The increasing demand for improved agricultural production will require more efficient breeding for traits that maintain yield under heterogeneous environments. The internal circadian oscillator is essential for perceiving and coordinating environmental cues such as day length, temperature, and abiotic stress responses within physiological processes. To investigate the contribution of the circadian clock to local adaptability, we have analyzed circadian period by leaf movement in natural populations of Mimulus guttatus and domesticated cultivars of Glycine max. We detected consistent variation in circadian period along a latitudinal gradient in annual populations of the wild plant and the selectively bred crop, and this provides novel evidence of natural and artificial selection for circadian performance. These findings provide new support that the circadian clock acts as a central regulator of plant adaptability and further highlight the potential of applying circadian clock gene variation to marker-assisted breeding programs in crops.
This article is part of a Journal of Biological Rhythms series exploring analysis and statistical topics relevant to researchers in biological rhythms and sleep research. The goal is to provide an overview of the most common issues that arise in the analysis and interpretation of data in these fields. In this article, we address issues related to the collection of multiple data points from the same organism or system at different times, since such longitudinal data collection is fundamental to the assessment of biological rhythms. Rhythmic longitudinal data require additional specific statistical considerations, ranging from curve fitting to threshold definitions to accounting for correlation structure. We discuss statistical analyses of longitudinal data including issues of correlational structure and stationarity, markers of biological rhythms, demasking of biological rhythms, and determining phase, waveform, and amplitude of biological rhythms.
The Journal of Biological Rhythms will be publishing articles exploring analysis and statistical topics relevant to researchers in biological rhythms and sleep research. The goal is to provide an overview of the most common issues that arise in the analysis and interpretation of data in these fields. By using case examples and highlighting the pearls and pitfalls of statistical inference, the authors will identify and explain ways in which experimental scientists can avoid common analytical and statistical mistakes and use appropriate analytical and statistical methods in their research. In this first article, we address the first steps in analysis of data: understanding the underlying statistical distribution of the data and establishing associative versus causal relationships. These ideas are then applied to sample size, power calculations, correlation testing, differences between description and prediction, and the narrative fallacy.
Circadian oscillators found across a variety of species are subject to periodic external light-dark forcing. Entrainment to light-dark cycles enables the circadian system to align biological functions with appropriate times of day or night. Phase response curves (PRCs) have been used for decades to gain valuable insights into entrainment; however, PRCs may not accurately describe entrainment to photoperiods with substantial amounts of both light and dark due to their reliance on a single limit cycle attractor. We have developed a new tool, called an entrainment map, that overcomes this limitation of PRCs and can assess whether, and at what phase, a circadian oscillator entrains to external forcing with any photoperiod. This is a 1-dimensional map that we construct for 3 different mathematical models of circadian clocks. Using the map, we are able to determine conditions for existence and stability of phase-locked solutions. In addition, we consider the dependence on various parameters such as the photoperiod and intensity of the external light as well as the mismatch in intrinsic oscillator frequency with the light-dark cycle. We show that the entrainment map yields more accurate predictions for phase locking than methods based on the PRC. The map is also ideally suited to calculate the amount of time required to achieve entrainment as a function of initial conditions and the bifurcations of stable and unstable periodic solutions that lead to loss of entrainment.
Circadian clocks keep organisms in synchrony with external day-night cycles. The free running period (FRP) of the clock, however, is usually only close to—not exactly—24 h. Here, we explored the geographical variation in the FRP of the linden bug, Pyrrhocoris apterus, in 59 field-lines originating from a wide variety of localities representing geographically different environments. We have identified a remarkable range in the FRPs between field-lines, with the fastest clock at ~21 h and the slowest close to 28 h, a range comparable to the collections of clock mutants in model organisms. Similarly, field-lines differed in the percentage of rhythmic individuals, with a minimum of 13.8% and a maximum of 86.8%. Although the FRP correlates with the latitude and perhaps with the altitude of the locality, the actual function of this FRP diversity is currently unclear. With the recent technological progress of massive parallel sequencing and genome editing, we can expect remarkable progress in elucidating the genetic basis of similar geographic variants in P. apterus or in similar emerging model species of chronobiology.
The daily timing of mammalian physiology is coordinated by circadian clocks throughout the body. Although measurements of clock gene expression indicate that these clocks in mice are normally in phase with each other, the situation in humans remains unclear. We used publicly available data from five studies, comprising over 1000 samples, to compare the phasing of circadian gene expression in human brain and human blood. Surprisingly, after controlling for age, clock gene expression in brain was phase-delayed by ~8.5 h relative to that of blood. We then examined clock gene expression in two additional human organs and in organs from nine other mammalian species, as well as in the suprachiasmatic nucleus (SCN). In most tissues outside the SCN, the expression of clock gene orthologs showed a phase difference of ~12 h between diurnal and nocturnal species. The exception to this pattern was human brain, whose phasing resembled that of the SCN. Our results highlight the value of a multi-tissue, multi-species meta-analysis, and have implications for our understanding of the human circadian system.
Female Drosophila melanogaster, like many other organisms, exhibit different behavioral repertoires after mating with a male. These postmating responses (PMRs) include increased egg production and laying, increased rejection behavior (avoiding further male advances), decreased longevity, altered gustation and decreased sleep. Sex Peptide (SP), a protein transferred from the male during copulation, is largely responsible for many of these behavioral responses, and acts through a specific circuit to induce rejection behavior and alter dietary preference. However, less is known about the mechanisms and neurons that influence sleep in mated females. In this study, we investigated postmating changes in female sleep across strains and ages and on different media, and report that these changes are robust and relatively consistent under a variety of conditions. We find that female sleep is reduced by male-derived SP acting through the canonical sex peptide receptor (SPR) within the same neurons responsible for altering other PMRs. This circuit includes the SPSN-SAG neurons, whose silencing by DREADD induces postmating behaviors including sleep. Our data are consistent with the idea that mating status is communicated to the central brain through a common circuit that diverges in higher brain centers to modify a collection of postmating sensorimotor processes.
The circadian master pacemaker in the suprachiasmatic nucleus (SCN) orchestrates peripheral clocks in various organs and synchronizes them with external time, including those in adipose tissue, which displays circadian oscillations in various metabolic and endocrine outputs. Because our knowledge about the instructive role of the SCN clock on peripheral tissue function is based mainly on SCN lesion studies, we here used an alternative strategy employing the Cre/loxP system to functionally delete the SCN clock in mice. We performed whole-genome microarray hybridizations of murine epididymal white adipose tissue (eWAT) RNA preparations to characterize the role of the SCN clock in eWAT circadian transcriptome regulation. Most of the rhythmic transcripts in control animals were not rhythmic in SCN mutants, but a significant number of transcripts were rhythmic only in mutant eWAT. Core clock genes were rhythmic in both groups, but as was reported before for other tissues, rhythms were dampened and phase advanced in mutant animals. In SCN-mutant mice, eWAT lost the rhythm of metabolic pathway–related transcripts, while transcripts gaining rhythms in SCN-mutant mice were associated with various immune functions. These data reveal a complex interaction of SCN-derived and local circadian signals in the regulation of adipose transcriptome programs.
While previous studies have demonstrated short-wavelength sensitivity to the acute alerting effects of light during the biological night, fewer studies have assessed the alerting effect of light during the daytime. This study assessed the wavelength-dependent sensitivity of the acute alerting effects of daytime light exposure following chronic sleep restriction in 60 young adults (29 men, 31 women; 22.5 ± 3.1 mean ± SD years). Participants were restricted to 5 h time in bed the night before laboratory admission and 3 h time in bed in the laboratory, aligned by wake time. Participants were randomized for exposure to 3 h total of either narrowband blue (max 458-480 nm, n = 23) or green light (max 551-555 nm, n = 25) of equal photon densities (2.8-8.4 x 1013 photons/cm2/sec), beginning 3.25 h after waking, and compared with a darkness control (0 lux, n = 12). Subjective sleepiness (Karolinska Sleepiness Scale), sustained attention (auditory Psychomotor Vigilance Task), mood (Profile of Mood States Bi-Polar form), working memory (2-back task), selective attention (Stroop task), and polysomnographic and ocular sleepiness measures (Optalert) were assessed prior to, during, and after light exposure. We found no significant effect of light wavelength on these measures, with the exception of a single mood subscale. Further research is needed to optimize the characteristics of lighting systems to induce alerting effects during the daytime, taking into account potential interactions between homeostatic sleep pressure, circadian phase, and light responsiveness.
Because of the complications in achieving the necessary long-term observations and experiments, the nature and adaptive value of seasonal time-keeping mechanisms in long-lived organisms remain understudied. Here we present the results of a 20-year-long study of the repeated seasonal changes in body mass, plumage state, and primary molt of 45 captive red knots Calidris canutus islandica, a High Arctic breeding shorebird that spends the nonbreeding season in temperate coastal areas. Birds kept outdoors and experiencing the natural photoperiod of the nonbreeding area maintained sequences of life-cycle stages, roughly following the timing in nature. For 6 to 8 years, 14 of these birds were exposed to unvarying ambient temperature (12 °C) and photoperiodic conditions (12:12 LD). Under these conditions, for at least 5 years they expressed free-running circannual cycles of body mass, plumage state, and wing molt. The circannual cycles of the free-running traits gradually became longer than 12 months, but at different rates. The prebreeding events (onset and offset of prealternate molt and the onset of spring body mass increase) occurred at the same time of the year as in the wild population for 1 or several cycles. As a result, after 4 years in 12:12 LD, the circannual cycles of prealternate plumage state had drifted less than the cycles of prebasic plumage state and wing molt. As the onset of body mass gain drifted less than the offset, the period of high body mass became longer under unvarying conditions. We see these differences between the prebreeding and postbreeding life-cycle stages as evidence for adaptive seasonal time keeping in red knots: the life-cycle stages linked to the initiation of reproduction rely mostly on endogenous oscillators, whereas the later stages rather respond to environmental conditions. Postbreeding stages are also prone to carryover effects from the earlier stages.
The pineal gland produces the hormone melatonin, and its volume may influence melatonin levels. We describe an innovative method for estimating pineal volume in humans and present the association of pineal parenchyma volume with levels of the primary melatonin metabolite, 6-sulfatoxymelatonin. We selected a random sample of 122 older Icelandic men nested within the AGES-Reykjavik cohort and measured their total pineal volume, their parenchyma volume, and the extent of calcification and cysts. For volume estimations we used manual segmentation of magnetic resonance images in the axial plane with simultaneous side-by-side view of the sagittal and coronal plane. We used multivariable adjusted linear regression models to estimate the association of pineal parenchyma volume and baseline characteristics, including 6-sulfatoxymelatonin levels. We used logistic regression to test for differences in first morning urinary 6-sulfatoxymelatonin levels among men with or without cystic or calcified glands. The pineal glands varied in volume, shape, and composition. Cysts were present in 59% of the glands and calcifications in 21%. The mean total pineal volume measured 207 mm3 (range 65-536 mm3) and parenchyma volume 178 mm3 (range 65-503 mm3). In multivariable-adjusted models, pineal parenchyma volume was positively correlated with 6-sulfatoxymelatonin levels (β = 0.52, p < 0.001). Levels of 6-sulfatoxymelatonin did not differ significantly by presence of cysts or calcification. By using an innovative method for pineal assessment, we found pineal parenchyma volume to be positively correlated with 6-sulfatoxymelatonin levels, in line with other recent studies.
Many people in our modern civilized society sleep later on free days compared to work days. This discrepancy in sleep timing will lead to so-called ‘social jetlag’ on work days with negative consequences for performance and health. Light therapy in the morning is often proposed as the most effective method to advance the circadian rhythm and sleep phase. However, most studies focus on direct effects on the circadian system and not on posttreatment effects on sleep phase and sleep integrity. In this placebo-controlled home study we investigated if blue light, rather than amber light therapy, can phase shift the sleep phase along with the circadian rhythm with preservation of sleep integrity and performance. We selected 42 participants who suffered from ‘social jetlag’ on workdays. Participants were randomly assigned to either high-intensity blue light exposure or amber light exposure (placebo) with similar photopic illuminance. The protocol consisted of 14 baseline days without sleep restrictions, 9 treatment days with either 30-min blue light pulses or 30-min amber light pulses in the morning along with a sleep advancing scheme and 7 posttreatment days without sleep restrictions. Melatonin samples were taken at days 1, 7, 14 (baseline), day 23 (effect treatment), and day 30 (posttreatment). Light exposure was recorded continuously. Sleep was monitored through actigraphy. Performance was measured with a reaction time task. As expected, the phase advance of the melatonin rhythm from day 14 to day 23 was significantly larger in the blue light exposure group, compared to the amber light group (84 min ± 51 (SD) and 48 min ± 47 (SD) respectively; t36 = 2.23, p < 0.05). Wake-up time during the posttreatment days was slightly earlier compared to baseline in the blue light group compared to slightly later in the amber light group (–21 min ± 33 (SD) and +12 min ± 33 (SD) respectively; F1,35 = 9.20, p < 0.01). The number of sleep bouts was significantly higher in the amber light group compared to the blue light group during sleep in the treatment period (F1,32 = 4.40, p < 0.05). Performance was significantly worse compared to baseline at all times during (F1,13 = 10.1, p < 0.01) and after amber light treatment (F1,13 = 17.1, p < 0.01), while only in the morning during posttreatment in the blue light condition (F1,10 = 9.8, p < 0.05). The data support the conclusion that blue light was able to compensate for the sleep integrity reduction and to a large extent for the performance decrement that was observed in the amber light condition, both probably as a consequence of the advancing sleep schedule. This study shows that blue light therapy in the morning, applied in a home setting, supports a sleep advancing protocol by phase advancing the circadian rhythm as well as sleep timing.
Circadian rhythmicity is a fundamental process that synchronizes behavioral cues with metabolic homeostasis. Disruption of daily cycles due to jet lag or shift work results in severe physiological consequences including advanced aging, metabolic syndrome, and even cancer. Our understanding of the molecular clock, which is regulated by intricate positive feedforward and negative feedback loops, has expanded to include an important metabolic transcriptional coregulator, Steroid Receptor Coactivator-2 (SRC-2), that regulates both the central clock of the suprachiasmatic nucleus (SCN) and peripheral clocks including the liver. We hypothesized that an environmental uncoupling of the light-dark phases, termed chronic circadian disruption (CCD), would lead to pathology similar to the genetic circadian disruption observed with loss of SRC-2. We found that CCD and ablation of SRC-2 in mice led to a common comorbidity of metabolic syndrome also found in humans with circadian disruption, non-alcoholic fatty liver disease (NAFLD). The combination of SRC-2–/– and CCD results in a more robust phenotype that correlates with human non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) gene signatures. Either CCD or SRC-2 ablation produces an advanced aging phenotype leading to increased mortality consistent with other circadian mutant mouse models. Collectively, our studies demonstrate that SRC-2 provides an essential link between the behavioral activities influenced by light cues and the metabolic homeostasis maintained by the liver.
An improvement to our current quantitative model of arousal state dynamics is presented that more accurately predicts sleep propensity as measured with sleep dynamics depending on circadian phase and prior wakefulness. A nonlinear relationship between the circadian variables within the dynamic circadian oscillator model is introduced to account for the skewed shape of the circadian rhythm of alertness that peaks just prior to the onset of the biological night (the "wake maintenance zone") and has a minimum toward the end of the biological night. The revised circadian drive thus provides a strong inhibitory input to the sleep-active neuronal population in the evening, counteracting the excitatory effects of the increased homeostatic sleep drive as originally proposed in the opponent process model of sleep-wake regulation. The revised model successfully predicts the sleep propensity profile as reflected in the dynamics of the total sleep time, sleep onset latency, wake/sleep ratio, and sleep efficiency during a wide range of experimental protocols. Specifically, all of these sleep measures are predicted for forced desynchrony schedules with day lengths ranging from 1.5 to 42.85 h and scheduled time in bed from 0.5 to 14.27 h. The revised model is expected to facilitate more accurate predictions of sleep under normal conditions as well as during circadian misalignment, for example, during shiftwork and jetlag.
Overlapping genetic influences have been implicated in diurnal preference and subjective sleep quality. Our overall aim was to examine overlapping concurrent and longitudinal genetic and environmental effects on diurnal preference and sleep quality over ~5 years. Behavioral genetic analyses were performed on data from the longitudinal British G1219 study of young adult twins and nontwin siblings. A total of 1556 twins and siblings provided data on diurnal preference (Morningness-Eveningness Questionnaire) and sleep quality (Pittsburgh Sleep Quality Index) at time 1 (mean age = 20.30 years, SD = 1.76; 62% female), and 862 participated at time 2 (mean age = 25.30 years, SD = 1.81; 66% female). Preference for eveningness was associated with poorer sleep quality at both time points (r = 0.25 [95% confidence intervals {CIs} = 0.20-0.30] and r = 0.21 [CI = 0.15-0.28]). There was substantial overlap in the genetic influences on diurnal preference and sleep quality individually, across time (genetic correlations [rAs]: 0.64 [95% CI = 0.59-0.67] and 0.48 [95% CI = 0.42-.053]). There were moderate genetic correlations between diurnal preference and sleep quality concurrently and longitudinally (rAs = 0.29-0.60). Nonshared environmental overlap was substantially smaller for all cross-phenotype associations (nonshared environmental correlations (rEs) = -0.02 to 0.08). All concurrent and longitudinal associations within and between phenotypes were largely accounted for by genetic factors (explaining between 60% and 100% of the associations). All shared environmental effects were nonsignificant. Nonshared environmental influences played a smaller role on the associations between phenotypes (explaining between -0.06% and 40% of the associations). These results suggest that to some extent, similar genes contribute to the stability of diurnal preference and sleep quality throughout young adulthood but also that different genes play a part over this relatively short time frame. While there was evidence of genetic overlap between phenotypes concurrently and longitudinally, the possible emergence of new genetic factors (or decline of previously associated factors) suggests that molecular genetic studies focusing on young adults should consider more tightly specified age groups, given that genetic effects may be time specific.
Our objectives were to investigate the period lengths (i.e., taus) of the endogenous core body temperature rhythm and melatonin rhythm in delayed sleep-wake phase disorder patients (DSWPD) and non-24-h sleep-wake rhythm disorder patients (N24SWD) compared with normally entrained individuals. Circadian rhythms were measured during an 80-h ultradian modified constant routine consisting of 80 ultrashort 1-h "days" in which participants had 20-min sleep opportunities alternating with 40 min of enforced wakefulness. We recruited a community-based sample of 26 DSWPD patients who met diagnostic criteria (17 males, 9 females; age, 21.85 ± 4.97 years) and 18 healthy controls (10 males, 8 females; age, 23.72 ± 5.10 years). Additionally, 4 full-sighted patients (3 males, 1 female; age, 25.75 ± 4.99 years) were diagnosed with N24SWD and included as a discrete study group. Ingestible core temperature capsules were used to record minute temperatures that were averaged to obtain 80 hourly data points. Salivary melatonin concentration was assessed every half-hour to determine time of dim light melatonin onset at the beginning and end of the 80-h protocol. DSWPD patients had significantly longer melatonin rhythm taus (24 h 34 min ± 17 min) than controls (24 h 22 min ± 15 min, p = 0.03, d = 0.70). These results were further supported by longer temperature rhythm taus in DSWPD patients (24 h 34 min ± 26 min) relative to controls (24 h 13 min ± 15 min, p = 0.01, d = 0.80). N24SWD patients had even longer melatonin (25 h ± 19 min) and temperature (24 h 52 min ± 17 min) taus than both DSWPD (p = 0.007, p = 0.06) and control participants (p < 0.001, p = 0.02, respectively). Between 12% and 19% of the variance in DSWPD patients’ sleep timing could be explained by longer taus. This indicates that longer taus of circadian rhythms may contribute to the DSWPD patients’ persistent tendency to delay, their frequent failure to respond to treatment, and their relapse following treatment. Additionally, other factors can contribute to misalignments in DSWPD and N24SWD disorders.
Phase response curves (PRCs) for light or temperature stimuli have been shown to be most valuable in understanding how circadian clocks are entrained to daily environmental cycles. Nowadays, PRC experiments in which clock neurons are manipulated in a temporally restricted manner by thermogenetic or optogenetic tools are also useful to comprehend clock network properties. Here, we temporally depolarized specific clock neurons of Drosophila melanogaster by activating temperature-sensitive dTrpA1 channels to unravel their role in phase shifting the flies’ activity rhythm. The depolarization of all clock neurons caused a PRC resembling the flies’ light PRC, with strong phase delays in the first half of the subjective night and modest phase advances in its second half. However, the activation of the flies’ pigment-dispersing factor (PDF)-positive morning (M) neurons (s-LNvs) only induced phase advances, and these reached into the subjective day, where the light PRC has its dead zone. This indicates that the M neurons are very potent in accelerating the clock, which is in line with previous observations. In contrast, the evening (E) neurons together with the PDF-positive l-LNvs appear to mediate phase delays. Most interestingly, the molecular clock (Period protein cycling) of the depolarized clock neurons was shifted in parallel to the behavior, and this shift was already visible within the first cycle after the temperature pulse. We identified cAMP response element binding protein B (CREB) as a putative link between membrane depolarization and the molecular clock.
Light is the primary signal that calibrates circadian neural circuits and thus coordinates daily physiological and behavioral rhythms with solar entrainment cues. Drosophila and mammalian circadian circuits consist of diverse populations of cellular oscillators that exhibit a wide range of dynamic light responses, periods, phases, and degrees of synchrony. How heterogeneous circadian circuits can generate robust physiological rhythms while remaining flexible enough to respond to synchronizing stimuli has long remained enigmatic. Cryptochrome is a short-wavelength photoreceptor that is endogenously expressed in approximately half of Drosophila circadian neurons. In a previous study, physiological light response was measured using real-time bioluminescence recordings in Drosophila whole-brain explants, which remain intrinsically light-sensitive. Here we apply analysis of real-time bioluminescence experimental data to show detailed dynamic ensemble representations of whole circadian circuit light entrainment at single neuron resolution. Organotypic whole-brain explants were either maintained in constant darkness (DD) for 6 days or exposed to a phase-advancing light pulse on the second day. We find that stronger circadian oscillators support robust overall circuit rhythmicity in DD, whereas weaker oscillators can be pushed toward transient desynchrony and damped amplitude to facilitate a new state of phase-shifted network synchrony. Additionally, we use mathematical modeling to examine how a network composed of distinct oscillator types can give rise to complex dynamic signatures in DD conditions and in response to simulated light pulses. Simulations suggest that complementary coupling mechanisms and a combination of strong and weak oscillators may enable a robust yet flexible circadian network that promotes both synchrony and entrainment. A more complete understanding of how the properties of oscillators and their signaling mechanisms facilitate their distinct roles in light entrainment may allow us to direct and augment the circadian system to speed recovery from jet lag, shift work, and seasonal affective disorder.
The Arabian oryx inhabits an environment where summer ambient temperatures can exceed 40 °C for extended periods of time. While the oryx uses a suite of adaptations that aid survival, the effects of this extreme environment on inactivity are unknown. To determine how the oryx manages inactivity seasonally, we measured the daily rhythm of body temperature and used fine-grain actigraphy, in 10 animals, to reveal when the animals were inactive in relation to ambient temperature and photoperiod. We demonstrate that during the cooler winter months, the oryx was inactive during the cooler parts of the 24-h day (predawn hours), showing a nighttime (nocturnal) inactivity pattern. In contrast, in the warmer summer months, the oryx displayed a bimodal inactivity pattern, with major inactivity bouts (those greater than 1 h) occurring equally during both the coolest part of the night (predawn hours) and the warmest part of the day (afternoon hours). Of note, the timing of the daily rhythm of body temperature did not vary seasonally, although the amplitude did change, leading to a seasonal alteration in the phase relationship between inactivity and the body temperature rhythm. Because during periods of inactivity the oryx were presumably asleep for much of the time, we speculate that the daytime shift in inactivity may allow the oryx to take advantage of the thermoregulatory physiology of sleep, which likely occurs when the animal is inactive for more than 1 h, to mitigate environmentally induced increases in body temperature.
In mammals, the circadian system is composed of a principal circadian oscillator located in the suprachiasmatic nucleus (SCN) and a number of subordinate oscillators in extra-SCN brain regions and peripheral tissues/organs. However, how the time-keeping functions of this multiple oscillator circuit are affected by aberrant lighting environments remains largely unknown. In the present study, we investigated the effects of chronic light exposure in the middle of the night on the circadian system by comparing the mice housed in a 12:4:4:4-h L:DLD condition with the controls in 12:12-h L:D condition. Daily rhythms in locomotor activity were analyzed and the expression patterns of protein products of clock genes Period1 and Period2 (PER1 and PER2) were examined in the SCN and extra-SCN brain regions, including the dorsal striatum, hippocampus, paraventricular nucleus (PVN), and basolateral amygdala (BLA). Following 2 weeks of housing in the L:DLD condition, animals showed disturbed daily rhythms in locomotor activity and lacked daily rhythms of PER1 and PER2 in the SCN. In the extra-SCN brain regions, the PER1 and PER2 rhythms were affected in a region-specific pattern, such that they were relatively undisturbed in the striatum and hippocampus, phase-shifted in the BLA, and abolished in the PVN. In addition, mice in the L:DLD condition showed increased anxiety-like behaviors and reduced brain-derived neurotropic factor messenger RNA expression in the hippocampus, amygdala, and medial prefrontal cortex, which are brain regions that are involved in emotional regulation. These results indicate that nighttime light exposure leads to circadian disturbances not only by abolishing the circadian rhythms in the SCN but also by inducing misalignment among brain oscillators and negatively affects emotional processing. These observations serve to identify risks associated with decisions regarding lifestyle in our modern society.
The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings.
Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIm/m:CKI+/m:Cry2m/m mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKI-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.
Odor discrimination behavior displays circadian fluctuations in mice, indicating that mammalian olfactory function is under control of the circadian system. This is further supported by the facts that odor discrimination rhythms depend on the presence of clock genes and that olfactory tissues contain autonomous circadian clocks. However, the molecular link between circadian function and olfactory processing is still unknown. To elucidate the molecular mechanisms underlying this link, we focused on the olfactory epithelium (OE), the primary target of odors and the site of the initial events in olfactory processing. We asked whether olfactory sensory neurons (OSNs) within the OE possess an autonomous circadian clock and whether olfactory pathways are under circadian control. Employing clock gene–driven bioluminescence reporter assays and time-dependent immunohistochemistry on OE samples, we found robust circadian rhythms of core clock genes and their proteins in OSNs, suggesting that the OE indeed contains an autonomous circadian clock. Furthermore, we performed a circadian transcriptome analysis and identified several OSN-specific components that are under circadian control, including those with putative roles in circadian olfactory processing, such as KIRREL2—an established factor involved in short-term OSN activation. The spatiotemporal expression patterns of our candidate proteins suggest that they are involved in short-term anabolic processes to rhythmically prepare the cell for peak performances and to promote circadian function of OSNs.
In mammals, circadian rhythms in peripheral organs are impaired when animals are maintained in abnormal environmental light-dark cycles such as constant light (LL). This conclusion is based on averaged data from groups of experimental animals sacrificed at each time point. To investigate the effect of LL housing on the peripheral clocks of individual mice, an in vivo imaging system was used to observe the circadian bioluminescence rhythm in peripheral tissues of the liver, kidney, and submandibular salivary gland in PER2::LUCIFERASE knock-in mice. Using this technique, we demonstrated that the majority of individual peripheral tissues still had rhythmic oscillations of their circadian clocks in LL conditions. However, LL housing caused decreased amplitudes and a broad distribution of peak phases in PER2::LUCIFERASE oscillations irrespective of the state of the animals’ behavioral rhythmicity. Because both scheduled feeding and scheduled exercise are effective recovery stimuli for circadian clock deficits, we examined whether scheduled feeding or scheduled exercise could reverse this impairment. The results showed that scheduled feeding or exercise could not restore the amplitude of peripheral clocks in LL. On the other hand, the LL-induced broad phase distribution was reversed, and peak phases were entrained to a specific time point by scheduled feeding but only slightly by scheduled exercise. The present results demonstrate that LL housing impairs peripheral circadian clock oscillations by altering both amplitude and phase in individual mice. The broad distribution of clock phases was clearly reversed by scheduled feeding, suggesting the importance of scheduled feeding as an entraining stimulus for impaired peripheral clocks.
To maintain synchrony with the environment, circadian clocks use a wide range of cycling sensory cues that provide input to the clock (zeitgebers), including environmental temperature cycles (TCs). There is some knowledge about which clock neuronal groups are important for temperature synchronization, but we currently lack knowledge on the temperature receptors and their signaling pathways that feed temperature information to the (neuronal) clock. Since TRPA1 is a well-known thermosensor that functions in a range of temperature-related behaviors, and it is potentially expressed in clock neurons, we set out to test the putative role of TRPA1 in temperature synchronization of the circadian clock. We found that flies lacking TRPA1 are still able to synchronize their behavioral activity to TCs comparable to wild-type flies, both in 16°C : 25°C and 20°C : 29°C TCs. In addition, we found that flies lacking TRPA1 show higher activity levels during the middle of the warm phase of 20°C : 29°C TCs, and we show that this TRPA1-mediated repression of locomotor activity during the "siesta" is caused by a lack of sleep. Based on these data, we conclude that the TRPA1 channel is not required for temperature synchronization in this broad temperature range but instead is required to repress activity during the warm part of the day.
The chicken pineal gland synthesizes and releases melatonin rhythmically in light/dark (LD) cycles, with high melatonin levels during the dark phase, and in constant darkness (DD) for several cycles before it gradually damps to arrhythmicity in DD. Daily administration of norepinephrine (NE) in vivo and in vitro prevents the damping and restores the melatonin rhythm. To investigate the role of the circadian clock on melatonin rhythm damping and of its restoration by NE, the effects of NE administration at different phases of the melatonin cycle revealed a robust rhythm in NE sensitivity in which NE efficacy in increasing melatonin amplitude peaked in late subjective night and early subjective day, suggesting a clock underlying NE sensitivity. However, NE itself had no effect on circadian phase or period of the melatonin rhythms. Transcriptional analyses indicated that even though the rhythm of melatonin output damped to arrhythmicity, messenger RNA (mRNA) encoding clock genes gper2, gper3, gBmal1, gclock, gcry1, and gcry2; enzymes associated with melatonin biosynthesis; and enzymes involved in cyclic nucleotide signaling remained robustly rhythmic. Of these, only gADCY1 (adenylate cyclase 1) and gPDE4D (cAMP-specific 3',5'-cyclic phosphodiesterase 4D) were affected by NE administration at the mRNA levels, and only ADCY1 was affected at the protein level. The data strongly suggest that damping of the melatonin rhythm in the chick pineal gland occurs at the posttranscriptional level and that a major role of the clock is to regulate pinealocytes’ sensitivity to neuronal input from the brain.