Effects of the Light/Dark Phase and Constant Light on Spatial Working Memory and Spine Plasticity in the Mouse Hippocampus.

Circadian rhythms in behavior and physiology such as rest/activity and hormones are driven by an internal clock and persist in the absence of rhythmic environmental cues. However, the period and phase of the internal clock are entrained by the environmental light/dark cycle. Consequently, aberrant lighting conditions, which are increasing in modern society, have a strong impact on rhythmic body and brain functions. Mice were exposed to three different lighting conditions, 12 h light/12 h dark cycle (LD), constant darkness (DD), and constant light (LL), to study the effects of the light/dark cycle and aberrant lighting on the hippocampus, a critical structure for temporal and spatial memory formation and navigation. Locomotor activity and plasma corticosterone levels were analyzed as readouts for circadian rhythms. Spatial working memory via Y-maze, spine morphology of Golgi-Cox-stained hippocampi, and plasticity of excitatory synapses, measured by number and size of synaptopodin and GluR1-immunreactive clusters, were analyzed. Our results indicate that the light/dark cycle drives diurnal differences in synaptic plasticity in hippocampus. Moreover, spatial working memory, spine density, and size and number of synaptopodin and GluR1 clusters were reduced in LL, while corticosterone levels were increased. This indicates that acute constant light affects hippocampal function and synaptic plasticity.

Effects of constant darkness on behaviour and physiology of male and female mice.

A healthy state of life suggests not only a disease-free condition but also normal psychological functioning and behaviour. To maintain a healthy life, the duration of light exposure is a crucial factor. Perturbation of the standard light-dark cycle (LD: 12 h light-12 h dark in mice) may result in brain, behavioural and physiological abnormalities. The current study determined the effects of 3 and 5 weeks of constant darkness (DD: 00 h light-24 h dark) on the behaviour, hormones, prefrontal cortex (PFC) and metabolome of male and female C57BL/6 J mice. We also studied 3 weeks of restoration in LD following 5 weeks of DD exposure. The results revealed that 3 weeks of DD affected male mice more than females, and 5 weeks of DD had a comparable impact on behaviour, hormones and the PFC of male and female mice. After restoration in LD, the DD-induced changes reverted to time-matched LD conditions in male and female mice. Furthermore, metabolome analysis corroborated male and female mice's behavioural and molecular kinetics. The present study laid the foundation for understanding how DD affects behaviour and the PFC as a function of (a) time and (b) sex and described the roles of stress and sex hormones, cytokines, neurotrophins and metabolic pathways.

Sex and Time: Important Variables for Understanding the Impact of Constant Darkness on Behavior, Brain, and Physiology.

The circadian clock can coordinate, regulate and predict physiology and behavior in response to the standard light-dark (LD: 12 h light and 12 h dark) cycle. If we alter the LD cycle by exposing mice to constant darkness (DD: 00 h light and 24 h dark), it can perturb behavior, the brain, and associated physiological parameters. The length of DD exposure and the sex of experimental animals are crucial variables that could alter the impact of DD on the brain, behavior, and physiology, which have not yet been explored. We exposed mice to DD for three and five weeks and studied their impact on (1) behavior, (2) hormones, (3) the prefrontal cortex, and (4) metabolites in male and female mice. We also studied the effect of three weeks of standard light-dark cycle restoration after five weeks of DD on the parameters mentioned above. We found that DD exposure was associated with anxiety-like behavior, increased corticosterone and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), downregulated neurotrophins (BDNF and NGF), and altered metabolites profile in a duration of DD exposure and sex-dependent manner. Females showed a more robust adaptation than males under DD exposure. Three weeks of restoration was adequate to establish homeostasis in both sexes. To the best of our knowledge, this study is the first of its kind to look at how DD exposure impacts physiology and behavior as a function of sex- and time. These findings would have translational value and may help in establishing sex-specific interventions for addressing DD-related psychological issues.

Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

Long-term environmental exposure of darkness induces hyperandrogenism in PCOS via melatonin receptor 1A and aromatase reduction.

Polycystic ovary syndrome (PCOS) is a common and complex disorder impairing female fertility, yet its etiology remains elusive. It is reported that circadian rhythm disruption might play a crucial role in PCOS pathologic progression. Here, in this research, we investigated the effect of environmental long-term circadian rhythm dysfunction and clarified its pathogenic mechanism in the development of PCOS, which might provide the targeted clinical strategies to patients with PCOS. Female SD rats were used to construct a circadian rhythm misalignment model with constant darkness (12/12-h dark/dark cycle), and the control group was kept under normal circadian rhythm exposure (12/12-h light/dark cycle) for 8 weeks. We measured their reproductive, endocrinal, and metabolic profiles at different zeitgeber times (ZTs). Different rescue methods, including melatonin receptor agonist and normal circadian rhythm restoration, and in vitro experiments on the KGN cell line were performed. We found that long-term darkness caused PCOS-like reproductive abnormalities, including estrous cycle disorder, polycystic ovaries, LH elevation, hyperandrogenism, and glucose intolerance. In addition, the expression of melatonin receptor 1A (Mtnr1a) in ovarian granulosa cells significantly decreased in the darkness group. Normal light/dark cycle and melatonin receptor agonist application relieved hyperandrogenism of darkness-treated rats. In vitro experiments demonstrated that decreased MTNR1A inhibited androgen receptor (AR) and CYP19A1 expression, and AR acted as an essential downstream factor of MTNR1A in modulating aromatase abundance. Overall, our finding demonstrates the significant influence of circadian rhythms on PCOS occurrence, suggests that MTNR1A and AR play vital roles in pathological progression of hyperandrogenism, and broadens current treatment strategies for PCOS in clinical practice.

Effects of Extreme Light Cycle and Density on Melatonin, Appetite, and Energy Metabolism of the Soft-Shelled Turtle (Pelodiscus sinensis).

Constant darkness and constant light exposure often disturb the circadian rhythm in the behavior and energy metabolism of vertebrates. Melatonin is known as the hormonal mediator of photoperiodic information to the central nervous system and plays a key role in food intake and energy balance regulation in vertebrates. The popularly cultured soft-shelled turtle Pelodiscus sinensis has been reported to grow better under constant darkness; however, the underlying physiological mechanism by which darkness benefits turtle growth is not clear yet. We hypothesized that increased melatonin levels induced by darkness would increase appetite and energy metabolism and thus promote growth in P. sinensis. In addition, in order to elucidate the interaction of photoperiod and density, juvenile turtles were treated under three photoperiods (light/dark cycle: 24L:0D, 12L:12D, 0L:24D, light density 900 lux) and two stocking densities (high density: 38.10 ind./m2, low density: 6.35 ind./m2) for 4 weeks, and then the blood and brain tissues of turtles were collected during the day (11:00-13:00) and at night (23:00-1:00) after 2 days of fasting. We examined changes in plasma melatonin levels, food intake (FI), and appetite-related hormones (plasma ghrelin and leptin), as well as growth and energy metabolism parameters such as specific growth rate (SGR), standard metabolic rate (SMR), plasma growth hormone (GH), and thyroid hormone/enzyme activity (plasma triiodothyronine T3, thyroxine T4, and T45'-deiodinase activity). Moreover, we also assessed the responses of mRNA expression levels of food intake-related genes (kisspeptin 1 (Kiss1); cocaine amphetamine-regulated transcript (CART); neuropeptide Y (NPY)) in the brain. The results showed that under high density, SGR was the lowest in 24L:0D and the highest in 0L:24D. FI was the highest in 0L:24D regardless of density. Plasma melatonin was the highest in 0L:24D under high density at night. SMR increased with decreasing light time regardless of density. Most expressions of the measured appetite-related genes (Kiss1, CART, and NPY) were not affected by photoperiod, nor were the related hormone levels, such as plasma leptin, ghrelin, and GH. However, thyroid hormones were clearly affected by photoperiod. T3 level in 0L:24D under high density during the day was the highest among all treatment groups. T4 in 24L:0D under high density during the day and T45'-deiodinase activity in 24L:0D under low density at night were significantly reduced compared with the control. Furthermore, the energy metabolism-related hormone levels were higher under higher density, especially during the day. Together, melatonin secretion is not only modulated by light but also likely to be regulated by unknown endogenous factors and density. Altered plasma melatonin induced by constant darkness and density seems to be involved in the modulation of energy metabolism rather than appetite in the soft-shelled turtle.

Long-Term Effect of a Single Dose of Caffeine on Sleep, the Sleep EEG and Neuronal Activity in the Peduncular Part of the Lateral Hypothalamus under Constant Dark Conditions.

BACKGROUND: Caffeine is a central nervous system stimulant that influences both the sleep-wake cycle and the circadian clock and is known to influence neuronal activity in the lateral hypothalamus, an important area involved in sleep-wake regulation. Light is a strong zeitgeber and it is known to interact with the effect of caffeine on the sleep-wake cycle. We therefore wanted to investigate the long-term effects of a single dose of caffeine under constant dark conditions. METHODS: We performed long-term (2 days) electroencephalogram (EEG)/electromyogram recordings combined with multi-unit neuronal activity recordings in the peduncular part of the lateral hypothalamus (PLH) under constant darkness in Brown Norway rats, and investigated the effect of a single caffeine treatment (15 mg/kg) or saline control given 1 h after the onset of the endogenous rest phase. RESULTS: After a reduction in sleep and an increase in waking and activity in the first hours after administration, also on the second recording day after caffeine administration, rapid eye movement (REM) sleep was still reduced. Analysis of the EEG showed that power density in the theta range during waking and REM sleep was increased for at least two days. Neuronal activity in PLH was also increased for two days after the treatment, particularly during non-rapid eye movement sleep. CONCLUSION: Surprisingly, the data reveal long-term effects of a single dose of caffeine on vigilance states, EEG, and neuronal activity in the PLH. The absence of a light-dark cycle may have enabled the expression of these long-term changes. It therefore may be that caffeine, or its metabolites, have a stronger and longer lasting influence, particularly on the expression of REM sleep, than acknowledged until now.

Beneficial esthetic lightening effects of Cuscuta extract on skin darkness in healthy individuals: A clinical trial study.

Background and Objective: Many biological activities and pharmacological usages have been revealed for the Cuscuta species. The present study aimed to assess the beneficial effects of Cuscuta on removing skin dark spots in healthy individuals, which is an important cosmetic concern, especially in women. Materials and Methods: This prospective, interventional before-after trial was conducted in 70 healthy individuals without any evidence of skin or systemic disorders who presented for consultation on removing skin darkening. The Cuscuta extract was prepared and then assessed for plant quality control and lack of microbial contamination. The content of melanin at baseline and at 1 and 3 months after intervention was assessed using Dermacatch, an accurate skin colorimetric measurement tool. Results: Comparison of the melanin content of the lesions and treated area to the surrounding normal area at baseline and at 1 month after treatment showed a significant reduction in melanin content from 519.61 ± 45.09 to 498.50 ± 39.35 (P < 0.001). This reducing trend remained significant from the first month to the third month after treatment (from 498.50 ± 39.35 to 483.53 ± 40.99, P < 0.001). This decreasing trend was persistent even after adjusting baseline characteristics including gender, age, and duration of skin lesions. Both patients and investigators had high satisfaction level with the anti-melanogenesis effect of Cuscuta extract. Conclusion: Cuscuta extract is useful for removing hyperpigmented lesions and for skin lightening in healthy individuals.

Physiological and behavioral responses of Orchestia gammarellus (Amphipoda, Talitridae) towards trace elements contamination soil.

Effects of trace elements on the hepatopancreas ultrastructure and on the locomotor activity rhythm were investigated in the amphipod talitrid Orchestia gammarellus collected from Bizerte lagoon banks situated in the north of Tunisia. Animals were exposed to a series of contaminated soil with different concentrations of cadmium and zinc. The locomotor activity rhythm was studied under constant darkness. Histopathological analysis showed that the trace elements, especially the Cd, induced significant changes in the morphology and in the ultrastructural organization of hepatopancreatic cells. The significant alterations obtained were dose dependent. Concerning the behavioral response, results revealed the presence of two components whatever the experimental conditions. In addition, a great inter-individual variability of the locomotor rhythm was observed. Patterns were in majority bimodal for the control individuals and became unimodal and multimodal when exposed under Cd and Zn, respectively. Furthermore, ultradian and circadian periods were determined. The circadian period lengthened after Zn exposure. In addition, the locomotor activity rhythm was more stable for control individuals. However, those exposed to Cd were less active.

Differential response of diurnal and nocturnal mammals to prolonged altered light-dark cycle: a possible role of mood associated endocrine, inflammatory and antioxidant system.

The circadian system maintains internal 24 h oscillation of behavior and physiology, and its misalignment with external light-dark (LD) cycle results in negative health outcomes. In order to elucidate the effect of prolonged constant condition and the differences in the response between nocturnal and diurnal species, we studied the effects of constant light (LL) and constant darkness (DD) on a diurnal (squirrel) and a nocturnal (mouse) rodent species, focusing on the endocrine, inflammatory and antioxidant systems associated with depression-like behavior. Squirrels and mice (n = 10/group) were placed in chronocubicle under 12:12 h LD cycle, LL and DD. After 4 weeks, animals were subjected to sucrose preference test and blood and brain tissues were collected for measuring melatonin, corticosterone, proinflammatory cytokine, tumor necrosis factor-α (TNF-α) and the activity of primary antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). The results show that in diurnal squirrels, prolonged constant darkness reduced sucrose preference, CAT, and SOD, increased corticosterone and TNF-α levels, but caused no significant change in the melatonin compared to LD condition. In contrast, in nocturnal mice constant darkness caused no significant changes in sucrose preference and corticosterone levels, increased melatonin, CAT and SOD levels but decreased TNF-α levels. Chronic LL caused a similar response in both squirrels and mice: it decreased sucrose preference, melatonin, CAT and SOD levels but increased corticosterone and TNF-α levels. Together, the study demonstrates differential effects of altered light-dark cycle in a diurnal and a nocturnal rodent on interrelated endocrine, inflammatory and antioxidant systems associated with depression-like behavior, with constant light having adverse effects on both species but constant darkness having a negative effect mainly in the diurnal squirrels.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

Altered circadian activity and sleep/wake rhythms in the stable tubule only polypeptide (STOP) null mouse model of schizophrenia.

Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.

Natural food intake patterns have little synchronizing effect on peripheral circadian clocks.

BACKGROUND: Circadian rhythms across mammalian tissues are coordinated by a master clock in the suprachiasmatic nucleus (SCN) that is principally entrained by light-dark cycles. Prior investigations have shown, however, that time-restricted feeding (TRF)-daily alternation of fasting and food availability-synchronizes peripheral clocks independent of the light-dark cycle and of the SCN. This has led to the idea that downstream peripheral clocks are entrained indirectly by food intake rhythms. However, TRF is not a normal eating pattern, and it imposes non-physiologic long fasts that rodents do not typically experience. Therefore, we tested whether normal feeding patterns can phase-shift or entrain peripheral tissues by measuring circadian rhythms of the liver, kidney, and submandibular gland in mPer2Luc mice under different food schedules. RESULTS: We employed home cage feeders to first measure ad libitum food intake and then to dispense 20-mg pellets on a schedule mimicking that pattern. In both conditions, PER2::LUC bioluminescence peaked during the night as expected. Surprisingly, shifting the scheduled feeding by 12 h advanced peripheral clocks by only 0-3 h, much less than predicted from TRF protocols. To isolate the effects of feeding from the light-dark cycle, clock phase was then measured in mice acclimated to scheduled feeding over the course of 3 months in constant darkness. In these conditions, peripheral clock phases were better predicted by the rest-activity cycle than by the food schedule, contrary to expectation based on TRF studies. At the end of both experiments, mice were exposed to a modified TRF with food provided in eight equally sized meals over 12 h. In the light-dark cycle, this advanced the phase of the liver and kidney, though less so than in TRF with ad libitum access; in darkness, this entrained the liver and kidney but had little effect on the submandibular gland or the rest-activity cycle. CONCLUSIONS: These data suggest that natural feeding patterns can only weakly affect circadian clocks. Instead, in normally feeding mice, the central pacemaker in the brain may set the phase of peripheral organs via pathways that are independent of feeding behavior.

Equation representing the dark-entrained transition from inaction to action in male and female mice.

The activation of behaviour in a daily rhythm governed by the light cycle is a universal phenomenon among humans, laboratory mammals and other vertebrates. For mice, the active period is during the dark. We have quantified the increase in activity when the lights shut off (Light to Dark, L to D) using a generalized CNS arousal assay with 20 ms resolution, rather than traditional running wheels. Data analysis yielded the rare demonstration of an equation which precisely tracks this behavioural transition and, surprisingly, its reverse during D to L. This behavioural dynamic survives in constant darkness (experiment 2) and is hormone-sensitive (experiment 3). Finally (experiment 4), mice on a light schedule analogous to one which proved troublesome for U.S. Navy sailors, had dysregulated activity bursts which did not conform to the transitions between D and L. These experiments show the lawfulness of a behavioural phase transition and the consequence of deviating from that dynamic pattern. And, in a new way, they bring mathematics to the realm of behavioural neuroscience.

Endogenous circadian time genes expressions in the liver of mice under constant darkness.

BACKGROUND: The circadian rhythms regulate physiological functions and metabolism. Circadian Time (CT) is a unit to quantify the rhythm of endogenous circadian clock, independent of light influence. To understand the gene expression changes throughout CT, C57BL/6 J mice were maintained under constant darkness (DD) for 6 weeks, and the liver samples were collected starting at 9:00 AM (CT1), and every 4 h in a 24-h cycle (CT5, CT9, CT13, CT17 and CT21). Total RNA was extracted and subjected to RNA-Seq data (deposited as GSE 133342, L-DD). To compare gene oscillation pattern under normal light-dark condition (LD, GSE114400) and short time (2 days) dark-dark condition (S-DD, GSE70497), these data were retried from GEO database, and the trimmed mean of M-values normalization was used to normalize the three RNA-seq data followed by MetaCycle analysis. RESULTS: Approximate 12.1% of the genes under L-DD exhibited significant rhythmically expression. The top 5 biological processes enriched in L-DD oscillation genes were mRNA processing, aromatic compound catabolic process, mitochondrion organization, heterocycle catabolic process and cellular nitrogen compound mitotic catabolic process. The endogenous circadian rhythms of clock genes, P450 genes and lipid metabolism genes under L-DD were further compared with LD and S-DD. The oscillation patterns were similar but the period and amplitude of those oscillation genes were slightly altered. RT-qPCR confirmed the selected RNA sequence findings. CONCLUSIONS: This is the first study to profile oscillation gene expressions under L-DD. Our data indicate that clock genes, P450 genes and lipid metabolism genes expressed rhythmically under L-DD. Light was not the necessary factor for persisting circadian rhythm but influenced the period and amplitude of oscillation genes.

Suppression of voluntary ethanol intake in mice under constant light and constant darkness.

Seasonal variations in photoperiod are associated with alterations in human mood and behavior. Similarly, manipulation of the environmental lighting regimen can exert pronounced effects on affective behavior in experimental animals. These observations may be due, in part, to light-induced alterations in circadian rhythms, but it seems likely that other, non-circadian factors also contribute. Several studies have shown that voluntary alcohol (ethanol) consumption can be affected by lighting conditions in rodents, suggesting that photoperiodic variation may account for seasonal and geographic patterns of human alcohol consumption. Nevertheless, the existing animal data are somewhat inconsistent, and little work in this area has been performed in mice. In the present study, we monitored circadian activity rhythms and voluntary ethanol consumption under standard 12:12 light-dark (LD) cycles, and in constant light (LL) and constant darkness (DD). Experiment 1 employed male C3H/He inbred mice, while Experiment 2 employed males and females from a genetically heterogeneous line (WSC). Relative to LD conditions, ethanol intake and ethanol preference were reduced under both LL and DD in both experiments. Because similar effects were seen in both LL and DD, neither circadian disruption nor a classical photoperiodic mechanism are likely to account fully for these findings. Instead, we suggest that the absence of circadian entrainment may function as a mild stressor, resulting in reduced ethanol consumption.

Circadian clock network desynchrony promotes weight gain and alters glucose homeostasis in mice.

OBJECTIVE: A network of endogenous circadian clocks adapts physiology and behavior to recurring changes in environmental demands across the 24-hour day cycle. Circadian disruption promotes weight gain and type 2 diabetes development. In this study, we aim to dissect the roles of different tissue clocks in the regulation of energy metabolism. METHODS: We used mice with genetically ablated clock function in the circadian pacemaker of the suprachiasmatic nucleus (SCN) under different light and feeding conditions to study peripheral clock resetting and the role of the peripheral clock network in the regulation of glucose handling and metabolic homeostasis. RESULTS: In SCN clock-deficient mice, behavioral and non-SCN tissue clock rhythms are sustained under rhythmic lighting conditions but deteriorate quickly in constant darkness. In parallel to the loss of behavioral and molecular rhythms, the animals develop adiposity and impaired glucose utilization in constant darkness. Restoring peripheral clock rhythmicity and synchrony by time-restricted feeding normalizes body weight and glucose metabolism. CONCLUSIONS: These data reveal the importance of an overall synchronized circadian clockwork for the maintenance of metabolic homeostasis.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption.

BACKGROUND: The astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation. METHODS: We analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors. RESULTS: Fluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption. CONCLUSION: Circadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Dim Light at Night and Constant Darkness: Two Frequently Used Lighting Conditions That Jeopardize the Health and Well-being of Laboratory Rodents.

The influence of light on mammalian physiology and behavior is due to the entrainment of circadian rhythms complemented with a direct modulation of light that would be unlikely an outcome of circadian system. In mammals, physiological and behavioral circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) of the hypothalamus. This central control allows organisms to predict and anticipate environmental change, as well as to coordinate different rhythmic modalities within an individual. In adult mammals, direct retinal projections to the SCN are responsible for resetting and synchronizing physiological and behavioral rhythms to the light-dark (LD) cycle. Apart from its circadian effects, light also has direct effects on certain biological functions in such a way that the participation of the SCN would not be fundamental for this network. The objective of this review is to increase awareness, within the scientific community and commercial providers, of the fact that laboratory rodents can experience a number of adverse health and welfare outcomes attributed to commonly-used lighting conditions in animal facilities during routine husbandry and scientific procedures, widely considered as "environmentally friendly." There is increasing evidence that exposure to dim light at night, as well as chronic constant darkness, challenges mammalian physiology and behavior resulting in disrupted circadian rhythms, neural death, a depressive-behavioral phenotype, cognitive impairment, and the deregulation of metabolic, physiological, and synaptic plasticity in both the short and long terms. The normal development and good health of laboratory rodents requires cyclical light entrainment, adapted to the solar cycle of day and night, with null light at night and safe illuminating qualities during the day. We therefore recommend increased awareness of the limited information available with regards to lighting conditions, and therefore that lighting protocols must be taken into consideration when designing experiments and duly highlighted in scientific papers. This practice will help to ensure the welfare of laboratory animals and increase the likelihood of producing reliable and reproducible results.

High Melatonin Conditions by Constant Darkness and High Temperature Differently Affect Melatonin Receptor mt1 and TREK Channel trek2a in the Brain of Zebrafish.

Ambient light and temperature affect reproductive function by regulating kisspeptin and gonadotrophin-releasing hormone (GnRH) in vertebrates. Melatonin and melatonin receptors, as well as the two-pore domain K+ channel-related K+ (TREK) channels, are affected by light and/or temperature; therefore, these molecules could modulate kisspeptin and GnRH against ambient light and temperature. In this study, we investigated the effect of light and temperature, which affect melatonin levels in gene expression levels of TREK channels, kisspeptin, and GnRH. We first investigated the effects of different light and temperature conditions on brain melatonin concentrations by ELISA. Fish were exposed to either constant darkness, constant light, high temperature (35°C), or low temperature (20°C) for 72 h. Brain melatonin levels were significantly high under constant darkness and high temperature. We further investigated the effects of high brain melatonin levels by constant darkness and high temperature on gene expression levels of melatonin receptors (mt1, mt2, and mel1c), TREK channels (trek1b, trek2a, and trek2b), gnrh3, and kiss2 in the adult zebrafish brain by real-time polymerase chain reaction. Fish were exposed to constant darkness or elevated temperatures (35°C) for 72 h. trek2a, kiss2, and gnrh3 levels were increased under constant darkness. High temperature decreased gene expression levels of mt1, mt2, mel1c, and gnrh3 in the preoptic area, whereas other genes remained unchanged. Melatonin receptors, TREK channels, gnrh3, and kiss2 responded differently under high melatonin conditions. The melatonin receptors and the TREK channels could play roles in the regulation of reproduction by environmental cues, especially ambient light and temperature.