Constant Darkness Research Articles

Heat-inactivated Streptococcus pneumoniae augments circadian clock gene expression in zebrafish cells.

The circadian clock is a cell-autonomous process that regulates daily internal rhythms by interacting with environmental signals. Reports across species show that infection can alter the expression of circadian genes; however, in teleosts, these effects are influenced by light exposure. Currently, no reports analyze the direct effects of bacterial exposure on the zebrafish clock. Using zebrafish Z3 cells, we demonstrate that exposure to heat-killed Streptococcus pneumoniae (HK-Spn) augments the expression of core repressive factors in a light- and time-dependent manner. In constant darkness, HK-Spn highly upregulated cry1a, per3, and per1b expression. In the presence of light, HK-Spn exposure rapidly and strongly upregulated per2 and cry1a, and this was proportionally increased with light intensity. The combinatorial effect of light and HK-Spn on per2 and cry1a was not duplicated with H2O2, a known byproduct of light exposure. However, the ROS inhibitor N-acetyl cysteine was sufficient to block HK-Spn augmentation of per2, cry1a, and per3. These findings demonstrate that exposure to an inactive bacteria influences the expression of zebrafish clock genes under different light conditions.

Exposure to constant artificial light alters honey bee sleep rhythms and disrupts sleep.

Artificial light at night (ALAN) changes animal behavior in multiple invertebrates and vertebrates and can result in decreased fitness. However, ALAN effects have not been studied in European honey bees (Apis mellifera), an important pollinator in which foragers show strong circadian rhythmicity. Colonies can be exposed to ALAN in swarm clusters, when bees cluster outside the nest on hot days and evenings, and, in limited cases, when they build nests in the open. We captured and maintained foragers in incubated cages and subjected them to constant light (LL), constant dark (DD), or 12h light:12h dark (LD) cycle, and observed them with infrared cameras. After 79h, there was a significant interaction of treatment and time because LL bees slept less. In detail, the bees maintained a regular sleep pattern for three days but LL bees showed a shift on the fourth day. LL bees had the largest sleep differences from LD controls, with trends of lengthened periods and increased phase misalignment from both LD and DD bees. LL bees also experienced significantly more disturbances from their nestmates and preferred to sleep in the lower portion of the cages, which had significantly lower light intensity. These findings suggest that ALAN can disrupt the sleep of honey bee foragers, which has implications for their behavior and overall colony health.

Circadian desynchrony in early life leads to enduring autistic-like behavioral changes in adulthood.

Circadian rhythm regulates a variety of biological processes in almost all living organisms. Modern lifestyles, e.g. transmeridian travel, night shift, light at night, etc., frequently disrupt people's regular sleep-wake cycles and create a misalignment (circadian desynchrony) between the natural environment and the endogenous body clock, and between different circadian oscillators within the body. The long-term consequences of circadian desynchrony on neurodevelopment and adult behavior remain elusive. Increasing clinical evidence supports a correlation between the disruption of the circadian system and neurodevelopmental disorders, such as autism spectrum disorders. Despite clinical correlations, experimental evidence is yet to establish a link between circadian disturbance in early life and adult behavioral changes. Here, using a "short day" (SD) mouse model, in which mice were exposed to an 8 h/8 h light/dark (LD) cycle mimicking a "shift work" schedule from gestation day 1 to postnatal day 21, we performed a battery of behavioral tests to assess changes in adult behaviors, including sociability, affective behaviors, stereotypy, cognition and locomotor functions. In contrast to the control mice kept in a 12 h/12 h LD cycle, the adult SD mice entrained to the 8 h/8 h LD cycle, but their free running rhythms remained normal in constant darkness. Interestingly, however, the SD mice displayed diminished sociability, a reduced preference for social novelty, excessive repetitive behaviors, and compromised cognitive functions, all of which resemble characteristics of autism-like behavioral alterations. In addition, the SD mice exhibited significant anxiety- and depressive-like behaviors and impaired motor functions. By western blotting and immunostaining analyses, hyperactivation of the mTORC1/S6K1 pathway was detected in multiple forebrain regions of SD mice. These findings underscore the enduring impact of early-life circadian disruption on neurochemical signaling and behavioral patterns into adulthood, highlighting a pivotal role for circadian regulation in neurodevelopment.

Microglia drive diurnal variation in susceptibility to inflammatory blood-brain barrier breakdown.

The blood-brain barrier (BBB) is critical for maintaining brain homeostasis but is susceptible to inflammatory dysfunction. While transporter-dependent efflux of some lipophilic substrates across the BBB shows circadian variation due to rhythmic transporter expression, basal transporter-independent permeability and leakage is nonrhythmic. Whether daily timing influences BBB permeability in response to inflammation is unknown. Here, we induced systemic inflammation through repeated LPS injections either in the morning (ZT1) or evening (ZT13) under standard lighting conditions; we then examined BBB permeability to a polar molecule that is not a transporter substrate, sodium fluorescein. We observed clear diurnal variation in inflammatory BBB permeability, with a striking increase in paracellular leak across the BBB specifically following evening LPS injection. Evening LPS led to persisting glia activation as well as inflammation in the brain that was not observed in the periphery. The exaggerated evening neuroinflammation and BBB disruption were suppressed by microglial depletion or through keeping mice in constant darkness. Our data show that diurnal rhythms in microglial inflammatory responses to LPS drive daily variability in BBB breakdown and reveal time of day as a key regulator of inflammatory BBB disruption.

Daily rhythms of locomotor activity and transcript levels of non-visual opsins in the brain of the blind Mexican cavefish (Astyanax mexicanus)

Most organisms possess endogenous circadian clocks that synchronise their physiology and behaviour with environmental cycles, with the light-dark (LD) cycle being the most potent synchronising signal. Consequently, it can be hypothesised that animals that have evolved in the dark, as in caves or deep sea, may no longer possess a functional light-entrained biological clock. In this research, the blind cavefish Astyanax mexicanus was selected as a model organism to investigate the potential effects of daily light conditions on the circadian timekeeping mechanisms. First, we focused on describing behavioural photic entrainment and the presence of a circadian endogenous rhythmicity by recording locomotor activity rhythms under different lighting regimes: LD 12:12, after a 6-h shift of LD, constant darkness (DD), and constant dim light (LLdim). Secondly, we aimed at characterising the mechanisms of photodetection by analysing the daily rhythms of expression of selected non-visual extraocular opsins (exo-rhod, opn3, rgra, rgrb, tmt1a and tmt1b) in the brain of this blind species using real-time quantitative PCR. Our results revealed that blind Mexican cavefish activity rhythms were entrained to the LD cycle, with a diurnal activity pattern that persisted in a circadian fashion under constant lighting conditions. Additionally, statistically significant daily variations and/or rhythms were observed in three out of the six non-visual opsin genes analysed (opn3, rgra and tmt1b), all of them displaying nocturnal acrophases. These findings suggest that daily rhythms in extraretinal non-visual opsins may be transducing daily photic cycles and contributing to the entrainment of locomotor activity and other light-synchronised rhythms in blind cavefish species.

Serum iron and transferrin saturation variation are circadian regulated and linked to the harmonic circadian oscillations of erythropoiesis and hepatic Tfrc expression in mice.

Serum iron has long been thought to exhibit diurnal variation and is subsequently considered an unreliable biomarker of systemic iron status. Circadian regulation (endogenous ~24-h periodic oscillation of a biologic function) governs many critical physiologic processes. It is unknown whether serum iron levels are regulated by circadian machinery; likewise, the circadian nature of key players of iron homeostasis is unstudied. Here we show that serum iron, transferrin saturation (TSAT), hepatic transferrin receptor (TFR1) gene (Tfrc) expression, and erythropoietic activity exhibit circadian rhythms. Daily oscillations of serum iron, TSAT, hepatic Tfrc expression, and erythropoietic activity are maintained in mice housed in constant darkness, where oscillation reflects an endogenous circadian period. Oscillations of serum iron, TSAT, hepatic Tfrc, and erythropoietic activity were ablated when circadian machinery was disrupted in Bmal1 knockout mice. Interestingly, we find that circadian oscillations of erythropoietic activity and hepatic Tfrc expression are maintained in opposing phase, likely allowing for optimized usage and storage of serum iron whilst maintaining adequate serum levels and TSAT. This study provides the first confirmatory evidence that serum iron is circadian regulated, discerns circadian rhythms of TSAT, a widely used clinical marker of iron status, and uncovers liver-specific circadian regulation of TFR1, a major player in cellular iron uptake.

Comparative Metabolomic Analysis Reveals the Impact of the Photoperiod on the Hepatopancreas of Chinese Grass Shrimp (Palaemonetes sinensis)

The photoperiod is a key environmental factor that in crustaceans influences development, feeding, and metabolism. In this study, liquid chromatography-tandem mass spectrometry was used to examine metabolic changes in Palaemonetes sinensis under different photoperiods. Our results showed that key metabolic pathways, such as linoleic acid metabolism, axon regeneration, pyrimidine metabolism, and cortisol synthesis, were significantly altered under both constant light (24L:0D) and constant darkness (0L:24D) compared with natural light conditions. The photoperiod notably affected the digestive and metabolic functions of P. sinensis. Most metabolic pathways were downregulated under full darkness and full light conditions, suggesting that inhibition of metabolism is a potential adaptive response. Furthermore, enzyme assays revealed significant variations in trypsin, lipase, and amylase activity across different photoperiods, highlighting the profound impact of light conditions on digestive functions. These findings suggest that extreme light conditions may negatively impact the metabolic and digestive functions of P. sinensis. This study provides new insights into the adaptive mechanisms of P. sinensis in response to photoperiod changes and offers valuable information for optimizing aquaculture practices to enhance the health and growth performance of this crustacean.

Integrity of the circadian clock determines regularity of high-frequency and diurnal LFP rhythms within and between brain areas.

Circadian clocks control most physiological processes of many species. We specifically wanted to investigate the influence of environmental and endogenous rhythms and their interplay on electrophysiological dynamics of neuronal populations. Therefore, we measured local field potential (LFP) time series in wild-type and Cryptochrome 1 and 2 deficient (Cry1/2-/-) mice in the suprachiasmatic nucleus and the nucleus accumbens under regular light conditions and constant darkness. Using refined descriptive and statistical analyses, we systematically profiled LFP time series activity. We show that both environmental and endogenous rhythms strongly influence the rhythmicity of LFP signals and their frequency components, but also shape neuronal patterns on much smaller time scales, as neuronal activity in Cry1/2-/- mice is significantly less regular but at each time more synchronous within and between brain areas than in wild-type mice. These results show that functional circadian rhythms are integral for both circadian and non-circadian coordination of neuronal ensemble dynamics.

Late-onset caloric restriction improves cognitive performance and restores circadian patterns of neurotrophic, clock and epigenetic factors in the hippocampus of male old rats.

Aging is a complex multifactorial process that results in a general functional decline, including cognitive impairment. Caloric restriction (CR) can positively influence the aging processes and delay cognitive decline. There is a rhythmic variation in memory and learning processes throughout the day, indicating the involvement of the circadian clock in the regulation of these processes. Despite growing evidence on the efficacy of CR, it has not yet been fully determined whether starting this strategy at an advanced age is beneficial for improving quality of life and eventually, for protection against age-related diseases. Here, we investigated the effect of late-onset CR on the temporal organization of the molecular clock machinery, molecules related to cognitive processes and epigenetic regulation, in the hippocampus of male old rats maintained under constant darkness conditions. Our results evidenced the existence of a highly coordinated temporal organization of Bmal1, Clock, Bdnf, Trkb, Dnmts, Sirt1, and Pgc-1α in the hippocampus of young adult rats. We observed that aging led to cognitive deficits and loss of circadian oscillations of all the above variables. Interestingly, CR restored circadian rhythmicity in all cases and, in addition, improved the cognitive performance of the old animals. This work would highlight the importance of the circadian clock and its synchronization with feeding signals, as the basis of the beneficial effects of CR. Thus, lifestyle modifications, such as CR, might be a powerful intervention to preserve hippocampal circadian organization and cognitive health during aging.

We prefer nighttime, they prefer daytime: Biological rhythm variability in cave‐dwelling whip spiders (Amblypygi: Charontidae) in the Neotropics

AbstractThe lack of understanding regarding how endogenous and behavioral factors affect the biological rhythms of amblypygid arachnids in cave environments underscores a gap in chronobiologic knowledge for this group. This study investigates the influence of specialization on subterranean habitats and the presence of biological rhythms on the locomotor activity patterns of the troglobitic and troglophilic species of the genus Charinus. Specimens collected from carbonate caves in Brazil were subjected to experimental treatments of constant light (LL), constant darkness (DD), and light–dark cycles (LD). The results revealed variations in the distribution of main periods among species, without a uniform pattern. Although some specimens showed greater variability in activity patterns in the DD and LL treatments, no significant differences were observed between troglobites and troglophiles. The lack of a clear distinction in rhythms between the two groups suggests the dynamic nature of circadian rhythms in these populations, where individual variations in activity patterns indicate this behavioral diversity. Additionally, intraspecific competition for food resources, probably intensified by the oligotrophic conditions of the cave environments, may play an important role in shaping these patterns and differences in activity phases. The presence of infradian rhythms and weak circadian rhythms in some individuals underscores the importance of considering non‐photic zeitgebers for a deeper understanding of these rhythms in cave organisms.

Clock Gene Expression in Eel Retina and Hypothalamus: Response to Photoperiod and Moonlight.

Assessment of the clock genes, Period (Per) 1, Per2, Per3, and Cryptochrome (Cry) 2, Cry3, and Cry4, can help better understand eel spawning ecology. In this study, the circadian rhythm and moonlight effects of these clock genes in the eel retina and hypothalamus were analyzed. We examined clock gene expression patterns under 12 h light:12 h darkness (12L12D), constant darkness (DD), and constant light (LL) conditions; under short photoperiod (SP; 9L15D) and long photoperiod (LP; 15L9D), and during the new moon (NM) and full moon in male eels. Per2 expression increased after sunrise, Cry2, and Cry4 expression increased around sunset, and Per1, Per3, and Cry3 expression increased before sunrise. Under SP conditions, oscillations of retinal Per3 and Cry4, which did not occur under LP conditions, were generated. In addition, retinal Cry4 oscillation was generated under NM conditions. These results suggest that the retina of the eel may play an important role in regulating circadian rhythm, and migration is initiated by the synchronization of clock genes by moonlight, suggesting that photic signals are closely related to the migratory activity of the eel.

7861 Estrogen Receptor Alpha Regulates the Sex Difference in Jet Lag in Mice

Abstract Disclosure: M.A. Venegas: None. N. Westray: None. J.S. Pendergast: None. The circadian system aligns 24-hour cycles of internal biological processes with the environmental light-dark cycle. Abrupt shifts in the timing of the light-dark cycle that occur during travel across time zones misalign internal circadian clocks with the environment and cause jet lag. The symptoms of jet lag subside when circadian clocks resynchronize to the new light-dark cycle. Female mice in proestrus resynchronize to shifted light-dark cycles faster than males. The objective of this study was to investigate the estrogen signaling mechanisms underlying this sex difference in jet lag in mice. We measured wheel-running activity rhythms in C57BL/6J male and female wild-type (WT) and ERα knockout (ERαKO) mice. Female WT mice had 2-fold greater daily activity than male mice. Disabling the function of ERα ablated the sex difference in daily activity; both male and female ERαKO mice had markedly reduced activity compared to WT mice. Next, we advanced the timing of the light-dark cycle by 6 hours to simulate eastward travel. Female WT mice resynchronized to the new light-dark cycle faster than WT males. This sex difference was abolished in ERαKO mice. The rate of resynchronization was slower in female ERaKO mice compared to WT females and was indistinguishable from males. We next investigated 2 putative mechanisms underlying the accelerated rate of resynchronization in WT females. First, mice with faster endogenous clocks resynchronize to new light-dark cycles faster. However, we found that the velocity of the endogenous clock (circadian period) did not differ between WT and ERαKO male and female mice. Second, mice with greater phase advances to light exposure in the late night also resynchronize faster. To determine the magnitude of phase advances, we housed mice in constant darkness and then exposed them to a 15-minute light pulse during the late subjective night. We found no differences in the magnitudes of phase shifts between WT and ERαKO male and female mice. Together these studies show that ERα regulates the rate of resynchronization to shifted light-dark cycles in female mice but does not do so by altering conventional circadian rhythm parameters. Uncovering the mechanisms underlying the sex difference in resynchronization to shifted light-dark cycles can be used to develop strategies to alleviate jet lag and shift work symptoms. Presentation: 6/3/2024

Characterization of Bipolaris bicolor germination: effects of a physical factor on fungal adaptability.

Studies on physiological responses to stimuli from physical factors are essential for understanding the dynamics of the microorganisms and higly important for the management of plant diseases. Besides, the development of an epidemiological model for pathogen populations requires studying their physiological responses to physical stimuli. The objective of this study was to evaluate the germination dynamics of spores from six isolates of Bipolaris bicolor under effects of light at 25°C. Suspensions of 1.6 × 105 conidia mL- 1 from the B. bicolor isolates were inoculated onto Petri dishes containing agar-water culture medium and incubated in a BOD chamber under two physical conditions: (a) constant darkness and (b) constant light for five hours. The study was conducted in a completely randomized design, with a 6 × 2 factorial arrangement (six B. bicolor isolates and two physical conditions) and five replications. The length of the germ tube was measured hourly. The constant darkness resulted in higher mean germ tube growth for the pathogen; however, differences in the final germination percentage were found among the isolates. The isolate F-24-02 exhibited the highest germination adaptability to constant darkness, presenting the longest germ tube length.

Photoperiod, food restriction and memory for objects and places in mice

The suprachiasmatic nucleus (SCN) contains a population of cell-autonomous circadian oscillators essential for entrainment to daily light–dark (LD) cycles. Synchrony among SCN oscillators is modified by photoperiod and determines functional properties of SCN clock cycling, including its amplitude, phase angle of entrainment, and free running periodicity (τ). For many species, encoding of daylength in SCN output is critical for seasonal regulation of metabolism and reproduction. C57BL/6 mice do not show seasonality in these functions, yet do show photoperiodic modulation of SCN clock output. The significance of this for brain systems and functions downstream from the SCN in these species is largely unexplored. C57BL/6 mice housed in a long-day photoperiod have been reported to perform better on tests of object, spatial and fear memory compared to mice in a standard 12 h photoperiod. We previously reported that encoding of photoperiod in SCN output, evident in τ in constant dark (DD), can be blocked by limiting food access to a 4 h mealtime in the light period. To determine whether this might also block the effect of long days on memory, mice entrained to 18 h:6 h (L18) or 6 h:18 h (L6) LD cycles were tested for 24 h object memory (novel object preference, NOP) and spatial working memory (Y-maze spontaneous alternation, SA), at 4 times of day, first with food available ad libitum and then during weeks 5–8 of daytime restricted feeding. Photoperiod modified τ as expected, but did not affect performance on the NOP and SA tests, either before or during restricted feeding. NOP performance did improve in the restricted feeding condition in both photoperiods, eliminating a weak time of day effect evident with food available ad-libitum. These results highlight benefits of restricted feeding on cognitive function, and suggest a dose–response relationship between photoperiod and memory, with no benefits at daylengths up to 18 h.

Capsaicin‐mediated repair of circadian disruption‐induced Intestinal barrier damage via the gut microbiota

AbstractCircadian clock serves as a regulator of gastrointestinal health, and the disruption of circadian rhythm may trigger the dysregulation of the intestinal barrier functions. This study investigated the effectiveness of capsaicin (CAP) in mitigating intestinal barrier dysfunction induced by circadian disruption in male SPF C57BL/6J mice. Our findings indicated that CAP supplementation attenuated abnormal weight gain, alterations in serum lipid levels, and misalignment in hepatic and colonic circadian clock gene expressions in mice caused by constant darkness (CD) treatment. The restoration of the intestinal barrier integrity was also observed through the increase in mucosal thickness and goblet cell numbers. Both the diversity and the circadian rhythm of gut microbiota were improved in CAP‐treated group. Correlation analysis indicated that CAP could modulate the gut microbiota by affecting intestinal barrier function and systemic metabolism in CD‐treated mice. These results highlight the potential of CAP as a functional food component in maintaining the intestinal barrier homeostasis through regulating gut microbiota.

Light sensitivity of the circadian system in the social Highveld mole-rat Cryptomys hottentotus pretoriae.

Highveld mole-rats (Cryptomys hottentotus pretoriae) are social rodents that inhabit networks of subterranean tunnels. In their natural environment, they are rarely exposed to light, and consequently their visual systems have regressed over evolutionary time. However, in the laboratory they display nocturnal activity, suggesting that they are sensitive to changes in ambient illumination. We examined the robustness of the Highveld mole-rat circadian system by assessing its locomotor activity under decreasing light intensities. Mole-rats were subjected to seven consecutive light cycles commencing with a control cycle (overhead fluorescent lighting at 150lx), followed by decreasing LED lighting (500, 300, 100, 10 and 1lx) on a 12h light:12h dark (L:D) photoperiod and finally a constant darkness (DD) cycle. Mole-rats displayed nocturnal activity under the whole range of experimental lighting conditions, with a distinct spike in activity at the end of the dark phase in all cycles. The mole-rats were least active during the control cycle under fluorescent light, locomotor activity increased steadily with decreasing LED light intensities, and the highest activity was exhibited when the light was completely removed. In constant darkness, mole-rats displayed free-running rhythms with periods (τ) ranging from 23.77 to 24.38 h, but was overall very close to 24 h at 24.07 h. Our findings confirm that the Highveld mole-rat has a higher threshold for light compared with aboveground dwelling rodents, which is congruent with previous neurological findings, and has implications for behavioural rhythms.

Maternal immune activation exerts long-term effects on activity and sleep in male offspring mice.

Exposure to infectious or non-infectious immune activation during early development is a serious risk factor for long-term behavioural dysfunctions. Mouse models of maternal immune activation (MIA) have increasingly been used to address neuronal and behavioural dysfunctions in response to prenatal infections. One commonly employed MIA model involves administering poly(I:C) (polyriboinosinic-polyribocytdilic acid), a synthetic analogue of double-stranded RNA, during gestation, which robustly induces an acute viral-like inflammatory response. Using electroencephalography (EEG) and infrared (IR) activity recordings, we explored alterations in sleep/wake, circadian and locomotor activity patterns on the adult male offspring of poly(I:C)-treated mothers. Our findings demonstrate that these offspring displayed reduced home cage activity during the (subjective) night under both light/dark or constant darkness conditions. In line with this finding, these mice exhibited an increase in non-rapid eye movement (NREM) sleep duration as well as an increase in sleep spindles density. Following sleep deprivation, poly(I:C)-exposed offspring extended NREM sleep duration and prolonged NREM sleep bouts during the dark phase as compared with non-exposed mice. Additionally, these mice exhibited a significant alteration in NREM sleep EEG spectral powerunder heightened sleep pressure. Together, our study highlights the lasting effects of infection and/or immune activation during pregnancy on circadian activity and sleep/wake patterns in the offspring.

Light and dopamine impact two circadian neurons to promote morning wakefulness

In both mammals and flies, circadian brain neurons orchestrate physiological oscillations and behaviors like wake and sleep-these neurons can be subdivided by morphology and by gene expression patterns. Recent single-cell sequencing studies identified 17 Drosophila circadian neuron groups. One of these includes only two lateral neurons (LNs), which are marked by the expression of the neuropeptide ion transport peptide (ITP). Although these two ITP+ LNs have long been grouped with five other circadian evening activity cells, inhibiting the two neurons alone strongly reduces morning activity, indicating that they also have a prominent morning function. As dopamine signaling promotes activity in Drosophila, like in mammals, we considered that dopamine might influence this morning activity function. Moreover, the ITP+ LNs express higher mRNA levels than other LNs of the type 1-like dopamine receptor Dop1R1. Consistent with the importance of Dop1R1, cell-specific CRISPR-Cas9 mutagenesis of this receptor in the two ITP+ LNs renders flies significantly less active in the morning, and exvivo live imaging shows Dop1R1-dependent cyclic AMP (cAMP) responses to dopamine in these two neurons. Notably, the response is more robust in the morning, reflecting higher morning Dop1R1 mRNA levels in the two neurons. As mRNA levels are not elevated in constant darkness, this suggests light-dependent upregulation of morning Dop1R1 transcript levels. Taken together with the enhanced morning cAMP response to dopamine, the data indicate how light and dopamine promote morning wakefulness in flies, mimicking the important effect of light on morning wakefulness in humans.

Non-chemical stresses do not strongly induce male offspring in Daphnia magna ascertained using the short-term juvenile hormone activity screening assay.

Juvenile hormone (JH), together with ecdysone, regulates molting, metamorphosis, growth, and reproduction in arthropods. The effects of its analogs used as insecticides on nontarget species are of concern. Since JH and JH analogs (JHAs) induce male offspring in daphnids, which generally reproduce by parthenogenesis, short-term JH activity screening assay (JHASA) using the male offspring ratio as an endpoint has been developed as a detection method for JHA. However, the production of male offspring is also induced by environmental stresses such as temperature, short-day length, overcrowding, and food limitation. Thus, it is vital to prevent non-chemical stresses from inducing male offspring during the test to detect chemicals with potential JH activity accurately. Therefore, we investigated the effects of temperature (low and high), hardness, high density with low feeding, and day length on male production utilizing JHASA. Male offspring were not strongly induced by any stresses in JHASA, although the male ratios of 4-12% were observed in the preculture under high density (≥70 daphnid/L) and constant darkness. The Clone A strain was relatively more sensitive to high density and day length compared with the strain from National Institute for Environmental Studies (NIES). The selection of strains that rarely produce males under non-chemical stresses and finding the culturing conditions for each strain appropriate for not-inducing male offspring are recommended to control and prevent male offspring induction during JHASA.

Abstract We114: Feeding Behavior Underlies the Circadian Rhythm in the Autonomic Input to the Heart and Heart Rate

Objective: The circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus drives circadian rhythms in feeding and activity. Several studies suggest the SCN regulates autonomic input to the heart to generate day-night rhythms in heart rate. Hypothesis: The circadian rhythm in feeding generates day-night rhythms in autonomic input to the heart and heart rate. Methodology: Mice (n=6/sex) were implanted with telemetry probes to continuously record heart rate, core body temperature, and activity in thermoneutral housing conditions. Mice were housed in a 12 h light: 12 h dark cycle (LD) with ad libitum access to food (ALF). In these conditions, mice primarily eat during the dark cycle. Mice were subjected to light phase time-restricted feeding (TRF) for 5 days. This forces the mice to eat during the light cycle. We quantified changes in autonomic input to the heart by injecting mice with the autonomic receptor inhibitors propranolol and methylatropine. To determine if circadian rhythms generate day-night rhythms in autonomic input to the heart, TRF mice were switched from LD to constant darkness (DD) and then returned to ALF in DD. Results: ALF mice housed in LD had 24-hour rhythms in autonomic input to the heart, heart rate, and activity that all peaked in alignment during the dark cycle. One day after starting TRF, the phase of the 24-hour rhythms in heart rate but not activity shifted by 8-10 hours to align with feeding during the light phase. Switching TRF mice in LD to DD did not alter the phase or amplitude of the day-night rhythms in autonomic input to the heart, heart rate, or activity. Returning the TRF mice in DD to ALF showed the autonomic input to the heart and heart rate re-aligned with the free running rhythm in activity. Conclusions: Day-night rhythms in heart rate and autonomic input to the heart align with activity in LD and DD in ad libitum fed but not time-restricted fed conditions. These data demonstrate that feeding behavior, not SCN-signaling or activity rhythms, drives day-night rhythms in autonomic input to the heart and heart rate.