that stress and stress hormones at certain times of day are capable of shifting peripheral oscillators in, e.g., liver, kidney and heart, which are normally under control of the SCN. Such shifts of peripheral oscillators may represent a temporary change in circadian organization that facilitates adaptation to repeated stress. Alternatively, these shifts of internal rhythms may represent an imbalance between precisely orchestrated physiological and behavioral processes that may have severe consequences for health and well-being.

Maximising survival requires animals to balance the competing demands of maintaining energy balance and avoiding predation. Here, quantitative modelling shows that optimising the daily timing of activity and rest based on the encountered environmental conditions enables small mammals to maximise survival. Our model shows that nocturnality is typically beneficial when predation risk is higher during the day than during the night, but this is reversed by the energetic benefit of diurnality when food becomes scarce. Empirical testing under semi‐natural conditions revealed that the daily timing of activity and rest in mice exposed to manipulations in energy availability and perceived predation risk is in line with the model’s predictions. Low food availability and decreased perceived daytime predation risk promote diurnal activity patterns. Overall, our results identify temporal niche switching in small mammals as a strategy to maximise survival in response to environmental changes in food availability and perceived predation risk.

Voles are small herbivorous rodents that can display both circadian activity rhythms (~24-h periodicity) and ultradian activity rhythms (~1- to 6-h periodicity). Ultradian rhythms are observed on an individual level, but also in synchronized populations. Ultradian rhythm period has been suggested to be influenced by energy balance, but the underlying mechanisms of ultradian rhythmicity are poorly understood. We manipulated energy balance by implementing the “work-for-food” paradigm, in which small rodents are exposed to increasing levels of food scarcity at different ambient temperatures in the laboratory. Photoperiodical spring-programmed voles on high workload changed their nocturnal circadian activity and body temperature rhythm to ultradian patterns, indicating that a negative energy balance induces ultradian rhythmicity. This interpretation was confirmed by the observation that ultradian patterns arose earlier at low temperatures. Interestingly, a positive relationship between ultradian period length and workload was observed in tundra voles. Spectral analysis revealed that the power of ultradian rhythmicity increased at high workload, whereas the circadian component of running wheel activity decreased. This study shows that the balance between circadian and ultradian rhythmicity is determined by energy balance, confirming flexible circadian and ultradian rhythms in females and males of 2 different vole species: the common vole ( Microtus arvalis) and the tundra vole ( Microtus oeconomus).

Seasonal timing of reproduction in voles is driven by photoperiod. Here we hypothesize that a negative energy balance can modify spring-programmed photoperiodic responses in the hypothalamus, controlling reproductive organ development. We manipulated energy balance by the ‘work-for-food’ protocol, in which voles were exposed to increasing levels of food scarcity at different ambient temperatures under long photoperiod. We reveal that common (Microtus arvalis) and tundra voles (Microtus oeconomus), reduce photoperiodic induced pars tuberalis thyroid-stimulating hormone β-subunit (Tshβ) expression to inhibit gonadal development when food is scarce. Reduction in gonadal size is more pronounced in tundra voles, in which the hypothalamic Kisspeptin (Kiss1) system seems involved in downregulating gonadal development, especially in males. Low temperature additionally leads to decreased hypothalamic RF-amide related peptide (Rfrp3) levels, which may facilitate further suppression of gonadal growth. Shutting off the photoperiodic-axis when food is scarce in spring may be an adaptive response to save energy, leading to delayed reproductive organ development until food resources are sufficient for reproduction, lactation and offspring survival. Defining the mechanisms through which metabolic cues modify photoperiodic responses will be important for a better understanding of how environmental cues impact reproduction.Summary statementThis study provides a better understanding of the molecular mechanism through which metabolic cues can modify photoperiodic responses, to adaptively adjust timing of reproductive organ development

Global warming is predicted to have major effects on the annual time windows during which species may successfully reproduce. At the organismal level, climatic shifts engage with the control mechanism for reproductive seasonality. In mammals, laboratory studies on neuroendocrine mechanism emphasize photoperiod as a predictive cue, but this is based on a restricted group of species. In contrast, field-oriented comparative analyses demonstrate that proximate bioenergetic effects on the reproductive axis are a major determinant of seasonal reproductive timing. The interaction between proximate energetic and predictive photoperiodic cues is neglected. Here, we focused on photoperiodic modulation of postnatal reproductive development in common voles (Microtus arvalis), a herbivorous species in which a plastic timing of breeding is well documented. We demonstrate that temperature-dependent modulation of photoperiodic responses manifest in the thyrotrophin-sensitive tanycytes of the mediobasal hypothalamus. Here, the photoperiod-dependent expression of type 2 deiodinase expression, associated with the summer phenotype was enhanced by 21°C, whereas the photoperiod-dependent expression of type 3 deiodinase expression, associated with the winter phenotype, was enhanced by 10°C in spring voles. Increased levels of testosterone were found at 21°C, whereas somatic and gonadal growth were oppositely affected by temperature. The magnitude of these temperature effects was similar in voles photoperiodical programmed for accelerated maturation (ie, born early in the breeding season) and in voles photoperiodical programmed for delayed maturation (ie, born late in the breeding season). The melatonin-sensitive pars tuberalis was relatively insensitive to temperature. These data define a mechanistic hierarchy for the integration of predictive temporal cues and proximate thermo-energetic effects in mammalian reproduction.

Light induced improvements in alertness are more prominent during night-time than during the day, indicating circadian regulation or wake duration related dependence. Relative contributions of both factors can be quantified using a forced desynchrony (FD) designs. Here we investigate alerting effects of light in a novel 4×18 hours FD protocol (5h sleep, 13h wake) under dim (6 mlux) and bright light (1159 mlux) conditions. Hourly saliva samples (melatonin and cortisol assessment) and 2-hourly test-sessions were used to assess effects of bright light on subjective and objective alertness (electroencephalography and performance). Results reveal (1) stable free-running cortisol rhythms with uniform phase progression under both light conditions, indicating that FD designs can be conducted under high intensity lighting, (2) subjective alerting effects of light depend on elapsed time awake, while (3) light consistently improves objective alertness independent of time awake. Three dimensional graphs reflecting light induced alertness improvements depending on wake duration related variation and circadian clock phase suggest that performance is improved during daytime, while subjective alertness remains unchanged. This suggests that light during office hours might be beneficial for performance, even though this may not be perceived as such.

Broad-spectrum light applied during the night has been shown to affect alertness in a dose-dependent manner. The goal of this experiment was to investigate whether a similar relationship could be established for light exposure during daytime. Fifty healthy participants were subjected to a paradigm (0730-1730 h) in which they were intermittently exposed to 1.5 h of dim light (

Significance Human retinas contain five light-responsive cell types: rods; short (S)-, mid (M)-, and long (L)-wavelength–sensitive cones; and melanopsin-expressing ganglion cells. Intrinsically light-sensitive retinal ganglion cells (RGCs) also relay rod and cone information to brain areas involved in nonvisual responses, such as pupil constriction. Cones allow for visual color discrimination. How cone-dependent color discrimination affects nonvisual responses is poorly understood. We show that selectively activating L-cones or melanopsin constricts the pupil whereas S- or M-cone activation paradoxically dilates the pupil. Intrinsically photosensitive RGCs therefore appear to signal color on yellow/blue and red/green scales, with blue and green cone shifts being processed as brightness decrements. These findings are crucial in understanding brightness coding for visual and nonvisual responses such as circadian entrainment and alerting effects of light., The human retina contains five photoreceptor types: rods; short (S)-, mid (M)-, and long (L)-wavelength–sensitive cones; and melanopsin-expressing ganglion cells. Recently, it has been shown that selective increments in M-cone activation are paradoxically perceived as brightness decrements, as opposed to L-cone increments. Here we show that similar effects are also observed in the pupillary light response, whereby M-cone or S-cone increments lead to pupil dilation whereas L-cone or melanopic illuminance increments resulted in pupil constriction. Additionally, intermittent photoreceptor activation increased pupil constriction over a 30-min interval. Modulation of L-cone or melanopic illuminance within the 0.25–4-Hz frequency range resulted in more sustained pupillary constriction than light of constant intensity. Opposite results were found for S-cone and M-cone modulations (2 Hz), mirroring the dichotomy observed in the transient responses. The transient and sustained pupillary light responses therefore suggest that S- and M-cones provide inhibitory input to the pupillary control system when selectively activated, whereas L-cones and melanopsin response fulfill an excitatory role. These findings provide insight into functional networks in the human retina and the effect of color-coding in nonvisual responses to light, and imply that nonvisual and visual brightness discrimination may share a common pathway that starts in the retina.

This study was supported by a UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant (BB/I019405/1) to DJS, grant 727.011.001 from the Netherlands Organization for Scientific Research (NWO) Forensic Science Program to MK and by Erasmus MC University Medical Centre Rotterdam. DJS is a Royal Society Wolfson Research Merit Award holder. RAH and IH were funded by the Dutch applied research foundation (STW Perspectief Program ‘OnTime’ project 12185). Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor’s alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose. Publisher PDF

Light is the most potent time cue that synchronizes (entrains) the circadian pacemaker to the 24-h solar cycle. This entrainment process is an interplay between an individual's daily light perception and intrinsic pacemaker period under free-running conditions. Establishing individual estimates of circadian phase and period can be time-consuming. We show that circadian phase can be accurately predicted (SD = 1.1 h for dim light melatonin onset, DLMO) using 9 days of ambulatory light and activity data as an input to Kronauer's limit-cycle model for the human circadian system. This approach also yields an estimated circadian period of 24.2 h (SD = 0.2 h), with longer periods resulting in later DLMOs. A larger amount of daylight exposure resulted in an earlier DLMO. Individuals with a long circadian period also showed shorter intervals between DLMO and sleep timing. When a field-based estimation of tau can be validated under laboratory studies in a wide variety of individuals, the proposed methods may prove to be essential tools for individualized chronotherapy and light treatment for shift work and jetlag applications. These methods may improve our understanding of fundamental properties of human circadian rhythms under daily living conditions.

The Darwinian fitness of mammals living in a rhythmic environment depends on endogenous daily (circadian) rhythms in behavior and physiology. Here, we discuss the mechanisms underlying the circadian regulation of physiology and behavior in mammals. We also review recent efforts to understand circadian flexibility, such as how the phase of activity and rest is altered depending on the encountered environment. We explain why shifting activity to the day is an adaptive strategy to cope with energetic challenges and show how this can reduce thermoregulatory costs. A framework is provided to make predictions about the optimal timing of activity and rest of non-model species for a wide range of habitats. This Review illustrates how the timing of daily rhythms is reciprocally linked to energy homeostasis, and it highlights the importance of this link in understanding daily rhythms in physiology and behavior.

Circadian (~24 h) rhythms in behavior and physiological functions are under control of an endogenous circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN directly drives some of these rhythms or serves as a coordinator of peripheral oscillators residing in other tissues and organs. Disruption of the circadian organization may contribute to disease, including stress-related disorders. Previous research indicates that the master clock in the SCN is resistant to stress, although it is unclear whether stress affects rhythmicity in other tissues, possibly mediated by glucocorticoids, released in stressful situations. In the present study, we examined the effect of uncontrollable social defeat stress and glucocorticoid hormones on the central and peripheral clocks, respectively in the SCN and liver. Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 (PER2) in SCN slices and liver tissue collected after 10 consecutive days of social defeat stress. The rhythmicity of PER2 expression in the SCN was not affected by stress exposure, whereas in the liver the expression showed a delayed phase in defeated compared to non-defeated control mice. In a second experiment, brain slices and liver samples were collected from transgenic mice and exposed to different doses of corticosterone. Corticosterone did not affect PER2 rhythm of the SCN samples, but caused a phase shift in PER2 expression in liver samples. This study confirms earlier findings that the SCN is resistant to stress and shows that clocks in the liver are affected by social stress, which might be due to the direct influence of glucocorticoids released from the adrenal gland., Highlights • Chronic social defeat does not affect phase or period of the circadian rhythm. • Chronic social defeat does not alter PER2 expression in the suprachiasmatic nucleus. • Chronic social defeat phase shifts PER2 expression in the liver. • PER2 expression in the liver is altered by corticosterone in vitro.

To optimally time reproduction, seasonal mammals use a photoperiodic neuroendocrine system (PNES) that measures photoperiod and subsequently drives reproduction. To adapt to late spring arrival at northern latitudes, a lower photoperiodic sensitivity and therefore a higher critical photoperiod for reproductive onset is necessary in northern species to arrest reproductive development until spring onset. Temperature–photoperiod relationships, and hence food availability–photoperiod relationships, are highly latitude dependent. Therefore, we predict PNES sensitivity characteristics to be latitude dependent. Here, we investigated photoperiodic responses at different times during development in northern (tundra or root vole, Microtus oeconomus) and southern vole species (common vole, Microtus arvalis) exposed to constant short (SP) or long photoperiod (LP). Although the tundra vole grows faster under LP, no photoperiodic effect on somatic growth is observed in the common vole. In contrast, gonadal growth is more sensitive to photoperiod in the common vole, suggesting that photoperiodic responses in somatic and gonadal growth can be plastic, and might be regulated through different mechanisms. In both species, thyroid-stimulating hormone β-subunit (Tshβ) and iodothyronine deiodinase 2 (Dio2) expression is highly increased under LP, whereas Tshr and Dio3 decrease under LP. High Tshr levels in voles raised under SP may lead to increased sensitivity to increasing photoperiods later in life. The higher photoperiodic-induced Tshr response in tundra voles suggests that the northern vole species might be more sensitive to thyroid-stimulating hormone when raised under SP. In conclusion, species differences in developmental programming of the PNES, which is dependent on photoperiod early in development, may form different breeding strategies as part of latitudinal adaptation.

The circadian system affects physiological, psychological, and molecular mechanisms in the body, resulting in varying physical performance over the day. The timing and relative size of these effects are important for optimizing sport performance. In this study, Olympic swim times (from 2004 to 2016) were used to determine time-of-day and circadian effects under maximal motivational conditions. Data of athletes who made it to the finals (N = 144, 72 female) were included and normalized on individual levels based on the average swim times over race types (heat, semifinal, and final) per individual for each stroke, distance and Olympic venue. Normalized swim times were analyzed with a linear mixed model and a sine fitted model. Swim performance was better during finals as compared to semi-finals and heats. Performance was strongly affected by time-of-day, showing fastest swim times in the late afternoon around 17:12 h, indicating 0.32% improved performance relative to 08:00 h. This study reveals clear effects of time-of-day on physical performance in Olympic athletes. The time-of-day effect is large, and exceeds the time difference between gold and silver medal in 40%, silver and bronze medal in 64%, and bronze or no medal in 61% of the finals.

In seasonal environments, organisms use biotic and abiotic cues to time various biological processes that are crucial for growth, survival and reproductive success. Photoperiod is the best-known cue used to regulate gonadal development, migration and moult of many animal species. In birds, the relationship between photoperiod and gonadal development is clearly established, but we have little understanding on whether photoperiod also regulates actual timing of egg laying under natural conditions. Elucidating the link between photoperiod and timing of breeding is however key to understand whether an evolutionary change in sensitivity to photoperiod is a possible mechanism through which organisms could adjust their seasonal timing in response to climate warming. Here, we investigated the causal relationship between photoperiod, gonadal growth and laying date in wild female great tits. We experimentally increased the photoperiod perceived by the birds in spring by clipping head feathers, and we subsequently monitored gonadal development in the lab and egg laying dates in the wild. We show that our manipulation increased the photoperiod perceived by the birds to a level that approximately corresponds to an advancement of ten calendar days. This increase in perceived photoperiod led to an acceleration of gonadal development, but not to an advancement of egg laying dates. Our results indicate that photoperiod sensitivity is not constraining the advancement of laying date under current environmental conditions and suggest that evolution of sensitivity to other supplementary cues is necessary to advance reproduction under global warming. This article is protected by copyright. All rights reserved.

Light significantly improves alertness during the night (Cajochen, Sleep Med Rev, 11, 2007 and 453; Ruger et al., AJP Regul Integr Comp Physiol, 290, 2005 and R1413), but results are less conclusive at daytime (Lok et al., J Biol Rhythms, 33, 2018 and 589). Melatonin and core body temperature levels at those times of day may contribute to differences in alerting effects of light. In this experiment, the combined effect of daytime exogenous melatonin administration and light intensity on alertness, body temperature, and skin temperature was studied. The goal was to assess whether (a) alerting effects of light are melatonin dependent, (b) soporific effects of melatonin are mediated via the thermoregulatory system, and (c) light can improve alertness after melatonin‐induced sleepiness during daytime. 10 subjects (5 females, 5 males) received melatonin (5 mg) in dim (10 lux) and, on a separate occasion, in bright polychromatic white light (2000 lux). In addition, they received placebo both under dim and bright light conditions. Subjects participated in all four conditions in a balanced order, yielding a balanced within‐subject design, lasting from noon to 04:00 pm. Alertness and performance were assessed half hourly, while body temperature and skin temperature were measured continuously. Saliva samples to detect melatonin concentrations were collected half hourly. Melatonin administration increased melatonin concentrations in all subjects. Subjective sleepiness and distal skin temperature increased after melatonin ingestion. Bright light exposure after melatonin administration did not change subjective alertness scores, but body temperature and proximal skin temperature increased, while distal skin temperature decreased. Light exposure did not significantly affect these parameters in the placebo condition. These results indicate that (a) exogenous melatonin administration during daytime increases subjective sleepiness, confirming a role for melatonin in sleepiness regulation, (b) bright light exposure after melatonin ingestion significantly affected thermoregulatory parameters without altering subjective sleepiness, therefore temperature changes seem nonessential for melatonin‐induced sleepiness, (c) subjective sleepiness was increased by melatonin ingestion, but bright light administration was not able to improve melatonin‐induced sleepiness feelings nor performance. Other (physiological) factors may therefore contribute to differences in alerting effects of light during daytime and nighttime.

Most organisms have an endogenous circadian clock with a period length of approximately 24 h that enables adaptation, synchronization and anticipation to environmental cycles. The circadian system (circa = about or around, diem = a day) may provide evolutionary benefits when entrained to the 24-h light-dark cycle. The more the internal circadian period (τ) deviates from the external light-dark cycle, the larger the daily phase shifts need to be to synchronize to the environment. In some species, large daily phase shifts reduce survival rate. Here we tested this 'resonance fitness hypothesis' on the diurnal wasp Nasonia vitripennis, which exhibits a large latitudinal cline in free-running period with longer circadian period lengths in the north than in the south. Longevity was measured in northern and southern wasps placed into light-dark cycles (T-cycles) with periods ranging from 20 h to 28 h. Further, locomotor activity was recorded to estimate range and phase angle of entrainment under these various T-cycles. A light pulse induced phase response curve (PRC) was measured in both lines to understand entrainment results. We expected a concave survival curve with highest longevity at T = τ and a reduction in longevity the further τ deviates from T (τ/T<>1). Our results do not support this resonance fitness hypothesis. We did not observe a reduction in longevity when τ deviates from T. Our results may be understood by the strong circadian light resetting mechanism (type 0 PRC) to single light pulses that we measured in Nasonia, resulting in: (1) the broad range of entrainment, (2) the wide natural variation in circadian free-running period, and (3) the lack of reduced survival when τ/T ratio's deviates from 1. Together this indicates that circadian adaption to latitude may lead to changes in circadian period and light response, without negative influences on survival.

Introduction Under real life conditions, increased light exposure during wakefulness seems associated with improved sleep quality, quantified as reduced time awake during bed time, increased time spent in non-REM (NREM) sleep or increased power in the EEG delta band (0.5–4 Hz). The causality of these important relationships and their dependency on circadian clock phase and/or time awake has not been studied in depth. To establish causality of light effects during wake time on subsequent sleep, and to disentangle possible circadian and homeostatic interactions, we employed a forced desynchrony (FD) protocol under dim light (6.5 lux) and bright light (1307 lux) during wakefulness. Methods The protocol consisted of a fast cycling sleep-wake schedule (13h wakefulness – 5h sleep; 4 cycles), followed by 3h recovery sleep in a within subject cross-over design. Individuals (7 men) were equipped with 10 polysomnography electrodes. Subjective sleep quality was measured immediately after wakening. Results Results indicated that circadian variation in delta power was only detected under dim light. Circadian variation in time in rapid eye movement (REM) sleep and wakefulness were uninfluenced by light. Prior light exposure increased accumulation of delta power and time in NREM sleep, while decreasing wakefulness, especially during the circadian wake phase. Subjective sleep quality scores showed that participants were only able to assess light induced improvement of sleep quality correctly when the circadian system promoted wakefulness. Conclusion This study presents significant effects of bright light exposure on sleep architecture, leading to sleep pressure related changes in objective sleep quality. At the end of the scheduled sleep phase after increased light exposure, more delta power and NREM sleep were detected, especially when sleep occurred outside the normal sleep phase. Subjective sleep quality scores showed light-induced improvements coinciding with increased delta power and time spend in NREM sleep, suggesting that light during wakefulness may improve subsequent sleep quality. These findings may have important implications for insomnia treatment and clinical applications of light therapy. Support (if any) This research was funded by the University of Groningen Campus Fryslân (Grant No. 01110939; co-financed by Philips Drachten and Provincie Fryslân). Additional financial support was obtained from a NWO-STW Program Grant “OnTime” (project 12185).

In modern society the night sky is lit up not only by the moon but also by artificial light devices. Both of these light sources can have a major impact on wildlife physiology and behaviour. For example, a number of bird species were found to sleep several hours less under full moon compared to new moon and a similar sleep-suppressing effect has been reported for artificial light at night (ALAN). Cloud cover at night can modulate the light levels perceived by wildlife, yet, in opposite directions for ALAN and moon. While clouds will block moon light, it may reflect and amplify ALAN levels and increases the night glow in urbanized areas. As a consequence, cloud cover may also modulate the sleep-suppressing effects of moon and ALAN in different directions. In this study we therefore measured sleep in barnacle geese (Branta leucopsis) under semi-natural conditions in relation to moon phase, ALAN and cloud cover. Our analysis shows that, during new moon nights stronger cloud cover was indeed associated with increased ALAN levels at our study site. In contrast, light levels during full moon nights were fairly constant, presumably because of moonlight on clear nights or because of reflected artificial light on cloudy nights. Importantly, cloud cover caused an estimated 24.8% reduction in the amount of night-time NREM sleep from nights with medium to full cloud cover, particularly during new moon when sleep was unaffected by moon light. In conclusion, our findings suggest that cloud cover can, in a rather dramatic way, amplify the immediate effects of ALAN on wildlife. Sleep appears to be highly sensitive to ALAN and may therefore be a good indicator of its biological effects., Environmental Pollution, 273, ISSN:0269-7491, ISSN:1878-2450, ISSN:1873-6424

Seasonal breeding is a universal reproductive strategy in many animals. Hypothalamic genes, especially type 2 and 3 iodothyronine deiodinases (Dio2/3), RFamide-related peptide 3 (Rfrp-3), kisspeptin (Kiss-1), and gonadotropin-releasing hormone (GnRH), are involved in a photoperiodic pathway that encodes seasonal signals from day length in many vertebrate species. However, the seasonal expression patterns of these genes in wild mammals are less studied. Here, we present a four-year field investigation to reveal seasonal rhythm and age-dependent reproductive activity in male Brandt's voles (Lasiopodomys brandtii) and to detect relationships among seasonal expression profiles of hypothalamic genes, testicular activity, age, and annual day length. From breeding season (April) to non-breeding season (October), adult male voles displayed a synchronous peak in gonadal activity with annual day length around summer solstice, which was jointly caused by age structure shifts and age-dependent gonadal development patterns. Overwintered males maintained reproductive activity until late in the breeding season, whereas most newborn males terminated gonadal development completely, except for a minority of males born early in spring. Consistently, the synchronous and opposite expression profiles of Dio2/3 suggest their central function to decode photoperiodic signals and to predict the onset of the non-breeding season. Moreover, changes in Dio2/3 signals may guide the actions of Kiss-1 and Rfrp-3 to regulate the age-dependent divergence of reproductive strategy in wild Brandt's vole. Our results provide evidence on how hypothalamic photoperiod genes regulate seasonal breeding in a natural rodent population. This article is protected by copyright. All rights reserved.

The mammalian circadian system encodes both absolute levels of light intensity and color to phase-lock (entrain) its rhythm to the 24-h solar cycle. The evolutionary benefits of circadian color-coding over intensity-coding per se are yet far from understood. A detailed characterization of sunlight is crucial in understanding how and why circadian photoreception integrates color and intensity information. To this end, we continuously measured 100 days of sunlight spectra over the course of a year. Our analyses suggest that circadian color-coding may have evolved to cope with cloud-induced variation in light intensity. We proceed to show how an integration of intensity and spectral composition reduces day-to-day variability in the synchronizing signal (Zeitgeber). As a consequence, entrained phase angle of the circadian clock will be more stable, which will be beneficial for the organism. The presented characterization of sunlight dynamics may become important in designing lighting solutions aimed at minimizing the detrimental effects of light at night in modern societies.

To maximise their fitness, organisms need to synchronise their phenology with the seasonal variation in environmental conditions. Most phenological traits are affected by environmental abiotic cues such as photoperiod, temperature and rainfall. When individuals with complex life cycles fail to match one of the stages with the favourable environment, the negative conditions experienced may lead to carry-over effects and, thus, influence fitness in subsequent stages. In the winter moth, an herbivorous insect with an annual life cycle, timing of egg-hatching in spring is strongly influenced by temperature and varies from year to year. To investigate whether the phenological variation in egg-hatching date affects subsequent stages, we analysed data on egg-hatching date and adult catching date (considered here to be a proxy for adult eclosion date) from our long-term study (1994–2014). Furthermore, we experimentally manipulated the photoperiod experienced by newly hatched larvae and recorded the phenology of their subsequent life cycle stages. In the long-term field study, we found that the timing of winter moth egg-hatching in spring varied strongly from year to year. Interestingly, however, the timing of adult eclosion date in winter showed little inter-annual variation. In line with these findings, our experimental data showed that the winter moth shortened the duration of their pupal development when they experienced a late spring photoperiod as a larva, and prolonged pupal development when experiencing early spring photoperiod. The effects of the larval photoperiodic treatments persisted during egg development in the following generation. The results show that a phenological shift that occurs during an early life stage is partially compensated during subsequent stages and suggest that the mechanism underlying this compensation is mediated by photoperiod. Winter moths regulated their phenology in such a way that the variation in the egg-hatching stage was not carried over to the next life cycle stages. This has strong effects on fitness as it (1) ensures the synchronisation of adult eclosion during the mating period and (2) is likely to reduce potentially negative fitness consequences of phenological mismatches in egg-hatching in the following generation. Overall, these findings stress the importance of understanding phenological carry-over effects to forecast the impact of global change in species with complex life cycles. A plain language summary is available for this article.

Study objectives To determine the effect of light exposure on subsequent sleep characteristics under ambulatory field conditions. Methods Twenty healthy participants were fitted with ambulatory polysomnography (PSG) and wrist-actigraphs to assess light exposure, rest–activity, sleep quality, timing, and architecture. Laboratory salivary dim-light melatonin onset was analyzed to determine endogenous circadian phase. Results Later circadian clock phase was associated with lower intensity (R2 = 0.34, χ2(1) = 7.19, p < .01), later light exposure (quadratic, controlling for daylength, R2 = 0.47, χ2(3) = 32.38, p < .0001), and to later sleep timing (R2 = 0.71, χ2(1) = 20.39, p < .0001). Those with later first exposure to more than 10 lux of light had more awakenings during subsequent sleep (controlled for daylength, R2 = 0.36, χ2(2) = 8.66, p < .05). Those with later light exposure subsequently had a shorter latency to first rapid eye movement (REM) sleep episode (R2 = 0.21, χ2(1) = 5.77, p < .05). Those with less light exposure subsequently had a higher percentage of REM sleep (R2 = 0.43, χ2(2) = 13.90, p < .001) in a clock phase modulated manner. Slow-wave sleep accumulation was observed to be larger after preceding exposure to high maximal intensity and early first light exposure (p < .05). Conclusions The quality and architecture of sleep is associated with preceding light exposure. We propose that light exposure timing and intensity do not only modulate circadian-driven aspects of sleep but also homeostatic sleep pressure. These novel ambulatory PSG findings are the first to highlight the direct relationship between light and subsequent sleep, combining knowledge of homeostatic and circadian regulation of sleep by light. Upon confirmation by interventional studies, this hypothesis could change current understanding of sleep regulation and its relationship to prior light exposure. Clinical trial details This study was not a clinical trial. The study was ethically approved and nationally registered (NL48468.042.14).

Under natural conditions, many aspects of the abiotic and biotic environment vary with time of day, season or even era, while these conditions are typically kept constant in laboratory settings. The timing information contained within the environment serves as critical timing cues for the internal biological timing system, but how this system drives daily rhythms in behaviour and physiology may also depend on the internal state of the animal. The disparity between timing of these cues in natural and laboratory conditions can result in substantial differences in the scheduling of behaviour and physiology under these conditions. In nature, temporal coordination of biological processes is critical to maximize fitness because they optimize the balance between reproduction, foraging and predation risk. Here we focus on the role of peripheral circadian clocks, and the rhythms that they drive, in enabling adaptive phenotypes. We discuss how reproduction, endocrine activity and metabolism interact with peripheral clocks, and outline the complex phenotypes arising from changes in this system. We conclude that peripheral timing is critical to adaptive plasticity of circadian organization in the field, and that we must abandon standard laboratory conditions to understand the mechanisms that underlie this plasticity which maximizes fitness under natural conditions. This article is part of the themed issue ‘Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals’.

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.

This review provides an overview of the interaction between the mammalian cholinergic system and circadian system, and its possible role in time memory. Several studies made clear that circadian (daily) fluctuations in acetylcholine (ACh) release, cholinergic enzyme activity and cholinergic receptor expression varies remarkably between species and even strains. Apparently, cholinergic features can be flexibly adjusted to the needs of a species or strain. Nevertheless, it can be generalized that circadian rhythmicity in the cholinergic system is characterized by high ACh release during the active phase of an individual. During the active phase, the activity of the ACh synthesizing enzyme Choline Acetyltransferase (ChAT) is enhanced, and the activity of the ACh degrading enzyme Acetylcholinesterase (AChE) is reduced. The number of free, unbound and thus available muscarinic acetylcholine receptors (mAChRs) is highest when ACh release is lowest. The cholinergic innervation of the suprachiasmatic nucleus (SCN), the hypothalamic circadian master clock, arises from the cholinergic forebrain and brain stem nuclei. The density of cholinergic fibers and terminals is modest as compared to other hypothalamic nuclei. This is the case for rat, hamster and mouse, three chronobiological model rodent species studied by us. A new finding is that the rat SCN contains some local cholinergic neurons. Hamster SCN contains less cholinergic neurons, whereas the mouse SCN is devoid of such cells. ACh has an excitatory effect on SCN cells (at least in vivo), and functions in close interaction with other neurotransmitters. Originally it was thought that ACh transferred retinal light information to the SCN. This turned out to be wrong. Thereafter, the phase shifting effects of ACh prompted researches to view ACh as an agent for nocturnal clock resetting. It is still not clear, however, what the function consequence is of SCN cholinergic neurotransmission. Here, we postulate the hypothesis that cholinergic neurotransmission in the SCN provides the brain with a mechanism to support the formation of time memory via 'time stamping'. We define time memory as the memory of a specific time of the day, for which an animal made an association with a certain event and/or location (for example in case of time-place association). We use the term 'time stamping' to refer to the process underlying the encoding of a specific time of day (the time stamp). Only relatively brief but arousing events seem to be time stamped at SCN level. This time stamping requires the engagement of mAChRs. New data suggests that the SCN uses the neuropeptide vasopressin (AVP) as an output system to transfer the specific time of day information to other brain regions such as hippocampus and neocortex where time memory is supposed to be acquired, consolidated and stored. Since time stamping is a cholinergically mediated function of the circadian system, the early disruption of the cholinergic and circadian systems as seen in Alzheimer's disease (AD) may contribute to the cognitive disruption of temporal organization of memory and behavior in these patients. (C) 2010 Elsevier B.V. All rights reserved.

Virtually all species have developed cellular oscillations and mechanisms that synchronize these cellular oscillations to environmental cycles. Such environmental cycles in biotic (e.g. food availability and predation risk) or abiotic (e.g. temperature and light) factors may occur on a daily, annual or tidal time scale. Internal timing mechanisms may facilitate behavioural or physiological adaptation to such changes in environmental conditions. These timing mechanisms commonly involve an internal molecular oscillator (a ‘clock’) that is synchronized (‘entrained’) to the environmental cycle by receptor mechanisms responding to relevant environmental signals (‘ Zeitgeber ’, i.e. German for time-giver). To understand the evolution of such timing mechanisms, we have to understand the mechanisms leading to selective advantage. Although major advances have been made in our understanding of the physiological and molecular mechanisms driving internal cycles ( proximate questions), studies identifying mechanisms of natural selection on clock systems ( ultimate questions) are rather limited. Here, we discuss the selective advantage of a circadian system and how its adaptation to day length variation may have a functional role in optimizing seasonal timing. We discuss various cases where selective advantages of circadian timing mechanisms have been shown and cases where temporarily loss of circadian timing may cause selective advantage. We suggest an explanation for why a circadian timing system has emerged in primitive life forms like cyanobacteria and we evaluate a possible molecular mechanism that enabled these bacteria to adapt to seasonal variation in day length. We further discuss how the role of the circadian system in photoperiodic time measurement may explain differential selection pressures on circadian period when species are exposed to changing climatic conditions (e.g. global warming) or when they expand their geographical range to different latitudes or altitudes.

Seasonal changes in day length are used by plants and animals to synchronize annual rhythms in reproduction, physiology, and behavior to the environment. Increasing day length during spring causes sudden changes in the mammalian reproductive system once the critical photoperiod is reached. The molecular mechanism behind this switch is now quickly being elucidated.

The circadian clock located within the suprachiasmatic nuclei (SCN) of the hypothalamus responds to changes in the duration of day length, i.e. photoperiod. Recently, changes in phase relationships among the SCN cell subpopulations, especially between the rostral and caudal region, were implicated in the SCN photoperiodic modulation. To date, the effect of abrupt, rectangular, light-to-dark transitions have been studied while in nature organisms experience gradual dawn and twilight transitions. The aim of this study was to compare the effect of a long (18 h of light) and a short (6 h of light) photoperiod with twilight relative to that with rectangular light-to-dark transition on the daily profiles of Per1 and Per2 mRNA (in situ hybridization) and PER1 and PER2 protein (immunohistochemistry) levels within the rostral, middle and caudal regions of the mouse SCN. Under the short but not under the long photoperiod, Per1, Per2 and PER1, PER2 profiles were significantly phase-advanced under the twilight relative to rectangular light-to-dark transition in all SCN regions examined. Under the photoperiods with rectangular light-to-dark transition, Per1 and Per2 mRNA profiles in the caudal SCN were phase-advanced as compared with those in the rostral SCN. The phase differences between the SCN regions were reduced under the long, or completely abolished under the short, photoperiods with twilight. The data indicate that the twilight photoperiod provides stronger synchronization among the individual SCN cell subpopulations than the rectangular one, and the effect is more pronounced under the short than under the long photoperiod.

The effect of twilight transitions on entrainment of C57BL/6JOlaHsd mice ( Mus musculus) was studied using light-dark cycles of different photoperiods (6, 12, and 18 h) and twilight transitions of different durations (0, 1, and 2 h). Phase angle differences of the onset, center of gravity, and offset of activity, activity duration (α), as well as free-running period (τ) in continuous darkness were analyzed. The main finding was that for all conditions the onset of activity was close to dusk or lights-off except for the short photoperiod with 2 h of twilight where activity onset was on average 5.3 (SEM 1.07) h after lights-off. This finding contrasts with the results of Boulos and Macchi for Syrian hamsters where a 5.9-h earlier activity onset was observed when similar photoperiod and twilight conditions are compared with a rectangular LD cycle. The authors suggest the opposite effects of 2 h of twilight in the 2 species may be related to their different free-running periods under DD conditions following entrainment to short photoperiod with 2-h twilight conditions. Since the authors observed larger variation in phase angle of entrainment in longer twilight conditions, twilight does not necessarily form a stronger zeitgeber.