Developmental patterning of peptide transcription in the central circadian clock in both sexes.

Introduction: Neuropeptide signaling modulates the function of central clock neurons in the suprachiasmatic nucleus (SCN) during development and adulthood. Arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) are expressed early in SCN development, but the precise timing of transcriptional onset has been difficult to establish due to age-related changes in the rhythmic expression of each peptide. Methods: To provide insight into spatial patterning of peptide transcription during SCN development, we used a transgenic approach to define the onset of Avp and Vip transcription. Avp-Cre or Vip-Cre males were crossed to Ai9+/+ females, producing offspring in which the fluorescent protein tdTomato (tdT) is expressed at the onset of Avp or Vip transcription. Spatial patterning of Avp-tdT and Vip-tdT expression was examined at critical developmental time points spanning mid-embryonic age to adulthood in both sexes. Results: We find that Avp-tdT and Vip-tdT expression is initiated at different developmental time points in spatial subclusters of SCN neurons, with developmental patterning that differs by sex. Conclusions: These data suggest that SCN neurons can be distinguished into further subtypes based on the developmental patterning of neuropeptide expression, which may contribute to regional and/or sex differences in cellular function in adulthood.

Somatostatin regulates central clock function and circadian responses to light.

Daily and annual changes in light are processed by central clock circuits that control the timing of behavior and physiology. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus processes daily photic inputs and encodes changes in day length (i.e., photoperiod), but the SCN circuits that regulate circadian and photoperiodic responses to light remain unclear. Somatostatin (SST) expression in the hypothalamus is modulated by photoperiod, but the role of SST in SCN responses to light has not been examined. Our results indicate that SST signaling regulates daily rhythms in behavior and SCN function in a manner influenced by sex. First, we use cell-fate mapping to provide evidence that SST in the SCN is regulated by light via de novo Sst activation. Next, we demonstrate that Sst  -/- mice display enhanced circadian responses to light, with increased behavioral plasticity to photoperiod, jetlag, and constant light conditions. Notably, lack of Sst  -/- eliminated sex differences in photic responses due to increased plasticity in males, suggesting that SST interacts with clock circuits that process light differently in each sex. Sst  -/- mice also displayed an increase in the number of retinorecipient neurons in the SCN core, which express a type of SST receptor capable of resetting the molecular clock. Last, we show that lack of SST signaling modulates central clock function by influencing SCN photoperiodic encoding, network after-effects, and intercellular synchrony in a sex-specific manner. Collectively, these results provide insight into peptide signaling mechanisms that regulate central clock function and its response to light.

Fluorescent Reporters for Studying Circadian Rhythms in Drosophila melanogaster.

Circadian rhythms are daily oscillations in physiology and gene expression that are governed by a molecular feedback loop known as the circadian clock. In Drosophila melanogaster, the core clock consists of transcription factors clock (Clk) and cycle (cyc) which form protein heterodimers that activate transcription of their repressors, period (per) and timeless (tim). Once produced, protein heterodimers of per/tim repress Clk/cyc activity. One cycle of activation and repression takes approximately ("circa") 24-h ("diem") and repeats even in the absence of external stimuli. The circadian clock is active in many cells throughout the body; however, tracking it dynamically represents a challenge. Traditional fluorescent reporters are slowly degraded and consequently cannot be used to assess dynamic temporal changes exhibited by the circadian clock. The use of rapidly degraded fluorescent protein reporters containing destabilized GFP (dGFP) that report transcriptional activity in vivo at a single-cell level with ~1-h temporal resolution can circumvent this problem. Here we describe the use of circadian clock reporter strains of Drosophila melanogaster, ClockPER and ClockTIM, to track clock transcriptional activity using the intestine as a tissue of interest. These methods may be extended to other tissues in the body.

BMAL1 Regulates the Daily Timing of Colitis.

Many physiological functions exhibit circadian rhythms: oscillations in biological processes that occur in a 24-hour period. These daily rhythms are maintained through a highly conserved molecular pacemaker known as the circadian clock. Circadian disruption has been proposed to cause increased risk of Inflammatory Bowel Disease (IBD) but the underlying mechanisms remain unclear. Patients with IBD experience chronic inflammation and impaired regeneration of intestinal epithelial cells. Previous animal-based studies have revealed that colitis models of IBD are more severe in mice without a circadian clock but the timing of colitis, and whether its inflammatory and regenerative processes have daily rhythms, remains poorly characterized. We tested circadian disruption using Bmal1-/- mutant mice that have a non-functional circadian clock and thus no circadian rhythms. Dextran Sulfate Sodium (DSS) was used to induce colitis. The disease activity of colitis was found to exhibit time-dependent variation in Bmal1+/+ control mice but is constant and elevated in Bmal1-/- mutants, who exhibit poor recovery. Histological analyses indicate worsened colitis severity in Bmal1-/- mutant colon, and colon infiltration of immune system cells shows a daily rhythm that is lost in the Bmal1-/- mutant. Similarly, epithelial proliferation in the colon has a daily rhythm in Bmal1+/+ controls but not in Bmal1-/- mutants. Our results support a critical role of a functional circadian clock in the colon which drives 24-hour rhythms in inflammation and healing, and whose disruption impairs colitis recovery. This indicates that weakening circadian rhythms not only worsens colitis, but delays healing and should be taken into account in the management of IBD. Recognition of this is important in the management of IBD patients required to do shift work.

Reduced VIP Expression Affects Circadian Clock Function in VIP-IRES-CRE Mice (JAX 010908).

Circadian rhythms are programmed by the suprachiasmatic nucleus (SCN), which relies on neuropeptide signaling to maintain daily timekeeping. Vasoactive intestinal polypeptide (VIP) is critical for SCN function, but the precise role of VIP neurons in SCN circuits is not fully established. To interrogate their contribution to SCN circuits, VIP neurons can be manipulated specifically using the DNA-editing enzyme Cre recombinase. Although the Cre transgene is assumed to be inert by itself, we find that VIP expression is reduced in both heterozygous and homozygous adult VIP-IRES-Cre mice (JAX 010908). Compared with wild-type mice, homozygous VIP-Cre mice display faster reentrainment and shorter free-running period but do not become arrhythmic in constant darkness. Consistent with this phenotype, homozygous VIP-Cre mice display intact SCN PER2::LUC rhythms, albeit with altered period and network organization. We present evidence that the ability to sustain molecular rhythms in the VIP-Cre SCN is not due to residual VIP signaling; rather, arginine vasopressin signaling helps to sustain SCN function at both intracellular and intercellular levels in this model. This work establishes that the VIP-IRES-Cre transgene interferes with VIP expression but that loss of VIP can be mitigated by other neuropeptide signals to help sustain SCN function. Our findings have implications for studies employing this transgenic model and provide novel insight into neuropeptide signals that sustain daily timekeeping in the master clock.

Circuit development in the master clock network of mammals.

Daily rhythms are generated by the circadian timekeeping system, which is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of mammals. Circadian timekeeping is endogenous and does not require exposure to external cues during development. Nevertheless, the circadian system is not fully formed at birth in many mammalian species and it is important to understand how SCN development can affect the function of the circadian system in adulthood. The purpose of the current review is to discuss the ontogeny of cellular and circuit function in the SCN, with a focus on work performed in model rodent species (i.e., mouse, rat, and hamster). Particular emphasis is placed on the spatial and temporal patterns of SCN development that may contribute to the function of the master clock during adulthood. Additional work aimed at decoding the mechanisms that guide circadian development is expected to provide a solid foundation upon which to better understand the sources and factors contributing to aberrant maturation of clock function.

Ontogeny of Circadian Rhythms and Synchrony in the Suprachiasmatic Nucleus.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism.SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN.

Hypothalamic expression of anorexigenic and orexigenic hormone receptors in obese females Neotomodon alstoni: effect of fasting.

Obesity is a world problem that requires a better understanding of its physiological and genetic basis, as well as the mechanisms by which the hypothalamus controls feeding behavior. The volcano mouse Neotomodon alstoni develops obesity in captivity when fed with regular chow diet, providing a novel model for the study of obesity. Females develop obesity more often than males; therefore, in this study, we analysed in females, in proestrous lean and obese, the differences in hypothalamus expression of receptors for leptin, ghrelin (growth hormone secretagogue receptor GHS-R), and VPAC, and correlates for plasma levels of total ghrelin. The main comparisons are between mice fed ad libitum and mice after 24 hours of fasting. Mice above 65 g body weight were considered obese, based on behavioral and physiological parameters such as food intake, plasma free fatty acids, and glucose tolerance. Hypothalamic tissue from obese and lean mice was analysed by western blot. Our results indicate that after ad libitum food access, obese mice show no significant differences in hypothalamic leptin receptors, but a significant increase of 60% in the GHS-R, and a nearly 62% decrease in VPAC2 was noted. After a 24-hour fast, plasma ghrelin increased nearly two fold in both lean and obese mice; increases of hypothalamic leptin receptors and GHS-R were also noted, while VPAC2 did not change significantly; levels of plasma free fatty acids were 50% less after fasting in obese than in lean animals. Our results indicate that in obese N. alstoni mice, the levels of orexigenic receptors in the hypothalamus correlate with overfeeding, and the fact that lean and obese females respond in different ways to a metabolic demand such as a 24-hour fast.

Obesity alters circadian behavior and metabolism in sex dependent manner in the volcano mouse Neotomodon alstoni.

The aim of the present study is to evaluate whether circadian locomotor activity, and the daily profile of plasma parameters related to metabolic syndrome (nutrients: glucose and triacylglycerides, and hormones: insulin and leptin), differ between male and female Neotomodon alstoni mice, both lean and obese. Young adult animals were captured in the field and kept at the laboratory animal facility. After 6 to 7 months feeding the animals ad libitum with a regular diet for laboratory rodents, 50-60% of mice became obese. Comparisons between sexes indicated that lean females were more active than males; however obese females reduced their nocturnal activity either in LD or DD, and advanced the phase of their activity-onset with respect to lights off. No differences in food intake between lean and obese mice, either during the day or night, were observed. Daily profiles of metabolic syndrome-related plasma parameters showed differences between sexes, and obesity was associated with increased values, especially leptin (500% in females and 273% in males) and insulin (150% in both females and males), as compared with lean mice. Our results indicate that lean mice display behavioral and endocrine differences between sexes, and obesity affects the parameters tested in a sex-dependent manner. The aforementioned leads us to propose N. alstoni, studied in captivity, could be an interesting model for the study of sex differences in the effects of obesity.