Constant Darkness Research Articles

Effect of Ambient Lighting on Intraocular Pressure Rhythms in Rats.

The purpose of this study was to determine the effects of ambient lighting on intraocular pressure (IOP) rhythmicity and variability. IOP was continuously recorded by wireless telemetry from rats under light/dark (LD), dark/light (DL), asymmetric (6L18D and 18D6L), constant dark (DD), and constant light (LL) cycles. In some DD experiments, 1-hour light pulses were presented at varying times. IOP rhythmicity and variability were respectively quantified via cosinor analysis and peak detection algorithms that identified transient and sustained fluctuations. Rat IOP peaked at night and troughed during the day with LD amplitude of 8.7 ± 3.4mm Hg. Rhythmicity persisted in DD and LL with a free-running period of 24.1 ± 0.3 and 25.2 ± 0.4 hours, respectively. Peak-to-trough amplitude was approximately 60% smaller in LL, often disappeared after 1 to 2 weeks as daytime IOP drifted 2.6 ± 1.5mm Hg higher, and returned to approximately 60% larger in LD. Rhythmicity was similarly impacted but resynchronized to DL over 4 to 6days. Rhythmicity was unaltered by short photoperiods (6L18D), but the nocturnal IOP elevation was markedly shortened by long photoperiods (18L6D) and temporarily lowered to daytime levels by light pulses during the subjective night. Transient and sustained event rate, amplitude, interval, and energy content were nearly identical in LD, DD, and LL. Aqueous humor dynamics of rat eyes are intrinsically configured to set IOP at daytime levels. Circadian clock input modulates these dynamics to elevate IOP at night. Light at night blocks this input, sending IOP back to daytime levels. Effects of abnormal lighting on IOP rhythmicity may contribute to pressure-related ocular neuropathies.

Synchronized Photoactivation of T4K Rhodopsin Causes a Chromophore-Dependent Retinal Degeneration That Is Moderated by Interaction with Phototransduction Cascade Components.

Multiple mutations in the Rhodopsin gene cause sector retinitis pigmentosa in humans and a corresponding light-exacerbated retinal degeneration (RD) in animal models. Previously we have shown that T4K rhodopsin requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in mixed male and female Xenopus laevis In this model, RD was prevented by rearing animals in constant darkness but surprisingly also in constant light. RD was maximized by light cycles containing at least 1 h of darkness and 20 min of light exposure, light intensities >750 lux, and by a sudden light onset. Under conditions of frequent light cycling, RD occurred rapidly and synchronously, with massive shedding of ROS fragments into the RPE initiated within hours and subsequent death and phagocytosis of rod cell bodies. RD was minimized by reduced light levels, pretreatment with constant light, and gradual light onset. RD was prevented by genetic ablation of the retinal isomerohydrolase RPE65 and exacerbated by ablation of phototransduction components GNAT1, SAG, and GRK1. Our results indicate that photoactivated T4K rhodopsin is toxic, that cell death requires synchronized photoactivation of T4K rhodopsin, and that toxicity is mitigated by interaction with other rod outer segment proteins regardless of whether they participate in activation or shutoff of phototransduction. In contrast, RD caused by P23H rhodopsin does not require photoactivation of the mutant protein, as it was exacerbated by RPE65 ablation, suggesting that these phenotypically similar disorders may require different treatment strategies.

Light- and Relative Humidity-Regulated Hypersensitive Cell Death and Plant Immunity in Chinese Cabbage Leaves by a Non-adapted Bacteria Xanthomonas campestris pv. vesicatoria.

Inoculation of Chinese cabbage leaves with high titer (107 cfu/ml) of the non-adapted bacteria Xanthomonas campestris pv. vesicatoria (Xcv) strain Bv5-4a.1 triggered rapid leaf tissue collapses and hypersensitive cell death (HCD) at 24 h. Electrolyte leakage and lipid peroxidation markedly increased in the Xcv-inoculated leaves. Defence-related gene expressions (BrPR1, BrPR4, BrChi1, BrGST1 and BrAPX1) were preferentially activated in the Xcv-inoculated leaves. The Xcv-triggered HCD was attenuated by continuous light but accelerated by a dark environment, and the prolonged high relative humidity also alleviated the HCD. Constant dark and increased relative humidity provided favorable conditions for the Xcv bacterial growth in the leaves. Pretreated fluridone (biosynthetic inhibitor of endogenous abscisic acid [ABA]) increased the HCD in the Xcv-inoculated leaves, but exogenous ABA attenuated the HCD. The pretreated ABA also reduced the Xcv bacterial growth in the leaves. These results highlight that the onset of HCD in Chinese cabbage leaves initiated by non-adapted pathogen Xcv Bv5-4a.1 and in planta bacterial growth was differently modulated by internal and external conditional changes.

Maize ZmSRO1e promotes mesocotyl elongation and deep sowing tolerance by inhibiting the activity of ZmbZIP61.

Deep sowing is a traditional method for drought resistance in maize production, and mesocotyl elongation is strongly associated with the ability of maize to germinate from deep soil. However, little is known about the functional genes and mechanisms regulating maize mesocotyl elongation. In the present study, we identified a plant-specific SIMILAR TO RCD-ONE (SRO) protein family member, ZmSRO1e, involved in maize mesocotyl elongation. The expression of ZmSRO1e is strongly inhibited upon transfer from dark to white light. The loss-of-function zmsro1e mutant exhibited a dramatically shorter mesocotyl than the wild-type in both constant light and darkness, while overexpression of ZmSRO1e significantly promoted mesocotyl elongation, indicating that ZmSRO1e positively regulates mesocotyl elongation. We showed that ZmSRO1e physically interacted with ZmbZIP61, an ortholog of Arabidopsis ELONGATED HYPOCOTYL 5 (HY5) and showed a function similar to that of HY5 in regulating photomorphogenesis. We found that ZmSRO1e repressed the transcriptional activity of ZmbZIP61 toward target genes involved in the regulation of cell expansion, such as ZmEXPB4 and ZmEXPB6, by interfering with the binding of ZmbZIP61 to the promoters of target genes. Our results provide a new understanding of the mechanism by which SRO regulates photomorphogenesis and highlight its potential application in deep sowing-resistant breeding.

The Estrous Cycle Coordinates the Circadian Rhythm of Eating Behavior in Mice.

The estrous cycle regulates rhythms of locomotor activity, body temperature, and circadian gene expression. In female mice, activity increases on the night of proestrus, when elevated estrogens cause ovulation. Exogenous estradiol regulates eating behavior rhythms in female mice fed a high-fat diet, but it is unknown whether endogenous estrogens regulate eating rhythms. In this study, we investigated whether diurnal and circadian eating behavior rhythms change systematically across the estrous cycle. We first studied diurnal eating behavior rhythms in female C57BL/6J mice in 12L:12D. Estrous cycle stages were determined by vaginal cytology while eating behavior and wheel revolutions were continuously measured. The mice had regular 4- to 5-day estrous cycles. Consistent with prior studies, the greatest number of wheel revolutions occurred on the night of proestrus into estrus when systemic levels of estrogens peak. The amplitude, or robustness, of the eating behavior rhythm also fluctuated with 4- to 5-day cycles and peaked primarily during proestrus or estrus. The phases of eating behavior rhythms fluctuated, but not at 4- or 5-day intervals, and phases did not correlate with estrous cycle stages. After ovariectomy, the eating behavior rhythm amplitude fluctuated at irregular intervals. In constant darkness, the amplitude of the circadian eating behavior rhythm peaked every 4 or 5 days and coincided with the circadian day that had the greatest number of wheel revolutions, a marker of proestrus. These data suggest that fluctuations of ovarian hormones across the estrous cycle temporally organize the robustness of circadian eating behavior rhythms so that it peaks during ovulation and sexual receptivity.

A Detailed Re-Examination of the Period Gene Rescue Experiments Shows That Four to Six Cryptochrome-Positive Posterior Dorsal Clock Neurons (DN1p) of Drosophila melanogaster Can Control Morning and Evening Activity.

Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit fly Drosophila melanogaster exhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. In this study, we re-examined the two-oscillator model for the M and E peaks of Drosophila by utilizing more than 50 Gal4 lines in conjunction with the UAS-period16 line, which enables the restoration of the clock function in specific cells in the period (per) null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LNv) is responsible for controlling the M peak, while the other group, consisting of the 5th ventrolateral neuron (5th LNv) and the three cryptochrome (CRY)-positive dorsolateral neurons (LNd), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN1p) is thought to also contain M and E oscillators. In this study, we found that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, we were able to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, we demonstrate that 4 to 6 CRY-positive DN1p cells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, our detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescue per in specific clock neurons.

Deciphering a Beetle Clock: Individual and Sex-Dependent Variation in Daily Activity Patterns.

Circadian clocks are inherent to most organisms, including cryptozoic animals that seldom encounter direct light, and regulate their daily activity cycles. A conserved suite of clock genes underpins these rhythms. In this study, we explore the circadian behaviors of the red flour beetle Tribolium castaneum, a significant pest impacting stored grain globally. We report on how daily light and temperature cues synchronize distinct activity patterns in these beetles, characterized by reduced morning activity and increased evening activity, anticipating the respective environmental transitions. Although less robust, rhythmicity in locomotor activity is maintained in constant dark and constant light conditions. Notably, we observed more robust rhythmic behaviors in males than females with individual variation exceeding those previously reported for other insect species. RNA interference targeting the Clock gene weakened locomotor activity rhythms. Our findings demonstrate the existence of a circadian clock and of clock-controlled behaviors in T. castaneum. Furthermore, they highlight substantial individual differences in circadian activity, laying the groundwork for future research on the relevance of individual variation in circadian rhythms in an ecological and evolutionary context.

Regulation of opsin and circadian clock genes on mate-finding behavior of the day-flying red moth, Phauda flammans (Walker)

ABSTRACT First, significantly higher mate-finding success was found under light condition than under constant darkness condition in Phauda flammans, a typical diurnal moth. We speculate that mate-finding behavior in P. flammans may be influenced by the light-sensitive opsin genes Long wavelength opsin (PfLW), Ultraviolet opsin (PfUV) and Blue opsin (PfBL), which are potentially regulated by both light-cues and endogenous circadian rhythms. Second, the circadian clock genes Period (PfPer), Timeless (PfTim), Cryptochrome1 (PfCry1), Cryptochrome2 (PfCRY2), Cryptochrome3 (PfCry-like), Clock (PfClk), Cycle (PfCyc), Vrille (PfVri), and Slimb (PfSli) were identified in P. flammans. Third, circadian rhythms in the relative expression levels of opsin and circadian clock genes were demonstrated via quantitative real-time PCR analysis, with peak expression coinciding with the mate-finding peak. Notably, the relative expression of PfLW in males P. flammans was significantly higher than that in females P. flammans at the mate-finding peaks Zeitgeber time (ZT) 8 and ZT 10 under light, while the expression of the opsin gene PfBL showed a similar pattern at ZT 10 under light. Additionally, the expression of the clock gene PfCry-like was significantly higher in males than in females at ZT 8 and ZT 10 under light, while PfPer, PfTim, PfClk and PfCyc exhibited similar male-biased expression patterns at ZT 10 under light. Conversely, PfCry1 and PfVri expression was significantly higher in females than in male at ZT 8 under light. In conclusion, sex differences were detected in the expression of opsin and circadian clock genes, which indicated that light-mediated regulation of these genes may contribute to the daytime mate-finding behavior of P. flammans.

Diel transcriptional responses of coral-Symbiodiniaceae holobiont to elevated temperature

Coral exhibits diel rhythms in behavior and gene transcription. However, the influence of elevated temperature, a key factor causing coral bleaching, on these rhythms remains poorly understood. To address this, we examined physiological, metabolic, and gene transcription oscillations in the Acropora tenuis-Cladocopium sp. holobiont under constant darkness (DD), light-dark cycle (LD), and LD with elevated temperature (HLD). Under LD, the values of photosystem II efficiency, reactive oxygen species leakage, and lipid peroxidation exhibited significant diel oscillations. These oscillations were further amplified during coral bleaching under HLD. Gene transcription analysis identified 24-hour rhythms for specific genes in both coral and Symbiodiniaceae under LD. Notably, these rhythms were disrupted in coral and shifted in Symbiodiniaceae under HLD. Importantly, we identified over 20 clock or clock-controlled genes in this holobiont. Specifically, we suggested CIPC (CLOCK-interacting pacemaker-like) gene as a core clock gene in coral. We observed that the transcription of two abundant rhythmic genes encoding glycoside hydrolases (CBM21) and heme-binding protein (SOUL) were dysregulated by elevated temperature. These findings indicate that elevated temperatures disrupt diel gene transcription rhythms in the coral-Symbiodiniaceae holobiont, affecting essential symbiosis processes, such as carbohydrate utilization and redox homeostasis. These disruptions may contribute to the thermal bleaching of coral.

A hypothalamic circuit for circadian regulation of corticosterone secretion.

The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for the daily active cycle and is impaired in chronic depression. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN) which governs the activity of neurons in the paraventricular nucleus of the hypothalamus that produce the corticotropin-releasing hormone (PVHCRH neurons). The dorsomedial nucleus of the hypothalamus (DMH) serves as a crucial intermediary, being innervated by the SCN both directly and via relays in the subparaventricular zone, and projecting axons to the PVH, thereby exerting influence over the cortisol/corticosterone rhythm. However, the role and synaptic mechanisms by which DMH neurons regulate the daily rhythm of Cort secretion has not been explored. We found that either ablating or acutely inhibiting the DMH glutamatergic (DMHVglut2) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMHVglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVHCRH neurons through glutamate release (the DMHVglut2 → PVHCRH pathway). Similarly, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMHVgat) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of DMHVgat neurons increased Cort, although with a lower magnitude compared to DMHVglut2 neuron stimulation, suggesting a role in disinhibiting PVHCRH neurons. Supporting this hypothesis, we found that rostral DMHVgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVHCRH neurons, and that activating the DMHVgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVHCRH neurons. Finally, ablation of cvPVHVgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMHVgat → cvPVHVgat → PVHCRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVHCRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between SCN, DMH, and PVH.

Circadian rhythms of antioxidant enzymes activity, clock, and inflammation factors are disrupted in the prefrontal cortex of aged rats. Potential targets for therapeutic strategies for a healthy aging.

Age impairs cognitive functions and antioxidant defenses, for example, by increasing oxidative stress and inflammation in the brain. However, so far, there is no report on the consequences of aging on temporal patterns of proteins and lipids oxidation, antioxidant enzymes activity, endogenous clock and proinflammatory cytokine, in the prefrontal cortex (PFC). Therefore, our objectives here were: 1) to investigate the endogenous nature of 24h-rhythms of lipoperoxidation, protein carbonyls levels, CAT and GPx activity, RORa, and TNFα, in the rat PFC, and 2) to study the consequences of aging on the circadian organization of those factors in the same brain area. To do that, 3- and 22-mo-old male Holtzman rats were maintained under constant darkness conditions during 15 days before reaching the corresponding age. PFC samples were isolated every 4 h, under dim-red light, during a 24h period. Our results revealed circadian patterns of antioxidant enzymes activity, oxidative stress, RORa and TNFα proteins levels, in the PFC of young rats. The circadian distribution of the rhythms’ phases suggests the existence of a reciprocal communication among the antioxidant defenses, the endogenous clock, and the inflammation, in the PFC. Noteworthy, such circadian organization disappears in the PFC of aged rats. An increased oxidative stress would make the redox environment to change into an oxidative status, which alters the endogenous clock activity and disrupts the circadian organization of, at least part, of the antioxidant defenses and the TNFα, in the PFC. These results might highlight novel chronobiological targets for the design of therapeutic strategies addressed to a healthy aging.

Temperature and LED lightning affect the regeneration of somatic embryo-derived sporophytes on the internode explants of the tree fern Cyathea delgadii Sternb.

The influence of the temperature and different light qualities emitted by light-emitting diodes (LEDs) and fluorescent lamps (Fl) on the micropropagation of the tree fern Cyathea delgadii was evaluated. The most efficient somatic embryo production was obtained on internode explants at 22 °C. The optimal temperature range for obtaining well-developed plants was 24–28 °C. This stimulated the elongation and development of the first leaf and the formation of the next leaf and roots primordia. Temperatures lower than 24 °C and higher than 28 °C inhibited the formation of young sporophyte organs and delayed their development. The RBUV (35% red, 15% blue, and 50% UV) and B (100% blue) lights and Fl light were beneficial for the sporophyte production on internode explants. However, plants obtained under RBUV light were undeveloped. The white LED light stimulated the number of explants capable of gametophyte production and development. The RB light (70% red and 30% blue) enhanced the number of roots of newly-formed plants. Most of the LED lights tested had a good impact on root elongation compared to Fl light and constant darkness. The R light (100% red) benefits leaf development and elongation. Research shows that temperature and LED lightning play a significant role in the process of morphogenesis in C. delgadii, significantly affecting the embryogenic competence of somatic cells and the development of sporophytes.

Potential impacts of titanium dioxide nanoparticles on the biochemical and behavioural status of the amphipods Talitrus saltator (Amphipoda, Talitridae)

ABSTRACT Titanium dioxide nanoparticles (TiO2 NPs) are among the most universally engineered nanomaterials and widely used nanomaterials in the world. In order to predict the environmental impact and interaction between these NPs and coastal organisms, the amphipod Talitrus saltator was exposed to TiO2 NPs C1 = 151.5 µg.L−1 and TiO2 NPs C2 = 303 µg.L−1. Catalase activity increased significantly and reaches a maximum of about 0.8 ± 0.02 µMol/min/mg proteins after exposure to TiO2 NPs C2 showing possible oxidative mechanisms. In addition, acetylcholinesterase activity reduced significantly (p < 0.05) after exposure to the highest TiO2 NPs concentration, indicating neurotransmission disturbance treated with 303 µg.L−1 of TiO2 NPs. Locomotor behaviour for untreated and treated specimens with TiO2 NPs was studied under constant darkness (DD). Results revealed the existence of ultradian and circadian components. In addition, locomotor activity patterns were generally bimodal for untreated specimens and became multimodal for treated ones. We noted also a slippage of the activity peak from the middle of the subjective night for the control until the subjective day after TiO2 NPs exposure. Furthermore, the most important stability was observed for control individuals who were more active than treated ones. Overall, this laboratory study provides preliminary toxicity data for Talitrus saltator exposed to TiO2 NPs.

Galaxy Formation in ΛCDM Cosmology

This is a golden age for galaxy formation: Existing and especially new telescopes are providing observations that challenge and illuminate rapidly improving theory and simulations. This review describes the formation of the cosmic web and the structure of the dark matter halos that provide the scaffolding of the Universe. It then summarizes how empirical models, semianalytic models, and hydrodynamic simulations attempt to account for key properties of the galaxy population, including the main sequence of star-forming galaxies, the inefficiency of star formation, the shape evolution and color bimodality of galaxies, and the phenomena that cause galaxies to quench their star formation. It concludes with a summary of observations that have challenged the cosmological constant cold dark matter (ΛCDM) paradigm of galaxy formation—including the Hubble and S 8 tensions, bright galaxies in the early Universe, an extragalactic background light mystery, missing satellite galaxies, the diversity of dwarf galaxies, the cusp–core problem, the too-big-to-fail problem, stellar clumps, planes of satellite galaxies, and galaxies without dark matter—and solutions that have been proposed.

Dietary acrylamide disrupts the functioning of the biological clock

Acrylamide (ACR) is a known carcinogen and neurotoxin. It is chronically consumed in carbohydrate-rich snacks processed at high temperatures. This calls for systematic research into the effects of ACR intake, best performed in an experimental model capable of detecting symptoms of its neurotoxicity at both high and low doses. Here, we study the influence of 10 µg/g (corresponding to the concentrations found in food products) and, for comparison, 60, 80 and 110 µg/g dietary ACR, on the fruit fly Drosophila melanogaster. We show that chronic administration of ACR affects lifespan, activity level and, most importantly, the daily and circadian pattern of locomotor activity of Drosophila. ACR-treated flies show well-defined and concentration-dependent symptoms of ACR neurotoxicity; a reduced anticipation of upcoming changes in light conditions and increased arrhythmicity in constant darkness. The results suggest that the rhythm-generating neural circuits of their circadian oscillator (biological clock) are sensitive to ACR even at low concentrations if the exposure time is sufficiently long. This makes the behavioural readout of the clock, the rhythm of locomotor activity, a useful tool for studying the adverse effects of ACR and probably other compounds.

Measuring honey bee feeding rhythms with the BeeBox, a platform for nectar foraging insects

In honey bees, most studies of circadian rhythms involve a locomotion test performed in a small tube, a tunnel, or at the hive entrance. However, despite feeding playing an important role in honey bee health or fitness, no demonstration of circadian rhythm on feeding has been performed until recently. Here, we present the BeeBox, a new laboratory platform for bees based on the concept of the Skinner box, which dispenses discrete controlled amounts of food (sucrose syrup) following entrance into an artificial flower. We compared caged groups of bees in 12 h-12 h light/dark cycles, constant darkness and constant light and measured average hourly syrup consumption per living bee. Food intake was higher in constant light and lower in constant darkness; mortality increased in constant light. We observed rhythmic consumption with a period longer than 24 h; this is maintained in darkness without environmental cues, but is damped in the constant light condition. The BeeBox offers many new research perspectives and numerous potential applications in the study of nectar foraging animals.

Circadian rhythm in pro-arrhythmic activity of pulmonary vein cardiomyocytes

Abstract Introduction Cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of ectopic activity driving atrial fibrillation (AF) and episodes of AF in patients are more prevalent at night. While it is known that circadian clocks within the heart regionally control 24-hour variation in pacemaking and ventricular repolarisation, the mechanisms underlying the nighttime preponderance in AF are unknown. Purpose This study addresses the hypothesis that circadian rhythms exist in the pro-arrhythmic activity of PV cardiomyocytes. Methods Male Wistar rats maintained in a 24-hour cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12) were subject to terminal general anaesthesia (140 mg/kg Na pentobarbital i.p.) at ZT=0, 6, 12 or 18 hr and the hearts removed. RNA was extracted from left atrial (LA) appendage (LAA) and proximal PV at the LA/PV junction (3 rats per ZT) and RNA sequencing conducted. Reads were mapped to the rat genome, counts normalised and models in which ZT was or was not included as a factor compared (Likelihood Ratio Test, adjusted-P&amp;lt;0.01). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=308) and the LAA (n=264) (N=74 rats). The effects of noradrenaline (NA, 1 µM) and acetylcholine (ACh, 1 µM) were examined. Data were plotted against the ZT of the time of recording and fitted to a sine wave to establish circadian rhythmicity (P&amp;lt;0.05, extra-sum-of-squares F-test). The effect of ≥24 hr constant dark in the period immediately before experiment was examined. Data are reported as mean ± standard error. Results The expression of 1368 genes varied significantly with ZT, including circadian clock components (e.g. Bmal1, Per1). PV cells were larger than LAA cells (73±1.6 pF vs 53±1.3 pF, P&amp;lt;0.0001) and had more depolarised resting membrane potential (-69±0.2 mV vs -72±0.1 mV, P&amp;lt;0.0001). Both cell types showed circadian variation in action potential duration at 90% repolarisation (APD90) and frequency of pro-arrhythmic activity. Pro-arrhythmic activity was greatest in PV cells and the frequency was greater during the rest phase (ZT0-12) in both cell types. In contrast, the circadian rhythm in APD90 differed between cell type, with the longest APD­­90 recorded during the active phase in PV cells (ZT12-24) but during the rest phase in LAA cells. Pro-arrhythmic activity was increased by NA and decreased by ACh in both cell types with maximal effect during the rest phase. Conclusion Circadian variation in APD90 and proarrhythmic-activity has been demonstrated in isolated proximal PV and LAA cardiomyocytes, with differences in rhythm between the two cell types. RNA sequencing suggests the presence of peripheral clocks in LAA and PV cardiomyocytes. Understanding these mechanisms is important in development of future therapeutic options for AF.

Influence of various light regimes on some circadian rhythms of transplantable melanoma B16

BACKGROUND: Today it is known that disturbance of the lighting regime, expressed both by lengthening of the light period and its reduction, can not only affect the regulation of circadian rhythms of the organism, but also contributes to the initiation of neoplasm growth. AIM: The aim of the study was to investigate circadian rhythmicity of melatonin level, some micromorphometric indices of tumor cells and expression of genes Bmal1, Clock and Per2 in them in mice with transplanted melanoma B16. MATERIALS AND METHODS: The study was conducted on 75 mice with subcutaneously transplanted melanoma B16, divided into 3 groups: control group, in which animals were kept under fixed light regime (light/darkness 10/14 hours with light on at 8:00 and off at 18:00), group under dark deprivation conditions, with animals kept under constant light 24 hours a day and group, in which animals were kept in constant darkness. The duration of the experiment was 2 weeks. RESULTS: It was shown that under conditions of fixed light there are reliable circadian rhythms for all studied parameters, except for the nuclear-cytoplasmic ratio, the circadian rhythms of which was not revealed in any group. Constant darkness leads to rearrangement of all identified rhythms, and constant light causes destruction of all circadian rhythms except the Clock expression rhythm. CONCLUSIONS: This study shows that light disturbances, whether constant light or constant darkness, lead to significant changes in the structure of the studied circadian rhythms.

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.