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

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

Late evening electric light exposure is associated with low sleep regularity in adolescents living in rural areas.

In this study, we tested the prediction that sleep regularity would be lower in adolescents exposed to late evening electric light (LEEL) than in those without exposure to it. The Sleep Regularity Index was calculated based on actigraph recordings from adolescents living in rural communities in Argentina and Brazil that were either exposed to LEEL or not. The effect of the LEEL on sleep variables was tested using linear models considering sex and age, as well as accounting for the differences between countries. Sleep onset was delayed, sleep duration shortened, and Sleep Regularity Index was 4 [1-8] points lower in the group exposed to LEEL (p = .0176, eta2 =0.13). Our results show that beyond sleep phase and duration, which are known to be affected by LEEL in this age group, sleep irregularity should also be considered as an important outcome variable when assessing the adverse effects of evening light on adolescents.

Expression of the vesicular GABA transporter within neuromedin S+ neurons sustains behavioral circadian rhythms.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.

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

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

Toward an indoor lighting solution for social jet lag.

There is growing interest in developing artificial lighting that stimulates intrinsically photosensitive retinal ganglion cells (ipRGCs) to entrain circadian rhythms to improve mood, sleep, and health. Efforts have focused on stimulating the intrinsic photopigment, melanopsin; however, recently, specialized color vision circuits have been elucidated in the primate retina that transmit blue-yellow cone-opponent signals to ipRGCs. We designed a light that stimulates color-opponent inputs to ipRGCs by temporally alternating short and longer wavelength components that strongly modulate short-wavelength sensitive (S) cones. Two-hour exposure to this S-cone modulating light produced an average circadian phase advance of one hour and twenty minutes in 6 subjects (mean age = 30 years) compared to no phase advance for the subjects after exposure to a 500-lux white light equated for melanopsin effectiveness. These results are promising for developing artificial lighting that is highly effective in controlling circadian rhythms by invisibly modulating cone-opponent circuits.

Sleep Under Preindustrial Conditions: What We Can Learn from It.

Human sleep is regulated by light in two fundamental ways: The light-dark (LD) cycle entrains a circadian clock that in turn regulates sleep timing, and light per se can acutely inhibit sleep. Throughout evolution, these sleep regulatory systems became highly sensitive to the effects of light and they can be affected by the relatively low light intensities that are used indoors. Thus, postindustrial living conditions have created built environments that have isolated humans from the natural LD cycle and exposed them to an artificial one that can affect daily sleep timing. Studying indigenous communities that have differential access to electricity, as well as communities living in highly urbanized areas, we and others have shown that human access to artificial light has delayed the daily onset of sleep but has had a smaller effect on its offset, leading to an overall reduction in sleep duration that is pervasive in modern societies. In this chapter we discuss these studies, highlight their main findings, and point to their limitations.

Moonstruck sleep: Synchronization of human sleep with the moon cycle under field conditions.

Before the availability of artificial light, moonlight was the only source of light sufficient to stimulate nighttime activity; still, evidence for the modulation of sleep timing by lunar phases is controversial. Here, we use wrist actimetry to show a clear synchronization of nocturnal sleep timing with the lunar cycle in participants living in environments that range from a rural setting with and without access to electricity in indigenous Toba/Qom communities in Argentina to a highly urbanized postindustrial setting in the United States. Our results show that sleep starts later and is shorter on the nights before the full moon when moonlight is available during the hours following dusk. Our data suggest that moonlight likely stimulated nocturnal activity and inhibited sleep in preindustrial communities and that access to artificial light may emulate the ancestral effect of early-night moonlight.

Access to electric light is associated with delays of the dim-light melatonin onset in a traditionally hunter-gatherer Toba/Qom community.

Key to the transition of humans from nomadic hunting-gathering groups to industrialized and highly urbanized societies was the creation of protected and artificially lit environments that extended the natural daylight hours and consolidated sleep away from nocturnal threats. These conditions isolated humans from the natural regulators of sleep and exposed them to higher levels of light during the evening, which are associated with a later sleep onset. Here, we investigated the extent to which this delayed timing of sleep is due to a delayed circadian system. We studied two communities of Toba/Qom in the northern region of Argentina, one with and the other without access to electricity. These communities have recently transitioned from a hunting-gathering subsistence to mixed subsistence systems and represent a unique model in which to study the potential effects of the access to artificial light on sleep physiology. We have previously shown that participants in the community with access to electricity had, compared to participants in the community without electricity, later sleep onsets, and shorter sleep bouts. Here, we show they also have a delayed dim-light melatonin onset (DLMO). This difference is present during the winter but not during the spring when the influence of evening artificial light is likely less relevant. Our results support the notion that the human transition into artificially lit environments had a major impact on physiological systems that regulate sleep timing, including the phase of the master circadian clock.

Sleep in university students prior to and during COVID-19 Stay-at-Home orders.

Sleep health has multiple dimensions including duration, regularity, timing, and quality [1-4]. The Coronavirus 2019 (COVID-19) outbreak led to Stay-at-Home orders and Social Distancing Requirements in countries throughout the world to limit the spread of COVID-19. We investigated sleep behaviors prior to and during Stay-at-Home orders in 139 university students (aged 22.2 ± 1.7 years old [±SD]) while respectively taking the same classes in-person and remotely. During Stay-at-Home, nightly time in bed devoted to sleep (TIB, a proxy for sleep duration with regard to public health recommendations [5]) increased by ∼30 min during weekdays and by ∼24 mins on weekends and regularity of sleep timing improved by ∼12 min. Sleep timing became later by ∼50 min during weekdays and ∼25 min on weekends, and thus the difference between weekend and weekday sleep timing decreased - hence reducing the amount of social jetlag [6,7]. Further, we find individual differences in the change of TIB devoted to sleep such that students with shorter TIB at baseline before the first COVID-19 cases emerged locally had larger increases in weekday and weekend TIB during Stay-at-Home. The percentage of participants that reported 7 h or more sleep per night, the minimum recommended sleep duration for adults to maintain health [5] - including immune health - increased from 84% to 92% for weekdays during Stay-at-Home versus baseline. Understanding the factors underlying such changes in sleep health behaviors could help inform public health recommendations with the goal of improving sleep health during and following the Stay-at-Home orders of the COVID-19 pandemic.

Evidence for Internal Desynchrony Caused by Circadian Clock Resetting.

Circadian disruption has been linked to markers for poor health outcomes in humans and animal models. What is it about circadian disruption that is problematic? One hypothesis is that phase resetting of the circadian system, which occurs in response to changes in environmental timing cues, leads to internal desynchrony within the organism. Internal desynchrony is understood as acute changes in phase relationships between biological rhythms from different cell groups, tissues, or organs within the body. Do we have strong evidence for internal desynchrony associated with or caused by circadian clock resetting? Here we review the literature, highlighting several key studies from measures of gene expression in laboratory rodents. We conclude that current evidence offers strong support for the premise that some protocols for light-induced resetting are associated with internal desynchrony. It is important to continue research to test whether internal desynchrony is necessary and/or sufficient for negative health impact of circadian disruption.

Mismatch between perceived family and individual chronotype and their association with sleep-wake patterns.

While social zeitgebers are known to shape diurnal preference, little research has been devoted to determining the contribution of the familiar group chronotype as social zeitgeber on individual circadian rhythms and sleep-wake patterns in adult subjects. The current study aimed to examine the matching between perceived family chronotype and individual chronotype and their relationship with sleep-wake patterns on weekdays and weekends, diurnal subjective somnolence, and substance consumption. Nine hundred and forty-two Colombian adults completed the Composite Scale of Morningness, the Epworth Sleepiness Scale, and responded to a questionnaire about circadian preferences of their family nucleus. We found evidence of a mismatch between perceived family and individual chronotype, mainly for morning-type individuals (Cohen's Kappa = -0.231; p < 0.001). This mismatch was associated with diurnal subjective somnolence (ß = 0.073; p < 0.001) and specific sleep-wake patterns (p < 0.01). In addition, subjects with evening-type families showed higher caffeine and alcohol consumption (p < 0.001). To our knowledge, this is the first study to assess and report the mismatching between perceived family and individual chronotypes, and it adds to the existing body of knowledge regarding the influence of social zeitgebers on circadian rhythms. This is particularly relevant since mismatching between circadian physiology and environmental cues have been shown to lead to diverse pathologies.

Mood rhythmicity is associated with depressive symptoms and caffeinated drinks consumption in South American young adults.

Among the factors that contribute to the onset and maintenance of depressive disorders, rhythmicity of symptoms and consumption of caffeine have recently gained attention. The current study aimed to examine the differential rhythmicity of relevant variables in a sample of young participants, considering the presence of depressive symptomatology and the frequency of caffeinated drinks consumption. A significant 24-hour differential rhythmicity of mood, cognitive and physiological variables was found indicating an evening peak pattern in the participants with depressive symptoms. Interestingly, caffeinated drinks consumption was differentially associated with self-perceived peaks, according to the presence of depressive symptomatology. Our findings are among the first reports about the potential association of the 24-hours rhythmicity of relevant mood-related variables, depressive symptoms, and caffeine intake. These results support the view that the identification of risk factors for depression, and the application of novel measurements and analysis methods in the development of new preventive strategies should be a public health priority.

Social Validation Influences Individuals' Judgments about Ownership.

In all domains, from informal to formal, there are conflicts about property and ownership which resolution demands consideration of alleged claims from more than one party. In this work we asked adults (N = 359) to judge cases in which a character held a property claim over an item, but is challenged by a second character who holds a different, subsequent claim over it. The specific goal of this work is to investigate how the resolution of such conflicts depends on the social endorsement of ownership claims. To achieve this aim, we designed variations of conflictive situations over property in which we manipulated details regarding the knowledge of the second agent of other third-parties about the first agent's actions. In essence, our questions were: if an agent claims ownership of something which has a previous property claim on (1) does it matter whether said agent knew of the first's agent actions or not? And (2) does it matter whether third parties were aware or notified of the first one's claim? The results confirm that adults resolve the settling of property rights based not only on the nature of ownership claims but also on the social acknowledgment of such claims, in accordance with what is stipulated in legal systems worldwide. Participants considered the second character in the stories to hold a lesser right over the object under dispute when she knew of the first character's claim. Participants also considered that the first character's claim was reinforced when there were witnesses for her actions, but not when third parties were merely communicated of such actions. This is the first study to our knowledge that studies how social validation of ownership claims drives adults' judgments on property claims.

Circadian and Metabolic Effects of Light: Implications in Weight Homeostasis and Health.

Daily interactions between the hypothalamic circadian clock at the suprachiasmatic nucleus (SCN) and peripheral circadian oscillators regulate physiology and metabolism to set temporal variations in homeostatic regulation. Phase coherence of these circadian oscillators is achieved by the entrainment of the SCN to the environmental 24-h light:dark (LD) cycle, coupled through downstream neural, neuroendocrine, and autonomic outputs. The SCN coordinate activity and feeding rhythms, thus setting the timing of food intake, energy expenditure, thermogenesis, and active and basal metabolism. In this work, we will discuss evidences exploring the impact of different photic entrainment conditions on energy metabolism. The steady-state interaction between the LD cycle and the SCN is essential for health and wellbeing, as its chronic misalignment disrupts the circadian organization at different levels. For instance, in nocturnal rodents, non-24 h protocols (i.e., LD cycles of different durations, or chronic jet-lag simulations) might generate forced desynchronization of oscillators from the behavioral to the metabolic level. Even seemingly subtle photic manipulations, as the exposure to a "dim light" scotophase, might lead to similar alterations. The daily amount of light integrated by the clock (i.e., the photophase duration) strongly regulates energy metabolism in photoperiodic species. Removing LD cycles under either constant light or darkness, which are routine protocols in chronobiology, can also affect metabolism, and the same happens with disrupted LD cycles (like shiftwork of jetlag) and artificial light at night in humans. A profound knowledge of the photic and metabolic inputs to the clock, as well as its endocrine and autonomic outputs to peripheral oscillators driving energy metabolism, will help us to understand and alleviate circadian health alterations including cardiometabolic diseases, diabetes, and obesity.

Effects of chronic forced circadian desynchronization on body weight and metabolism in male mice.

Metabolic functions are synchronized by the circadian clock setting daily patterns of food intake, nutrient delivery, and behavioral activity. Here, we study the impact of chronic jet-lag (CJL) on metabolism, and test manipulations aimed to overcome potential alterations. We recorded weight gain in C57Bl/6 mice under chronic 6 h advances or delays of the light-dark cycle every 2 days (ChrA and ChrD, respectively). We have previously reported ChrA, but not ChrD, to induce forced desynchronization of locomotor activity rhythms in mice (Casiraghi et al. 2012). Body weight was rapidly increased under ChrA, with animals tripling the mean weight gain observed in controls by day 10, and doubling it by day 30 (6% vs. 2%, and 15% vs. 7%, respectively). Significant increases in retroperitoneal and epidydimal adipose tissue masses (172% and 61%, respectively), adipocytes size (28%), and circulating triglycerides (39%) were also detected. Daily patterns of food and water intake were abolished under ChrA In contrast, ChrD had no effect on body weight. Wheel-running, housing of animals in groups, and restriction of food availability to hours of darkness prevented abnormal increase in body weight under ChrA Our findings suggest that the observed alterations under ChrA may arise either from a direct effect of circadian disruption on metabolism, from desynchronization between feeding and metabolic rhythms, or both. Direction of shifts, timing of feeding episodes, and other reinforcing signals deeply affect the outcome of metabolic function under CJL Such features should be taken into account in further studies of shift working schedules in humans.

The times they're a-changing: effects of circadian desynchronization on physiology and disease.

Circadian rhythms are endogenous and need to be continuously entrained (synchronized) with the environment. Entrainment includes both coupling internal oscillators to external periodic changes as well as synchrony between the central clock and peripheral oscillators, which have been shown to exhibit different phases and resynchronization speed. Temporal desynchronization induces diverse physiological alterations that ultimately decrease quality of life and induces pathological situations. Indeed, there is a considerable amount of evidence regarding the deleterious effect of circadian dysfunction on overall health or on disease onset and progression, both in human studies and in animal models. In this review we discuss the general features of circadian entrainment and introduce diverse experimental models of desynchronization. In addition, we focus on metabolic, immune and cognitive alterations under situations of acute or chronic circadian desynchronization, as exemplified by jet-lag and shiftwork schedules. Moreover, such situations might lead to an enhanced susceptibility to diverse cancer types. Possible interventions (including light exposure, scheduled timing for meals and use of chronobiotics) are also discussed.

Effect of experimental diabetic retinopathy on the non-image-forming visual system.

Diabetic retinopathy is a leading cause of blindness. Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, are involved in non-image-forming visual responses such as photoentrainment of circadian rhythms and pupillary light reflex. Since several reports indicate that retinal ganglion cells are affected by diabetes, we investigated the non-image-forming visual system in an advanced stage of experimental diabetes in rats induced by streptozotocin. After 15 wks of diabetes induction, clear alterations in the visual function were observed and all animals developed mature cataracts. At this time point, concomitantly with a significant decrease in the number of Brn3a(+) retinal ganglion cells, no differences in the number of melanopsin-containing cells, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were observed. At high light intensity, afferent pupil light reflex appears to be conserved in diabetic animals. After 15 wks of diabetes induction, a significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was found. In diabetic animals, the locomotor activity pattern was conserved, although a delay in the time needed for re-entrainment after a phase delay was observed. In diabetic animals, lensectomy reversed the alterations in c-Fos expression and in the locomotor activity rhythm. These results suggest that the neuronal substrate of the non-image-forming visual system remained largely unaffected at advanced stages of diabetes, and that lensectomy, a relatively easy and safe surgery, could partially restore circadian alterations induced by diabetes.

Forced desynchronization of activity rhythms in a model of chronic jet lag in mice.

We studied locomotor activity rhythms of C57/Bl6 mice under a chronic jet lag (CJL) protocol (ChrA(6/2) ), which consisted of 6-hour phase advances of the light-dark schedule (LD) every 2 days. Through periodogram analysis, we found 2 components of the activity rhythm: a short-period component (21.01 ± 0.04 h) that was entrained by the LD schedule and a long-period component (24.68 ± 0.26 h). We developed a mathematical model comprising 2 coupled circadian oscillators that was tested experimentally with different CJL schedules. Our simulations suggested that under CJL, the system behaves as if it were under a zeitgeber with a period determined by (24 - [phase shift size/days between shifts]). Desynchronization within the system arises according to whether this effective zeitgeber is inside or outside the range of entrainment of the oscillators. In this sense, ChrA(6/2) is interpreted as a (24 - 6/2 = 21 h) zeitgeber, and simulations predicted the behavior of mice under other CJL schedules with an effective 21-hour zeitgeber. Animals studied under an asymmetric T = 21 h zeitgeber (carried out by a 3-hour shortening of every dark phase) showed 2 activity components as observed under ChrA(6/2): an entrained short-period (21.01 ± 0.03 h) and a long-period component (23.93 ± 0.31 h). Internal desynchronization was lost when mice were subjected to 9-hour advances every 3 days, a possibility also contemplated by the simulations. Simulations also predicted that desynchronization should be less prevalent under delaying than under advancing CJL. Indeed, most mice subjected to 6-hour delay shifts every 2 days (an effective 27-hour zeitgeber) displayed a single entrained activity component (26.92 ± 0.11 h). Our results demonstrate that the disruption provoked by CJL schedules is not dependent on the phase-shift magnitude or the frequency of the shifts separately but on the combination of both, through its ratio and additionally on their absolute values. In this study, we present a novel model of forced desynchronization in mice under a specific CJL schedule; in addition, our model provides theoretical tools for the evaluation of circadian disruption under CJL conditions that are currently used in circadian research.

"Time sweet time": circadian characterization of galectin-1 null mice.

BACKGROUND: Recent evidence suggests a two-way interaction between the immune and circadian systems. Circadian control of immune factors, as well as the effect of immunological variables on circadian rhythms, might be key elements in both physiological and pathological responses to the environment. Among these relevant factors, galectin-1 is a member of a family of evolutionarily-conserved glycan-binding proteins with both extracellular and intracellular effects, playing important roles in immune cell processes and inflammatory responses. Many of these actions have been studied through the use of mice with a null mutation in the galectin-1 (Lgals1) gene. To further analyze the role of endogenous galectin-1 in vivo, we aimed to characterize the circadian behavior of galectin-1 null (Lgals1-/-) mice. METHODS: We analyzed wheel-running activity in light-dark conditions, constant darkness, phase responses to light pulses (LP) at circadian time 15, and reentrainment to 6 hour shifts in light-dark schedule in wild-type (WT) and Lgals1-/- mice. RESULTS: We found significant differences in free-running period, which was longer in mutant than in WT mice (24.02 vs 23.57 h, p < 0.005), phase delays in response to LP (2.92 vs 1.90 circadian h, p < 0.05), and also in alpha (14.88 vs. 12.35 circadian h, p < 0.05). CONCLUSIONS: Given the effect of a null mutation on circadian period and entrainment, we indicate that galectin-1 could be involved in the regulation of murine circadian rhythmicity. This is the first study implicating galectin-1 in the mammalian circadian system.