Cryptochrome-Timeless structure reveals circadian clock timing mechanisms.

Circadian rhythms influence many behaviours and diseases1,2. They arise from oscillations in gene expression caused by repressor proteins that directly inhibit transcription of their own genes. The fly circadian clock offers a valuable model for studying these processes, wherein Timeless (Tim) plays a critical role in mediating nuclear entry of the transcriptional repressor Period (Per) and the photoreceptor Cryptochrome (Cry) entrains the clock by triggering Tim degradation in light2,3. Here, through cryogenic electron microscopy of the Cry-Tim complex, we show how a light-sensing cryptochrome recognizes its target. Cry engages a continuous core of amino-terminal Tim armadillo repeats, resembling how photolyases recognize damaged DNA, and binds a C-terminal Tim helix, reminiscent of the interactions between light-insensitive cryptochromes and their partners in mammals. The structure highlights how the Cry flavin cofactor undergoes conformational changes that couple to large-scale rearrangements at the molecular interface, and how a phosphorylated segment in Tim may impact clock period by regulating the binding of Importin-α and the nuclear import of Tim-Per4,5. Moreover, the structure reveals that the N terminus of Tim inserts into the restructured Cry pocket to replace the autoinhibitory C-terminal tail released by light, thereby providing a possible explanation for how the long-short Tim polymorphism adapts flies to different climates6,7.

Circadian Responses to Light in the BTBR Mouse.

Animals with altered freerunning periods are valuable in understanding properties of the circadian clock. Understanding the relationship between endogenous clock properties, entrainment, and influence of light in terms of parametric and non-parametric models can help us better understand how different populations adapt to external light cycles. Many clinical populations often show significant changes in circadian properties that in turn cause sleep and circadian problems, possibly exacerbating their underlying clinical condition. BTBR T+Itpr3tf/J (BTBR) mice are a model commonly used for the study of autism spectrum disorders (ASD). Adults and adolescents with ASD frequently exhibit profound sleep and circadian disruptions, including increased latency to sleep, insomnia, advanced and delayed sleep phase disorders, and sleep fragmentation. Here, we investigated the circadian phenotype of BTBR mice in freerunning and light-entrained conditions and found that this strain of mice showed noticeably short freerunning periods (~22.75 h). In addition, when compared to C57BL/6J controls, BTBR mice also showed higher levels of activity even though this activity was compressed into a shorter active phase. Phase delays and phase advances to light were significantly larger in BTBR mice. Despite the short freerunning period, BTBR mice exhibited normal entrainment in light-dark cycles and accelerated entrainment to both advanced and delayed light cycles. Their ability to entrain to skeleton photoperiods of 1 min suggests that this entrainment cannot be attributed to masking. Period differences were also correlated with differences in the number of vasoactive intestinal polypeptide-expressing cells in the suprachiasmatic nucleus (SCN). Overall, the BTBR model, with their unique freerunning and entrainment properties, makes an interesting model to understand the underlying circadian clock.

Light intensity alters the effects of light-induced circadian disruption on glucose and lipid metabolism in mice.

Circadian disruption induced by rotating light cycles has been linked to metabolic disorders. However, how the interaction of light intensity and light cycle affects metabolism under different diets remains to be explored. Eighty mice were first randomly stratified into the low-fat diet (LFD, n = 40) or high-fat diet (HFD, n = 40) groups. Each group was further randomly subdivided into four groups (n = 8-12 per group) in terms of different light intensities [lower (LI, 78 lx) or higher intensity (HI, 169 lx)] and light cycles [12-h light:12-h dark cycle or circadian-disrupting (CD) light cycle consisting of repeated 6-h light phase advancement]. Body weight was measured weekly. At the end of the 16-wk experiment, mice were euthanized for serum and pathological analysis. Glucose and insulin tolerance tests were performed during the last 2 wk. The CD cycle increased body weight gain, adipocyte area, glucose intolerance, and insulin resistance of LFD as well as HFD mice under HI but not LI condition. Moreover, the serum and hepatic triglyceride levels increased with LFD-HI treatment, regardless of light cycle. In addition, the CD cycle improved lipid and glucose metabolism under HFD-LI condition. In summary, the detrimental effects of the CD cycle on metabolism were alleviated under LI condition, especially in HFD mice. These results indicate that modulating light intensity is a potential strategy to prevent the negative metabolic consequences associated with jet lag or shift work.NEW & NOTEWORTHY Glucose and lipid homeostasis is altered by the CD cycles in a light-intensity-dependent manner. Lower-intensity light reverses the negative metabolic effects of the CD cycles, especially under HFD feeding. The interaction of light intensity and light cycle on metabolism is independent of energy intake and eating pattern. Glucose metabolic disorders caused by rotating light cycles occur along with compensatory ß-cell mass expansion.

Light sets the brain's daily clock by regional quickening and slowing of the molecular clockworks at dawn and dusk.

How daily clocks in the brain are set by light to local environmental time and encode the seasons is not fully understood. The suprachiasmatic nucleus (SCN) is a central circadian clock in mammals that orchestrates physiology and behavior in tune with daily and seasonal light cycles. Here, we have found that optogenetically simulated light input to explanted mouse SCN changes the waveform of the molecular clockworks from sinusoids in free-running conditions to highly asymmetrical shapes with accelerated synthetic (rising) phases and extended degradative (falling) phases marking clock advances and delays at simulated dawn and dusk. Daily waveform changes arise under ex vivo entrainment to simulated winter and summer photoperiods, and to non-24 hr periods. Ex vivo SCN imaging further suggests that acute waveform shifts are greatest in the ventrolateral SCN, while period effects are greatest in the dorsomedial SCN. Thus, circadian entrainment is encoded by SCN clock gene waveform changes that arise from spatiotemporally distinct intrinsic responses within the SCN neural network.

Circadian autophagy drives iTRF-mediated longevity.

Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.

The Impact of the Circadian Clock on Skin Physiology and Cancer Development.

Skin cancers are growing in incidence worldwide and are primarily caused by exposures to ultraviolet (UV) wavelengths of sunlight. UV radiation induces the formation of photoproducts and other lesions in DNA that if not removed by DNA repair may lead to mutagenesis and carcinogenesis. Though the factors that cause skin carcinogenesis are reasonably well understood, studies over the past 10-15 years have linked the timing of UV exposure to DNA repair and skin carcinogenesis and implicate a role for the body's circadian clock in UV response and disease risk. Here we review what is known about the skin circadian clock, how it affects various aspects of skin physiology, and the factors that affect circadian rhythms in the skin. Furthermore, the molecular understanding of the circadian clock has led to the development of small molecules that target clock proteins; thus, we discuss the potential use of such compounds for manipulating circadian clock-controlled processes in the skin to modulate responses to UV radiation and mitigate cancer risk.

Phytochrome A elevates plant circadian-clock components to suppress shade avoidance in deep-canopy shade.

Shade-avoiding plants can detect the presence of neighboring vegetation and evoke escape responses before canopy cover limits photosynthesis. Rapid stem elongation facilitates light foraging and enables plants to overtop competitors. A major regulator of this response is the phytochrome B photoreceptor, which becomes inactivated in light environments with a low ratio of red to far-red light (low R:FR), characteristic of vegetational shade. Although shade avoidance can provide plants with a competitive advantage in fast-growing stands, excessive stem elongation can be detrimental to plant survival. As such, plants have evolved multiple feedback mechanisms to attenuate shade-avoidance signaling. The very low R:FR and reduced levels of photosynthetically active radiation (PAR) present in deep canopy shade can, together, trigger phytochrome A (phyA) signaling, inhibiting shade avoidance and promoting plant survival when resources are severely limited. The molecular mechanisms underlying this response have not been fully elucidated. Here, we show that Arabidopsis thaliana phyA elevates early-evening expression of the central circadian-clock components TIMING OF CAB EXPRESSION 1 (TOC1), PSEUDO RESPONSE REGULATOR 7 (PRR7), EARLY FLOWERING 3 (ELF3), and ELF4 in photocycles of low R:FR and low PAR. These collectively suppress stem elongation, antagonizing shade avoidance in deep canopy shade.

Light-induced changes in the suprachiasmatic nucleus transcriptome regulated by the ERK/MAPK pathway.

The mammalian master circadian pacemaker within the suprachiasmatic nucleus (SCN) maintains tight entrainment to the 24 hr light/dark cycle via a sophisticated clock-gated rhythm in the responsiveness of the oscillator to light. A central event in this light entrainment process appears to be the rapid induction of gene expression via the ERK/MAPK pathway. Here, we used RNA array-based profiling in combination with pharmacological disruption methods to examine the contribution of ERK/MAPK signaling to light-evoked gene expression. Transient photic stimulation during the circadian night, but not during the circadian day, triggered marked changes in gene expression, with early-night light predominately leading to increased gene expression and late-night light predominately leading to gene downregulation. Functional analysis revealed that light-regulated genes are involved in a diversity of physiological processes, including DNA transcription, RNA translation, mRNA processing, synaptic plasticity and circadian timing. The disruption of MAPK signaling led to a marked reduction in light-evoked gene regulation during the early night (32/52 genes) and late night (190/191 genes); further, MAPK signaling was found to gate gene expression across the circadian cycle. Together, these experiments reveal potentially important insights into the transcriptional-based mechanisms by which the ERK/MAPK pathway regulates circadian clock timing and light-evoked clock entrainment.

Reversible modulation of circadian time with chronophotopharmacology.

The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

Circadian rhythms driving a fast-paced root clock implicate species-specific regulation in Medicago truncatula.

Plants have a hierarchical circadian structure comprising multiple tissue-specific oscillators that operate at different speeds and regulate the expression of distinct sets of genes in different organs. However, the identity of the genes differentially regulated by the circadian clock in different organs, such as roots, and how their oscillations create functional specialization remain unclear. Here, we profiled the diurnal and circadian landscapes of the shoots and roots of Medicago truncatula and identified the conserved regulatory sequences contributing to transcriptome oscillations in each organ. We found that the light-dark cycles strongly affect the global transcriptome oscillation in roots, and many clock genes oscillate only in shoots. Moreover, many key genes involved in nitrogen fixation are regulated by circadian rhythms. Surprisingly, the root clock runs faster than the shoot clock, which is contrary to the hierarchical circadian structure showing a slow-paced root clock in both detached and intact Arabidopsis thaliana (L.) Heynh. roots. Our result provides important clues about the species-specific circadian regulatory mechanism, which is often overlooked, and possibly coordinates the timing between shoots and roots independent of the current prevailing model.

The singularity response reveals entrainment properties of the plant circadian clock.

Circadian clocks allow organisms to synchronize their physiological processes to diurnal variations. A phase response curve allows researchers to understand clock entrainment by revealing how signals adjust clock genes differently according to the phase in which they are applied. Comprehensively investigating these curves is difficult, however, because of the cost of measuring them experimentally. Here we demonstrate that fundamental properties of the curve are recoverable from the singularity response, which is easily measured by applying a single stimulus to a cellular network in a desynchronized state (i.e. singularity). We show that the singularity response of Arabidopsis to light/dark and temperature stimuli depends on the properties of the phase response curve for these stimuli. The measured singularity responses not only allow the curves to be precisely reconstructed but also reveal organ-specific properties of the plant circadian clock. The method is not only simple and accurate, but also general and applicable to other coupled oscillator systems as long as the oscillators can be desynchronized. This simplified method may allow the entrainment properties of the circadian clock of both plants and other species in nature.

Light at Night and Risk of Pancreatic Cancer in the NIH-AARP Diet and Health Study.

Circadian disruption may play a role in carcinogenesis. Recent research suggests that light at night (LAN), a circadian disruptor, may be a risk factor for cancer. Moreover, LAN has been linked to obesity and diabetes, two risk factors for pancreatic ductal adenocarcinoma (PDAC). Here we examine the relationship between LAN and PDAC in an epidemiologic study of 464,371 participants from the NIH-AARP Diet and Health Study. LAN was estimated from satellite imagery at baseline (1996), and incident primary PDAC cases were ascertained from state cancer registries. Cox proportional hazards models were used to estimate HRs and two-sided 95% confidence intervals (CI) for the association between quintiles of LAN and PDAC in the overall population stratified by sex. Over up to 16.2 years of follow-up, a total of 2,502 incident PDAC were identified in the cohort. Higher estimated LAN exposure was associated with an elevated PDAC risk. Compared with those living in areas in the lowest LAN quintile, those in areas in the highest quintile had a 27% increase PDAC risk [HR (95% CI), 1.24 (1.03-1.49)], with similar risk for men [1.21 (0.96-1.53)] and women [1.28 (0.94-1.75)]. In addition, stronger associations were observed in normal and overweight groups compared with the obese group (P interaction = 0.03). Our results support the hypothesis that LAN and circadian disruption may be risk factors for PDAC. SIGNIFICANCE: Our study suggests that higher LAN is a risk factor for pancreatic cancer, contributing to the growing literature that demonstrates the potentially adverse health effects of light pollution.

Chronic circadian shift leads to adipose tissue inflammation and fibrosis.

The circadian clock exerts temporal coordination of metabolic pathways. Clock disruption is intimately linked with the development of obesity and insulin resistance, and our previous studies found that the essential clock transcription activator, Brain and Muscle Arnt-like 1 (Bmal1), is a key regulator of adipogenesis. However, the metabolic consequences of chronic shiftwork on adipose tissues have not been clearly defined. Here, using an environmental lighting-induced clock disruption that mimics rotating shiftwork schedule, we show that chronic clock dysregulation for 6 months in mice resulted in striking adipocyte hypertrophy with adipose tissue inflammation and fibrosis. Both visceral and subcutaneous depots display enlarged adipocyte with prominent crown-like structures indicative of macrophage infiltration together with evidence of extracellular matrix remodeling. Global transcriptomic analyses of these fat depots revealed that shiftwork resulted in up-regulations of inflammatory, adipogenic and angiogenic pathways with disruption of normal time-of-the-day-dependent regulation. These changes in adipose tissues are associated with impaired insulin signaling in mice subjected to shiftwork, together with suppression of the mTOR signaling pathway. Taken together, our study identified the significant adipose depot dysfunctions induced by chronic shiftwork regimen that may underlie the link between circadian misalignment and insulin resistance.

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

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

The Role of Light Sensitivity and Intrinsic Circadian Period in Predicting Individual Circadian Timing.

There is large interindividual variability in circadian timing, which is underestimated by mathematical models of the circadian clock. Interindividual differences in timing have traditionally been modeled by changing the intrinsic circadian period, but recent findings reveal an additional potential source of variability: large interindividual differences in light sensitivity. Using an established model of the human circadian clock with real-world light recordings, we investigated whether changes in light sensitivity parameters or intrinsic circadian period could capture variability in circadian timing between and within individuals. Healthy participants (n = 12, aged 18-26 years) underwent continuous light monitoring for 3 weeks (Actiwatch Spectrum). Salivary dim-light melatonin onset (DLMO) was measured each week. Using the recorded light patterns, a sensitivity analysis for predicted DLMO times was performed, varying 3 model parameters within physiological ranges: (1) a parameter determining the steepness of the dose-response curve to light (p), (2) a parameter determining the shape of the phase-response curve to light (K), and (3) the intrinsic circadian period (tau). These parameters were then fitted to obtain optimal predictions of the three DLMO times for each individual. The sensitivity analysis showed that the range of variation in the average predicted DLMO times across participants was 0.65 h for p, 4.28 h for K, and 3.26 h for tau. The default model predicted the DLMO times with a mean absolute error of 1.02 h, whereas fitting all 3 parameters reduced the mean absolute error to 0.28 h. Fitting the parameters independently, we found mean absolute errors of 0.83 h for p, 0.53 h for K, and 0.42 h for tau. Fitting p and K together reduced the mean absolute error to 0.44 h. Light sensitivity parameters captured similar variability in phase compared with intrinsic circadian period, indicating they are viable targets for individualizing circadian phase predictions. Future prospective work is needed that uses measures of light sensitivity to validate this approach.

Constant Light Dysregulates Cochlear Circadian Clock and Exacerbates Noise-Induced Hearing Loss.

Noise-induced hearing loss is one of the major causes of acquired sensorineural hearing loss in modern society. While people with excessive exposure to noise are frequently the population with a lifestyle of irregular circadian rhythms, the effects of circadian dysregulation on the auditory system are still little known. Here, we disturbed the circadian clock in the cochlea of male CBA/CaJ mice by constant light (LL) or constant dark. LL significantly repressed circadian rhythmicity of circadian clock genes Per1, Per2, Rev-erbα, Bmal1, and Clock in the cochlea, whereas the auditory brainstem response thresholds were unaffected. After exposure to low-intensity (92 dB) noise, mice under LL condition initially showed similar temporary threshold shifts to mice under normal light-dark cycle, and mice under both conditions returned to normal thresholds after 3 weeks. However, LL augmented high-intensity (106 dB) noise-induced permanent threshold shifts, particularly at 32 kHz. The loss of outer hair cells (OHCs) and the reduction of synaptic ribbons were also higher in mice under LL after noise exposure. Additionally, LL enhanced high-intensity noise-induced 4-hydroxynonenal in the OHCs. Our findings convey new insight into the deleterious effect of an irregular biological clock on the auditory system.

Distinct Components of Photoperiodic Light Are Differentially Encoded by the Mammalian Circadian Clock.

Seasonal light cycles influence multiple physiological functions and are mediated through photoperiodic encoding by the circadian system. Despite our knowledge of the strong connection between seasonal light input and downstream circadian changes, less is known about the specific components of seasonal light cycles that are encoded and induce persistent changes in the circadian system. Using combinations of 3 T cycles (23, 24, 26 h) and 2 photoperiods per T cycle (long and short, with duty cycles scaled to each T cycle), we investigate the after-effects of entrainment to these 6 light cycles. We measure locomotor behavior duration (α), period (τ), and entrained phase angle (ψ) in vivo and SCN phase distribution (σφ), τ, and ψ ex vivo to refine our understanding of critical light components for influencing particular circadian properties. We find that both photoperiod and T-cycle length drive determination of in vivo ψ but differentially influence after-effects in α and τ, with photoperiod driving changes in α and photoperiod length and T-cycle length combining to influence τ. Using skeleton photoperiods, we demonstrate that in vivo ψ is determined by both parametric and nonparametric components, while changes in α are driven nonparametrically. Within the ex vivo SCN, we find that ψ and σφ of the PER2∷LUCIFERASE rhythm follow closely with their likely behavioral counterparts (ψ and α of the locomotor activity rhythm) while also confirming previous reports of τ after-effects of gene expression rhythms showing negative correlations with behavioral τ after-effects in response to T cycles. We demonstrate that within-SCN σφ changes, thought to underlie α changes in vivo, are induced primarily nonparametrically. Taken together, our results demonstrate that distinct components of seasonal light input differentially influence ψ, α, and τ and suggest the possibility of separate mechanisms driving the persistent changes in circadian behaviors mediated by seasonal light.

Effect of monochromatic light on the circadian clock of cultured chick retinal tissue.

The central biological clock system of bird is formed by hypothalamus suprachiasmatic nucleus, pineal gland and retina thereby interacting with each other in a neuroendocrine loop. Previous results have confirmed that monochromatic light can influence the clock genes in the pineal gland, hypothalamus and retina of chicks in vivo. The present work was conducted to study whether the cultured retinal tissue of chick could maintain the circadian oscillation and whether the monochromatic light affect the expression level of cultured retinal circadian clock in vitro. Retinal tissues of 0-day-old chicks were cultured in vitro under 4 light treatments (white, red, green and blue lights) with light dark cycle 12:12 and constant dark. The tissues and culture medium were collected every each 4 h. Melanopsin, clock genes, cAanat, the positive-regulating clock proteins and melatonin were measured. The results showed that cOpn4-1, cOpn4-2, cBmal1, cCry1, cPer2, cPer3, cAanat and melatonin concentrations possessed a significant circadian rhythm in cultured chick retina tissues under different monochromatic lights; while, in constant dark, cBmal1, cCry1, cPer2, cPer3, cAanat and melatonin concentration possessed a significant circadian rhythm. Green light promoted the circadian expression level of cOpn4-1, cOpn4-2, cBmal1, cAanat and BMAL1 proteins and the circadian rhythm of melatonin secretion of retina by increasing the mesors and amplitudes. In addition, green light significantly increased the average expression levels of cClock, cBmal2 and CLOCK proteins which were expressed arrhythmically. Results suggested that the retina is a central oscillator with autonomous circadian rhythm. In isolated retina tissues, green light activated the expression of melanopsin and promoted the expression of positive-regulating clock genes, thereby up-regulating the expression of cAanat and resulting the increasing of the synthesis and secretion of melatonin.

A standardized method to assess the endogenous activity and the light-response of the retinal clock in mammals.

Purpose: The bioluminescence reporter PER2::Luciferase (PER2::Luc) provides a powerful tool to study the regulation of biological clocks in explant tissues, including the retinal clock. However, the establishment of a standardized procedure to replicate experimental conditions and to enable meaningful comparisons between findings from different studies is still lacking. In addition, different parameters may affect the retinal circadian bioluminescence signal and its dynamic in in vitro assays. In the present study, we first evaluated the effect of sex and age on the main parameters of the mouse retinal clock. We then examined the impact of medium change on PER2::Luc rhythm and compared two light stimulation protocols of the retinal clock. Methods: In a first set of experiments, retinal explants from both male and female Per2Luc mice of different ages (1 to 8 months) are cultured and the period, phase, amplitude, and rhythmic power of PER2::Luc oscillations are analyzed. In a second set of experiments, we quantified the effect of a medium change done after 4, 6, 8, 9, or 10 days of culture on the phase and period of retinal explants. Finally, we compared the phase shift and the period change resulting from two methods of light stimulations of retinal explants: the first involved the transfer of the cultured tissues from the Lumicycle into a light stimulation chamber, while the second used a light delivery apparatus embedded in the Lumicycle. Results: We do not observe any sex-dependent effects on the amplitude, period, phase, and rhythmic power of the in vitro retinal PER2::Luc oscillations in animals aged of 2 to 3 months. The most remarkable effect of age is on the amplitude of PER2::Luc oscillations that significantly decrease from 1 to 4-5 months, whereas the endogenous period and rhythmic power increase slightly until 2 to 3 months and then do not change until 8 months. The phase is not affected by age. We then show that a medium change occurring after 4 days of culture does not alter the phase of PER2::Luc rhythm by comparison with day 0, whereas a medium change done after 6, 8, 9, or 10 days in culture advances the phase and lengthens the period. Finally, we observe that the physical displacement of the culture dishes containing retinal explants, even in complete darkness, induces a strong phase shift of PER2::Luc oscillations. Conclusions: Our work shows that the retina cultures are particularly sensitive to some aspects of the culture procedure, and it provides an accurate standard protocol to avoid biases due to artifactually induced phase shifts resulting from the medium change or physical displacement.

Light Perception: A Matter of Time.

Optimizing the perception of external cues and regulating physiology accordingly help plants to cope with the constantly changing environmental conditions to which they are exposed. An array of photoreceptors and intricate signaling pathways allow plants to convey the surrounding light information and synchronize an endogenous timekeeping system known as the circadian clock. This biological clock integrates multiple cues to modulate a myriad of downstream responses, timing them to occur at the best moment of the day and the year. Notably, the mechanism underlying entrainment of the light-mediated clock is not clear. This review addresses known interactions between the light-signaling and circadian-clock networks, focusing on the role of light in clock entrainment and known molecular players in this process.