LD Cycle Research Articles

Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the abnormal expansion of CGG repeats in the fragile X mental retardation 1 (FMR1) gene. Many FXS patients experience sleep disruptions, and we sought to explore these symptoms along with the possible benefits of a scheduled feeding intervention using the Fmr1 knockout (KO) mouse model. These mutants displayed clear evidence for sleep and circadian disturbances including delay in the onset of sleep and fragmented activity rhythms with increases in cycle-to-cycle variability. The Fmr1 KO mice exhibited deficits in their circadian behavioral response to light with reduced masking, longer time to resetting to shifts in the LD cycle, altered synchronization to a skeleton photoperiod and lower magnitude light-induced phase shifts of activity rhythms. Investigation of the retinal input to the surprachiasmatic nucleus (SCN) with the neurotracer cholera toxin (β subunit) and quantification of the light-evoked cFos expression in the SCN revealed an abnormal retinal innervation of the SCN in the Fmr1 KO, providing a possible mechanistic explanation for the observed behavioral deficits. Interestingly, disruptions in social and repetitive behavior correlated with sleep duration and fragmentation. Understanding the nature of the deficits, we decided to apply a scheduled feeding regimen (6-hr/18-hr feed/fast cycle) as a circadian-based strategy to boast circadian rhythms independently of light. This intervention significantly improved the activity rhythms and sleep in the mutants. Strikingly, the scheduled feeding ameliorated social interactions and reduced repetitive behaviors as well as the levels of Interferon-gamma and Interleukin-12 in the Fmr1 KO mutants, suggesting that timed eating may be an effective way to reduce inflammation. Collectively, this work adds support to efforts to develop circadian based interventions to help with symptoms of neurodevelopmental disorders.

Effects of light regimes on circadian gene co‐expression networks in Arabidopsis thaliana

AbstractLight/dark (LD) cycles are responsible for oscillations in gene expression, which modulate several aspects of plant physiology. Those oscillations can persist under constant conditions due to regulation by the circadian oscillator. The response of the transcriptome to light regimes is dynamic and allows plants to adapt rapidly to changing environmental conditions. We compared the transcriptome of Arabidopsis under LD and constant light (LL) for 3 days and identified different gene co‐expression networks in the two light regimes. Our studies yielded unforeseen insights into circadian regulation. Intuitively, we anticipated that gene clusters regulated by the circadian oscillator would display oscillations under LD cycles. However, we found transcripts encoding components of the flavonoid metabolism pathway that were rhythmic in LL but not in LD. We also discovered that the expressions of many stress‐related genes were significantly increased during the dark period in LD relative to the subjective night in LL, whereas the expression of these genes in the light period was similar. The nocturnal pattern of these stress‐related gene expressions suggested a form of “skotoprotection.” The transcriptomics data were made available in a web application named Cyclath, which we believe will be a useful tool to contribute to a better understanding of the impact of light regimes on plants.

Restoration of sleep-wake behavior following short photoperiod exposure in ventral subicular lesioned male Wistar rats: A 24-h sleep-wake electroencephalographical study.

The ventral subiculum regulates emotion, stress responses, and spatial and social cognition. In our previous studies, we have demonstrated anxiety- and depression-like symptoms, deficits in spatial and social cognition in ventral subicular lesioned (VSL) rats, and restoration of affective and cognitive behaviors following photoperiod manipulation (short photoperiod regime, SPR; 6:18 LD cycle). In the present study, we have studied the impact of VSL on sleep-wake behavioral patterns and the effect of SPR on sleep-wakefulness behavior. Adult male Wistar rats subjected to VSL demonstrated decreased wake duration and enhanced total sleep time due to increased non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS). Power spectral analysis indicated increased delta activity during NREMS and decreased sigma band power during all vigilance states. Light is one of the strongest entrainers of the circadian rhythm, and its manipulation may have various physiological and functional consequences. We investigated the effect of 21-day exposure to SPR on sleep-wakefulness (S-W) behavior in VSL rats. We observed that SPR exposure restored S-W behavior in VSL rats, resulting in an increase in wake duration and a significant increase in theta power during wake and REMS. This study highlights the crucial role of the ventral subiculum in maintaining normal sleep-wakefulness patterns and highlights the effectiveness of photoperiod manipulation as a non-pharmacological treatment for reversing sleep disturbances reported in mood and neuropsychiatric disorders like Alzheimer's disease, bipolar disorder, and major depressive disorder, which also involve alterations in circadian rhythm.

Circadian rhythm and surface activity in soil-dwelling caecilians (Amphibia: Gymnophiona)

The degree to which burrowing, soil-dwelling caecilian amphibians spend time on the surface is little studied, and circadian rhythm has not been investigated in multiple species of this order or by manipulating light–dark cycles. We studied surface-activity rhythm of the Indian caecilians Ichthyophis cf. longicephalus and Uraeotyphlus cf. oxyurus (Ichthyophiidae) and Gegeneophis tejaswini (Grandisoniidae), under LD, DD and DL cycles. We examined daily surface activity and the role of light–dark cycles as a zeitgeber. All three species were strictly nocturnal and G. tejaswini displayed the least surface activity. Four out of thirteen individuals, two I. cf. longicephalus, one G. tejaswini and one U. cf. oxyurus, displayed a more or less distinct surface-activity rhythm in all three cycles, and for the nine other animals the activity patterns were not evident. An approximately 24 h free-run period was observed in the three species. When the light–dark cycle was inverted, surface activity in the three species shifted to the dark phase. The findings of this study suggest that caecilians have a weak circadian surface-activity rhythm, and that the absence of light can act as a prominent zeitgeber in these burrowing, limbless amphibians.

Design of an optical system equipped with blue LEDs for the irradiation of Drosophila melanogaster cultures

Drosophila melanogaster, better known as the fruit fly, has become a widely used model organism that has allowed us to understand many biological behaviors, from sleep to neurological diseases, behavioral patterns, reproduction, and the circadian cycle, which coordinates biological rhythms in a 24-hour daily cycle through its main Zeitgerber, light, especially blue light. Therefore, the aim of this work was to build an optical setup with a hexagonal design that allowed a large number of Drosophila melanogaster cultures to be irradiated homogeneously with blue light simultaneously. This array can cover an illuminance range from 0 to approximately 600 lux by applying a current variation from 0 to approximately 1 A. It also has a real-time timer to turn the lights on and off, programmed in a 12:12 LD cycle for 24 hours. The optical setup with its unique design can become a very useful tool for developing experiments and understanding paradigms related to blue light at genetic, behavioral and neuronal levels, among others that are still unanswered.

Predatory Performance of <i>Microvelia douglasi</i> Scott (Hemiptera: Veliidae) with Reference to Diel Periodicity

The present work centers on the predatory performance of Microvelia douglasi adults with reference to diel periodicity. This experiment attempts to determine on whether the foraging efficiency was more at diurnal or nocturnal period, and was there an endogenous rhythm available within them to activate foraging response. The study was conducted in the laboratory for 24 hr with an interval of every three hr. The experiment was divided into Phase I (LD 12:12) and Phase II (DL 12:12). The predatory efficiency of M. douglasi adults was investigated on the first and second instars of Anopheles stephensi at prey densities of 25 and 50, and the experiment was conducted separately for male, female, and for both male and female, in 500 mℓ and 1000 mℓ containers. The bugs showed predatory activity both in diurnal and nocturnal periods. In LD cycle, maximum predatory activity was at 15:00 hr by the female bugs, and a total of 350.0 An. stephensi larvae were predated with 144.6 and 205.4 prey predated at 25 and 50 prey density, respectively. The male bugs predated 110.4 prey, and their response was less than that of females, which showed the highest rate of predation as they predated 129.4 prey. The prey predated when both male and female were put together was 110.2. In DL cycle, maximum predatory activity occurred at 24:00 hr again by the female bugs, and a maximum of 327.8 larval instars were predated with 153.4 and 174.4 prey predated at 25 and 50 prey density, respectively. Female bugs predated (121.2) more prey than male (99.4). However, the prey predated when both male and female were put together was 107.2, which was higher than prey predated by male. In LD cycle, the bugs predated more first instar (186.0) than the second instar (164.0), and in DL cycle, there was not much difference as 163.2 and 164.6 first and second instar, respectively were predated. Overall, the bugs showed more predatory activity during light than in dark, though natural light was changed to dark and dark to light. Predator’s sex, prey size, and different photoperiods testified the predatory performance of M. douglasi, and it was noted that the cumulative interactions of these three parameters were significant. The photoperiods were highly significant. Relatively high statistical significance was also derived in the interaction between the prey size and photoperiod. There was no statistical significance between predator’s sex and prey size and predators’ sex and photoperiod, and when all three parameters interacted, very less significance occurred.

Homeostatic crosstalk among gut microbiome, hypothalamic and hepatic circadian clock oscillations, immunity and metabolism in response to different light-dark cycles: a multi-omics study.

The accelerated pace of life at the present time has resulted in tremendous alterations in living patterns. Changes in diet and eating patterns, in particular, coupled with irregular light-dark cycles (LD) will further induce circadian misalignment and lead to disease. Emerging data has highlighted the regulatory effects of diet and eating patterns on the host microbe interactions with the circadian clock (CC), immunity, and metabolism. Herein, we studied how LD cycles regulate the homeostatic crosstalk among the gut microbiome (GM), hypothalamic and hepatic CC oscillations, and immunity and metabolism using multi-omics approaches. Our data demonstrated that central CC oscillations lost rhythmicity under irregular LD cycles, but LD cycles had minimal effects on diurnal expression of peripheral CC genes in the liver including Bmal1. We further demonstrated that the GM could regulate hepatic circadian rhythms under irregular LD cycles, the candidate bacteria including Limosilactobacillus, Actinomyces, Veillonella, Prevotella, Campylobacter, Faecalibacterium, Kingella, Clostridia vadinBB60, Veillonella. A comparative transcriptomic study of innate immune genes indicated that different LD cycles had varying effects on immune functions, while irregular LD cycles had greater impacts on hepatic innate immune functions than those in the hypothalamus. Extreme LD cycle alterations (LD0/24 and LD24/0) had worse impacts than slight alterations (LD8/16 and LD16/8), and led to gut dysbiosis in mice receiving antibiotics. Metabolome data also demonstrated that hepatic tryptophan metabolism mediated the homeostatic crosstalk among GM-liver-brain axis in response to different LD cycles. These research findings highlighted that GM could regulate immune and metabolic disorders induced by circadian dysregulation. Further, the data provided potential targets for developing probiotics for individuals with circadian disruption such as shift workers. This article is protected by copyright. All rights reserved.

Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus

ABSTRACT Circadian rhythms of physiology, behavior, and metabolism have an endogenous 24 h period that synchronizes with environmental cycles of light/dark and food availability. Alterations in light cycles are stressful and disrupt such diurnal oscillations. Recently, we witnessed a sudden rise in studies describing the mechanisms behind the interaction between the key characteristics of mitochondrial functions, peripheral clocks, and stress responses. To our knowledge, there is no study in the suprachiasmatic nuclei (SCN) describing the dysregulated mitochondrial bioenergetics under abnormal lighting conditions, which is common in today’s modern world. Thus, we aimed to investigate the existence of daily changes in mitochondrial bioenergetics (respiratory control rate, RCR), mitochondrial abundance (mtDNA/nDNA), plasma corticosterone, and to test whether disturbances in the lighting conditions might influence such rhythms. To confirm this, mice were sacrificed, mitochondria were isolated from the suprachiasmatic nuclei in the brain and blood was collected, every 3 h at various time points zeitgeber time/circadian time, (0, 3, 6, 9, 12, 15, 18, 21, and 24 h) under 12:12 h light-dark (LD, 150 lux L: 0 lux D) cycle and chronic artificial dim lighting (LL, 5 lux: 5lux) conditions, of a 24 h period, respectively. Our results demonstrate the existence of robust daily rhythmicity in RCR, mtDNA/nDNA and plasma CORT under a normal LD cycle. However, these rhythms were significantly disrupted and clock genes expressions were dysregulated under chronic dim LL. Furthermore, mitochondrial abundance was significantly reduced during LL compared to their numbers under LD cycle. Our data demonstrate that the circadian clock regulates mitochondrial functions (RCR, number), essential for accomplishing daily energy demands and supply by the SCN neurons. Abnormal light exposure dysregulates mitochondrial functions in the SCN and may alter metabolism, resulting in obesity, diabetes, and other metabolic disorders. Therefore, properly designing lighting conditions in workplaces is essential to mitigate the adverse consequences of light on humans.

Evidence for Sex-dependent Bmal1 Control of Mitochondrial Dynamics in Rat Kidney

The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The clock gene, Bmal1, is known to regulate mitochondrial O 2 consumption, but whether or how this occurs in the kidney has not been established. An estimated 95% of O 2 consumption in the kidney is linked to sodium transport. We developed and used a novel Bmal1 knockout rat on a Sprague Dawley background. Male Bmal1 -/- rats do not have the typical night-day difference in Na + excretion, while female rats maintain this pattern. We recently observed that ADP-dependent O 2 consumption in permeabilized kidney tissue was significantly higher in both sexes of Bmal1 -/- rats but only during the active/dark period. We also observed a significant increase in cytochrome c oxidase activity in Bmal1 -/- rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s·mg; p=0.0031, t-test). Since these effects were observed in both male and female rats, these changes in mitochondrial activity cannot explain the difference in diurnal sodium excretion. Since mitochondrial morphology can impact oxidative phosphorylation and energy availability, we hypothesized that Bmal1 regulates mitochondrial morphology in the kidney. Male and female global Bmal1 +/+ and Bmal1 -/- rats at 12-14 weeks of age were maintained in regular 12:12 LD cycles. Outer renal medullary tissue was dissected and prepared for mRNA expression and transmission electron microscopy (EM). For EM, tissues were obtained at light (ZT2-4) or dark (ZT14-16) periods to correspond with the minimum and maximum whole-body energy consumption and peak and trough Bmal1 protein expression. Digital droplet PCR mRNA expression was conducted from tissue collected at 4hr intervals over 24hrs. EM images appear to show an elongation of mitochondrial morphology in females but not male Bmal1 -/- during the active time of day. Mitofusion1 (Mfn1) and fission 1 (Fis1) are genes that regulate mitochondrial structural dynamics. Mfn1 and Fis1 mRNA expression over 24hrs showed a significant interaction between genotype and time of day in male rats (p=0.004 and p=0.020, respectively, 2way ANOVA). In contrast, there were no significant differences in overall Mfn1 or Fis1 expression between female Bmal1 +/+ and Bmal1 -/- rats. Our findings demonstrate that Bmal1 is crucial in maintaining mitochondrial respiration coupling and ATP generation in the kidney. Furthermore, we suggest that female Bmal1 -/- rats can better compensate for impaired respiration by mitochondrial morphological changes when the molecular clock is dysfunctional. We further propose that the rhythmicity of Na + excretion in females, but not male, Bmal1 -/- rats is due to adjustments in mitochondrial morphology. T32 HL007457, P01HL136267, AHA 908953 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

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-1β), 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.

Brain-derived neurotrophic factor Val66Met polymorphism modulates the effects of circadian desynchronization on activity and sleep in male mice.

Understanding how environmental interact challenges with genetic predispositions modulate health and wellbeing is an important area of biomedical research. Circadian rhythms play an important role in coordinating the multitude of cellular and tissue processes that organisms use to predict and adapt to regular changes in the environment, and robust circadian rhythms contribute to optimal physiological and behavioral responses to challenge. However, artificial lighting and modern round-the-clock lifestyles can disrupt the circadian system, leading to desynchronization of clocks throughout the brain and body. When coupled with genetic predispositions, circadian desynchronization may compound negative outcomes. Polymorphisms in the brain-derived neurotrophic (BDNF) gene contribute to variations in neurobehavioral responses in humans, including impacts on sleep, with the common Val66Met polymorphism linked to several negative outcomes. We explored how the Val66Met polymorphism modulates the response to environmental circadian desynchronization (ECD) in a mouse model. ECD was induced by housing adult male mice in a 20 h light-dark cycle (LD10:10; 10 h light, 10 h dark). Sleep and circadian activity were recorded in homozygous (Met) mice and their wild-type (Val) littermates in a standard 24 h LD cycle (LD12:12), then again after 20, 40, and 60 days of ECD. We found ECD significantly affected the sleep/wake timing in Val mice, however, Met mice maintained appropriate sleep timing after 20 days ECD, but not after 40 and 60 days of ECD. In addition, the rise in delta power at lights on was absent in Val mice but was maintained in Met mice. To elucidate the circadian and homeostatic contribution to disrupted sleep, mice were sleep deprived by gentle handling in LD12:12 and after 20 days in ECD. Following 6 h of sleep deprivation delta power was increased for both Val and Met mice in LD12:12 and ECD conditions. However, the time constant was significantly longer in the Val mice during ECD compared to LD12:12, suggesting a functioning but altered sleep homeostat. These data suggest the Val66Met mutation is associated with an ability to resist the effects of LD10:10, which may result in carriers suffering fewer negative impacts of ECD.

Clock genes regulate mating activity rhythms in the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

Endogenous circadian rhythms result from genetically-encoded molecular clocks, whose components and downstream output factors cooperate to generate cyclic changes in activity. Mating is an important activity of mosquitoes, however, the key aspects of mating rhythm patterns and their regulatory mechanisms in two vector mosquito species, Aedes albopictus and Culex quinquefasciatus, remain unclear. We determined and compared the diel mating activity rhythms of these two mosquito species and discovered that Ae. albopictus had mating peaks in the light/dark transition periods (ZT0-3 and ZT9-12), while Cx. quinquefasciatus only had a mating peak at ZT12-15. Knockouts of the clock (clk) orthologous genes (Aalclk and Cxqclk) resulted in phase delay or phase reversal of the mating peaks in Ae. albopictus and Cx. quinquefasciatus, respectively. In addition, the temporal expression pattern of the desaturase orthologous genes, desat1, in both mosquito species was also different in respective wild-type strains and showed phase changes similar to the mating rhythms in clk mutant strains. Inhibition of desat1 expression resulted in decreased mating activity in male mosquitoes of both species but not females. In addition, desat1 regulated cuticular hydrocarbons' synthesis in both species. Silencing desat1 in male Ae. albopictus resulted in decreases of nonadecane and tricosane, which promoted mating, with concomitant increases of heptacosane, which inhibited mating. Silencing desat1 in male Cx. quinquefasciatus also resulted in decreases of tricosane, which promoted mating. These results suggest that Aalclk and Cxqclk have significant roles in the mating activity rhythms in both Ae. albopictus and Cx. quinquefasciatus by regulating the temporal expression of the desat1 gene under LD cycles, which affects sex pheromone synthesis and mating. This work provides insights into the molecular regulatory mechanism of distinct mating rhythm of Ae. albopictus and Cx. quinquefasciatus and may provide a basis for the control of these two important vector mosquitoes.

Coupling Between Subregional Oscillators Within the Suprachiasmatic Nucleus Determines Free-Running Period in the Rat.

Rats housed in a 22-h light-dark cycle (11:11, T22) exhibit 2 distinct circadian locomotor activity (LMA) bouts simultaneously: one is entrained to the LD cycle and a second dissociated bout maintains a period greater than 24 h. These 2 activity bouts are associated with independent clock gene oscillations in the ventrolateral (vl-) and dorsomedial (dm-) suprachiasmatic nucleus (SCN), respectively. Previous results in our laboratory have shown that the vl- and dm-SCN oscillators are weakly coupled under T22 and that the period of the dissociated bout depends on coupling between the 2 subdivisions. Here, we sought to study the behavior of the T22 SCN pacemaker upon release into free-running conditions and compare it to the behavior of the system upon release from typical 24-h (12:12, T24) entrainment. T22-desynchronized rats or T24-entrained rats were released into constant darkness (DD). Activity rhythms in T22 rats rapidly resynchronized upon release into DD, and the free-running period (FRP) of the fused rhythm was longer than the FRP of T24 rats. We then asked whether the in vivo period changes were also present in the ex vivo SCN. Per1-luc rats were desynchronized in T22 for assessment of SCN Per1-luc ex vivo. Similar to behavioral FRP, the period of ex vivo SCN explanted from T22 rats was longer than that for T24 animals. Mathematical models supported the observed behavior of the dual oscillator system as the result of mutual coupling between the vl- and dm-SCN oscillators. This bidirectionally coupled model predicted both the FRP of the T22 system and its phase-shifting response to light. Together, these data support a model of pacemaker organization in which a light-sensitive vl-SCN oscillator is mutually coupled with a light-insensitive dm-SCN oscillator, and together they determine the period of the coupled system as a whole and its response to light pulses.

Abstract 094: Bmal1 Is Necessary For Food-Based Entrainment Of Blood Pressure And Temperature Rhythms In The Rat

Blood pressure (BP) follows a circadian rhythm, changing throughout the day. This change is crucial for cardiovascular health and in patient populations, as loss of these rhythms are associated with increased mortality and morbidity. Though BP rhythms have been recognized as important, the factors that control and entrain them are not well-described. Since the molecular clock is ubiquitously expressed in all cells and required for many circadian processes, we tested the hypothesis that the molecular clock contributes to the timing of BP rhythms in rats. Male and female rats with whole body loss of brain and muscle ARNTL-like protein ( Bmal1 ) and wild type (WT) controls (Sprague-Dawley background) were instrumented with telemeters in the abdominal aorta and placed in 12:12h light dark cycle (LD) to measure BP and core body temperature (TMP). Next, animals were placed in constant darkness (DD) for four weeks and then placed on a 12-hour time restricted feeding regimen (DD-TRF) for five days. Cosinor analysis was used to analyze BP and TMP rhythms. In a standard LD cycle, systolic BP (SBP) and TMP rhythms of both Bmal1 -knockout ( Bmal1- KO) and WT rats have similar amplitudes (2-way ANOVA for SBP and TMP, both p>0.05) which peak around zeitgeber time (ZT)18. During DD, WTs have SBP and TMP rhythms peak at circadian time (CT)19, while Bmal1 -KO rats have SBP and TMP rhythms that peak at CT 12. SBP amplitude is lower in all groups during DD compared to LD (WT males: 6.0±0.7 vs 3.8±0.7 mmHg; WT females: 7.2±0.9 vs 5.6±0.3 mmHg; KO males: 5.4±0.6 vs 4.7±0.4 mmHg; KO females: 6.2±0.6 vs 5.3±0.8 mmHg; 3-way ANOVA, p<0.05 for light cycle). During DD-TRF (CT 14.5 – 2.5), SBP and TMP rhythms of WTs peak at CT22. Bmal1 -KO rats have SBP and TMP rhythms that peak during CT16. SBP amplitude is lowered in Bmal1 -KO rats during DD-TRF compared to DD (KO males: 2.0±0.7 mmHg; KO females: 3.8±0.6 mmHg) but not in WT rats (WT males: 3.8±0.3 mmHg; WT females: 5.1±0.4 mmHg; 3-way ANOVA, p<0.05 for feeding time, sex, and feeding time*genotype). These data suggest that the molecular clock component Bmal1 is necessary for food-based entrainment of SBP and temperature rhythms in the absence of a regular light cue. Food, light cycle, and the molecular clock may all contribute to the timing and amplitude of SBP and TMP rhythms.

Strain and Age Dependent Entrainable Range of Circadian Behavior in C57BL/6 and BALB/c Mice

The mammalian circadian system has a plasticity in a certain range, rather than a strict 24-hour cycle, with considerable variations among species, strains, and ages. As the most widely used mouse strains in circadian research, C57BL/6 and BALB/c mice were well known to have different internal periods and responses to various non-24-hour light-dark cycles. However, their entrainable range of circadian behavior was not specifically studied, neither was the effect of aging. Besides, it is not well known if mice with appeared behavioral adaptation are really healthy. In the current study, we exposed C57BL/6 and BALB/c mice at 3 months and 18 months old to a series of short (T cycles < 24 h) and long (T cycles > 24 h) light-dark cycles. Wheel running activities were monitored continuously for calculation of the entrainable range and glucose homeostasis was investigated to reflect their health status. Our results showed that the range in both young and old C57BL/6 mice is between T23 and T26. By contrast, due to the strong adaptability to extreme LD cycles, the entrainable range on a circadian scale in both young and old BALB/c mice cannot be well determined. Despite the adaptation appeared at the behavioral level, glucose homeostasis revealed by glucose tolerance test and insulin tolerance test was impaired in mice upon T cycle treatment. In summary, our study explored the entrainment range in two popular mouse strains and suggested that behavioral adaptation may not well reflect their health status.

Sex differences in the diathetic effects of shift work schedules on circulating cytokine levels and pathological outcomes of ischemic stroke during middle age

Shift work is associated with increased risk for vascular disease, including stroke- and cardiovascular-related mortality. However, evidence from these studies is inadequate to distinguish the effect of altered circadian rhythms in isolation from other risk factors for stroke associated with shift work (e.g., smoking, poor diet, lower socioeconomic status). Thus, the present study examined the diathetic effects of exposure to shifted LD cycles during early adulthood on circadian rhythmicity, inflammatory signaling and ischemic stroke pathology during middle age, when stroke risk is high and outcomes are more severe. Entrainment of circadian activity was stable in all animals maintained on a fixed light:dark 12:12 cycle but was severely disrupted during exposure to shifted LD cycles (12hr advance/5d). Following treatment, circadian entrainment in the shifted LD group was distinguished by increased daytime activity and decreased rhythm amplitude that persisted into middle-age. Circadian rhythm desynchronization in shifted LD males and females was accompanied by significant elevations in circulating levels of the inflammatory cytokine IL-17A and gut-derived inflammatory mediator lipopolysaccharide (LPS) during the post-treatment period. Middle-cerebral artery occlusion, 3 months after exposure to shifted LD cycles, resulted in greater post-stroke mortality in shifted LD females. In surviving subjects, sensorimotor performance, assessed 2- and 5-days post-stroke, was impaired in males of both treatment groups, whereas in females, recovery of function was observed in fixed but not shifted LD rats. Overall, these results indicate that early exposure to shifted LD cycles promotes an inflammatory phenotype that amplifies stroke impairments, specifically in females, later in life.

Fragmented day-night cycle induces reduced light avoidance, excessive weight gain during early development, and binge-like eating during adulthood in mice

Fragmented day-night (FDN) cycles are environments in which multiple periods of light and dark alternate across a 24 h period. Exposure to FDN cycles disrupts circadian rhythms, resulting in period lengthening and alterations to mood in mice. A constant light environment, which also induces period lengthening, is linked to mood and metabolic disturbances and disruption to the development of the circadian clock. This study aims to determine how exposure to the FDN cycle impacts development in mice, with the hypothesis that there would be similar and adverse effects as observed in constant light conditions. Our study used CD-1 mice reared under the FDN cycle compared to the commonly used 12 h light: 12 h dark consolidated day-night cycle. During the first week of development, mouse pups reared under the FDN cycle gained bodyweight at a faster rate and did not avoid aberrant light exposure in comparison to 12:12 LD reared mouse pups. Developmental exposure to the FDN cycle lasted two weeks, and then mice were transferred to the 12:12 LD cycle, where after 2 weeks, bodyweight was similar between FDN reared and 12:12 LD reared mice at 1-month and 2-months old. When re-exposed to the FDN cycle during adulthood, FDN reared pups exhibited binge-like eating behaviors and reduced light avoidance. This study shows that the unnatural distribution of light and dark across the 24 h day can cause disruptions during early development that can reappear during adulthood when placed in the same stressful light-dark environment as adults.

High fat diet disrupts diurnal interactions between small intestinal host innate immune factor REG3γ and gut microbiota resulting in metabolic dysfunction

BackgroundCircadian rhythms are ubiquitous in nature, driving many bodily processes and behaviors, including sleep‐wake cycles and feeding patterns over 24 hours. We and others revealed gut microbes and their functional outputs also exhibit diurnal rhythms that are responsive to how much, what, and when food is consumed. These microbial cues are integrated into host circadian networks, serving as key regulators of metabolism. High fat (HF) diet disrupts diurnal microbial oscillations, impacting diet‐induced obesity (DIO). Apart from feeding, host factors that drive microbial oscillations, specifically in the small intestine, are complex and remain poorly understood. We hypothesized that HF diet disrupts coordination of diurnal rhythms between host‐derived antimicrobial peptides, particularly the host C‐type lectin Regenerating islet‐derived 3 gamma(Reg3γ), and gut microbial community membership, contributing to DIO and metabolic dysfunction.ResultsDistal ileal tissue and luminal contents were collected every 4 hours over a 12:12 LD cycle from regular chow (RC) vs. HF‐fed germ‐free (GF) and conventionally raised (CONV) C57Bl/6 age and sex‐matched mice. Ileal tissue gene expression analysis reveals diurnal Reg3γ expression is only observed in RC‐fed, but not HF‐fed, CONV mice. Illumina MiSeq 16S rRNA gene amplicon sequencing of ileal luminal contents indicates that HF diet significantly shifts microbial community membership with a corresponding reduction in oscillations relative to RC. Specific Lactobacillaceae bacteria selected by RC oscillate and exhibit positive correlation with Reg3γ expression, while HF promotes expansion of Clostridiales bacteria that negatively correlate with Reg3γ. Using both in vitro intestinal organoid and in vivomonoassociation of GF mice, we identified that exposure to bacterial strains representative of those selected by RC or HF diet elicit a bi‐directional interaction with Reg3γ; only RC‐driven Lactobacillus rhamnosus GG (LGG) induces diurnal Reg3γ expression, suggesting a bacteria‐specific effect. While dietary composition remains the primary driver of microbial oscillators, host factors such as Reg3γ provide secondary cues to drive abundance and oscillation of key gut microbes that are essential for host metabolic homeostasis.ConclusionsTogether, these results demonstrate transkingdom co‐evolved biological rhythms that are primarily influenced by diet, and reciprocal sensor‐effector signals between host and microbial components. The diurnal dynamics of host innate immune factors and specific diet‐induced ileal gut microbes are key for the maintenance of regional intestinal host‐microbe interactions and metabolic homeostasis.

Daylength Shapes Entrainment Patterns to Artificial Photoperiods in a Subterranean Rodent.

Photoperiodism plays an important role in the synchronization of seasonal phenomena in various organisms. In mammals, photoperiod encoding is mediated by differential entrainment of the circadian system. The limits of daily light entrainment and photoperiodic time measurement can be verified in organisms that inhabit extreme photic environments, such as the subterranean. In this experimental study, we evaluated entrainment of circadian wheel-running rhythms in South American subterranean rodents, the Anillaco tuco-tucos (Ctenomys aff. knighti), exposed to different artificial photoperiods, from extremely long to extremely short photophases (LD 21:3, LD 18:6, LD 15:9, LD 9:15, LD 6:18 and LD 3:21). Artificial photoperiods synchronized their activity/rest rhythms and clear differences occurred in (a) phase angles of entrainment relative to the LD cycle and (b) duration of the daily activity phase α. These photoperiod-dependent patterns of entrainment were similar to those reported for epigeous species. Release into constant darkness conditions revealed aftereffects of entrainment to different photoperiods, observed in α but not in the free-running period τ. We also verified if animals coming from summer and winter natural photoperiods entrained equally to the artificial photoperiods by evaluating their phase angle of entrainment, α and τ aftereffects. To this end, experimental animals were divided into "Matching" and "Mismatching" groups, based on whether the experimental photoperiod (short-day [L < 12 h] or long-day [L > 12 h]) matched or not the natural photoperiod to which they had been previously exposed. No significant differences were found in the phase angle of entrainment, α and τ aftereffects in each artificial photoperiod. Our results indicate that the circadian clocks of tuco-tucos are capable of photoperiodic time measurement despite their natural subterranean habits and that the final entrainment patterns achieved by the circadian clock do not depend on the photoperiodic history.