Neuroendocrine effects of the duper mutation in Syrian hamsters: a role for Cryptochrome 1.

Molecular and physiological determinants of the timing of reproductive events, including the pre-ovulatory LH surge and seasonal fluctuations in fertility, are incompletely understood. We used the Cryptochrome 1-deficient duper mutant to examine the role of this core circadian clock gene in Syrian hamsters. We find that the phase of the LH surge and its stability upon shifts of the light: dark cycle are altered in duper mutants. The intensity of immunoreactive PER1 in GnRH cells of the preoptic area peaks earlier in the day in duper than wild type hamsters. We note that GnRH fibers coursing through the suprachiasmatic nucleus (SCN) contact vasopressin- and VIP-immunoreactive cells, suggesting a possible locus of circadian control of the LH surge. Unlike wild types, duper hamsters do not regress their gonads within 8 weeks of constant darkness, despite evidence of melatonin secretion during the subjective night. In light of the finding that the duper allele is a stop codon in Cryptochrome 1, our results suggest important neuroendocrine functions of this core circadian clock gene.

Adult Neurogenesis Is Altered by Circadian Phase Shifts and the Duper Mutation in Female Syrian Hamsters.

Cell birth and survival in the adult hippocampus are regulated by a circadian clock. Rotating shift work and jet lag disrupt circadian rhythms and aggravate disease. Internal misalignment, a state in which abnormal phase relationships prevail between and within organs, is proposed to account for adverse effects of circadian disruption. This hypothesis has been difficult to test because phase shifts of the entraining cycle inevitably lead to transient desynchrony. Thus, it remains possible that phase shifts, regardless of internal desynchrony, account for adverse effects of circadian disruption and alter neurogenesis and cell fate. To address this question, we examined cell birth and differentiation in the duper Syrian hamster (Mesocricetus auratus), a Cry1-null mutant in which re-entrainment of locomotor rhythms is greatly accelerated. Adult females were subjected to alternating 8 h advances and delays at eight 16 d intervals. BrdU, a cell birth marker, was given midway through the experiment. Repeated phase shifts decreased the number of newborn non-neuronal cells in WT, but not in duper hamsters. The duper mutation increased the number of BrdU-IR cells that stained for NeuN, which marks neuronal differentiation. Immunocytochemical staining for proliferating cell nuclear antigen indicated no overall effect of genotype or repeated shifts on cell division rates after 131 days. Cell differentiation, assessed by doublecortin, was higher in duper hamsters but was not significantly altered by repeated phase shifts. Our results support the internal misalignment hypothesis and indicate that Cry1 regulates cell differentiation. Phase shifts may determine neuronal stem cell survival and time course of differentiation after cell birth. Figure created with BioRender.

The duper mutation reveals previously unsuspected functions of Cryptochrome 1 in circadian entrainment and heart disease.

The Cryptochrome 1 (Cry1)-deficient duper mutant hamster has a short free-running period in constant darkness (τDD) and shows large phase shifts in response to brief light pulses. We tested whether this measure of the lability of the circadian phase is a general characteristic of Cry1-null animals and whether it indicates resistance to jet lag. Upon advance of the light:dark (LD) cycle, both duper hamsters and Cry1-/- mice re-entrained locomotor rhythms three times as fast as wild types. However, accelerated re-entrainment was dissociated from the amplified phase-response curve (PRC): unlike duper hamsters, Cry1-/- mice show no amplification of the phase response to 15' light pulses. Neither the amplified acute shifts nor the increased rate of re-entrainment in duper mutants is due to acceleration of the circadian clock: when mutants drank heavy water to lengthen the period, these aspects of the phenotype persisted. In light of the health consequences of circadian misalignment, we examined effects of duper and phase shifts on a hamster model of heart disease previously shown to be aggravated by repeated phase shifts. The mutation shortened the lifespan of cardiomyopathic hamsters relative to wild types, but this effect was eliminated when mutants experienced 8-h phase shifts every second week, to which they rapidly re-entrained. Our results reveal previously unsuspected roles of Cry1 in phase shifting and longevity in the face of heart disease. The duper mutant offers new opportunities to understand the basis of circadian disruption and jet lag.

Anatomical Methods to Study the Suprachiasmatic Nucleus.

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker. In order to examine mechanisms by which it keeps time, entrains to periodic environmental signals (zeitgebers), and regulates subordinate oscillators elsewhere in the brain and in the periphery, a variety of molecular methods have been applied. Multiple label immunocytochemistry and in situ hybridization provide anatomical insights that complement physiological approaches (such as ex vivo electrophysiology and luminometry) widely used to study the SCN.The anatomical methods require interpretation of data gathered from groups of individual animals sacrificed at different time points. This imposes constraints on the design of the experiments that aim to observe changes that occur with circadian phase in free-running conditions. It is essential in such experiments to account for differences in the periods of the subjects. Nevertheless, it is possible to resolve intracellular colocalization and regional expression of functionally important transcripts and/or their peptide products that serve as neuromodulators or neurotransmitters. Armed with these tools and others, understanding of the mechanisms by which the hypothalamic pacemaker regulates circadian function is progressing apace.

duper is a null mutation of Cryptochrome 1 in Syrian hamsters.

The duper mutation is a recessive mutation that shortens the period length of the circadian rhythm in Syrian hamsters. These animals show a large phase shift when responding to light pulses. Limited genetic resources for the Syrian hamster (Mesocricetus auratus) presented a major obstacle to cloning duper. This caused the duper mutation to remain unknown for over a decade. In this study, we did a de novo genome assembly of Syrian hamsters with long-read sequencing data from two different platforms, Pacific Biosciences and Oxford Nanopore Technologies. Using two distinct ecotypes and a fast homozygosity mapping strategy, we identified duper as an early nonsense allele of Cryptochrome 1 (Cry1) leading to a short, unstable protein. CRY1 is known as a highly conserved component of the repressive limb of the core circadian clock. The genome assembly and other genomic datasets generated in this study will facilitate the use of the Syrian hamster in biomedical research.

Circadian Function in Multiple Cell Types Is Necessary for Proper Timing of the Preovulatory LH Surge.

The timing of the preovulatory surge of luteinizing hormone (LH), which occurs on the evening of proestrus in female mice, is determined by the circadian system. The identity of cells that control the phase of the LH surge is unclear: evidence supports a role of arginine vasopressin (AVP) cells of the suprachiasmatic nucleus (SCN), but it is not known whether vasopressinergic neurons are necessary or sufficient to account for circadian control of ovulation. Among other cell types, evidence also suggests important roles of circadian function of kisspeptin cells of the anteroventral periventricular nucleus (AvPV) and gonadotropin-releasing hormone (GnRH) neurons of the preoptic area (POA), whose discharge is immediately responsible for the discharge of LH from the anterior pituitary. The present studies used an ovariectomized, estradiol-treated preparation to determine critical cell types whose clock function is critical to the timing of LH secretion. As expected, the LH surge occurred at or shortly after ZT12 in control mice. In further confirmation of circadian control, the surge was advanced by 2 h in tau mutant animals. The timing of the surge was altered to varying degrees by conditional deletion of Bmal1 in AVPCre, KissCreBAC, and GnRHCreBAC mice. Excision of the mutant Cnsk1e (tau) allele in AVP neurons resulted in a reversion of the surge to the ZT12. Conditional deletion of Bmal1 in Kiss1 or GnRH neurons had no noticeable effect on locomotor rhythms, but targeting of AVP neurons produced variable effects on circadian period that did not always correspond to changes in the phase of LH secretion. The results indicate that circadian function in multiple cell types is necessary for proper timing of the LH surge.

Suprachiasmatic function in a circadian period mutant: Duper alters light-induced activation of vasoactive intestinal peptide cells and PERIOD1 immunostaining.

Mammalian circadian rhythms are entrained by photic stimuli that are relayed by retinal projections to the core of the suprachiasmatic nucleus (SCN). Neuronal activation, as demonstrated by expression of the immediate early gene c-fos, leads to transcription of the core clock gene per1. The duper mutation in hamsters shortens circadian period and amplifies light-induced phase shifts. We performed two experiments to compare the number of c-FOS immunoreactive (ir) and PER1-ir cells, and the intensity of staining, in the SCN of wild-type (WT) and duper hamsters at various intervals after presentation of a 15-min light pulse in the early subjective night. Light-induced c-FOS-ir within 1 hr in the dorsocaudal SCN of duper, but not WT hamsters. In cells that express vasoactive intestinal peptide (VIP), which plays a critical role in synchronization of SCN cellular oscillators, light-induced c-FOS-ir was greater in duper than WT hamsters. After the light pulse, PER1-ir cells were found in more medial portions of the SCN than FOS-ir, and appeared with a longer latency and over a longer time course, in VIP cells of duper than wild-type hamsters. Our results indicate that the duper allele alters SCN function in ways that may contribute to changes in free running period and phase resetting.

Circadian Rhythms: Understanding the SCN Connectome.

A new study utilizes transgenic mice to elucidate the coupling between cells of a neuronal pacemaker that determines circadian period.

Timing in the Testis.

The testis provides not just one but several models of temporal organization. The complexity of its rhythmic function arises in part from its compartmentalization and diversity of cell types: not only does the testis produce gametes, but it also serves as the major source of circulating androgens. Within the seminiferous tubules, the germ cells divide and differentiate while in intimate contact with Sertoli cells. The tubule is highly periodic: a spermatogenic wave travels along its length to determine the timing of the commitment of spermatogonia to differentiate, the phases of meiotic division, and the rate of differentiation of the postmeiotic germ cells. Recent evidence indicates that oscillations of retinoic acid play a major role in determining periodicity of the seminiferous epithelium. In the interstitial space, Leydig cells produce the steroid hormones required both for the completion of spermatogenesis and the development and maintenance of male sexual characteristics throughout the body. This endocrine output also oscillates; although the pulse generator lies outside the gonad, the steroidogenic function of Leydig cells is tuned to a regular episodic input. While the oscillations of the intratubular and interstitial cells have multihour (ultradian) and multiday (infradian) periodicities, respectively, the functions of both compartments also display dramatic seasonal rhythms. Furthermore, circadian rhythms are evident in some of the cell types, although their amplitude and pervasiveness are not as great as in many other tissues of the same organism, and their detection may require methods that recognize the heterogeneity of the testis. This review examines the periodicity of testicular function along multiple time scales.

Phase resetting in duper hamsters: specificity to photic zeitgebers and circadian phase.

The duper mutation in Syrian hamsters shortens the free-running period of locomotor activity (τDD) to about 23 h and results in a type 0 phase-response curve (PRC) to 15-min light pulses. To determine whether exaggerated phase shifts are specific to photic cues and/or restricted to subjective night, we subjected hamsters to novel wheel confinements and dark pulses during subjective day. Small phase shifts elicited by the nonphotic cue were comparable in mutant and wild-type (WT) hamsters, but dark pulses triggered larger shifts in dupers. To assess further the effects of the duper mutation on light-dark transitions, we transferred hamsters between constant light (LL) and constant dark (DD) or between DD and LL at various circadian phases. Duper hamsters displayed significantly larger phase shifts than WT hamsters when transferred from LL to DD during subjective day and from DD to LL during subjective night. The variability of phase shifts in response to all light/dark transitions was significantly greater in duper hamsters at all time points. In addition, most duper hamsters, but none of the WTs, displayed transient ultradian wheel-running patterns for 5 to 12 days when transferred from light to dark at CT 18. The χ(2) periodogram and autocorrelation analyses indicate that these ultradian patterns differ from the disruption of rhythmicity by SCN lesions or exposure to constant bright light. We conclude that the duper mutation specifically amplifies phase shifts to photic cues and may destabilize coupling of circadian organization upon photic challenge due to weakened coupling among components of the circadian pacemaker. Mathematical modeling of the circadian pacemaker supports this hypothesis.

Effects of the duper mutation on responses to light: parametric and nonparametric responses, range of entrainment, and masking.

The duper mutation in Syrian hamsters shortens the free-running period (τDD) of locomotor activity by approximately 1 h when expressed on the wild-type background and by 2 h on the tau mutant background ("super duper"). In either case, duper markedly amplifies the phase response curve (PRC) of the light pulse. This work examined whether the duper mutation alters parametric as well as nonparametric properties, intensity thresholds, and noncircadian responses to light. Furthermore, it assessed the effects of duper on the range of entrainment and circadian aftereffects. In the first study, duper mutant and wild-type (wt) hamsters showed a similar intensity threshold for light-induced phase shifts. In the second, wt, tau mutant, and super duper hamsters were exposed to LD cycles whose period (T) progressively shortened. Regardless of whether the light phase was held at 50% of T or fixed at 3 h, super duper mutants entrained to a wider range of T cycles and showed aftereffects upon release into DD. In the third study, τLL was measured in mutant and wt hamsters that were maintained for 30-day intervals in constant light of progressively greater intensities. With increasing light intensity, the circadian period shortened in duper mutants. Circadian rhythms of super duper hamsters were disrupted at light intensities considerably below those that induced arrhythmicity in wt, tau heterozygote, or duper homozygote hamsters. In the fourth study, hamsters that were wt or homozygous for duper received two 15-min light pulses: the first at CT14 to CT16 or CT17 to CT19 and the second 2 h later. As expected, wt and duper mutants showed weak and strong resetting, respectively. Light pulses in early subjective night had an additive effect in mutant but not in wt hamsters, indicating that larger phase shifts of the pacemaker take longer to complete. Finally, super duper hamsters showed slightly but not significantly more negative masking than did wt or duper mutant hamsters. These results indicate that the duper mutation affects the properties of the central circadian pacemaker. The mutant allele affects not only the PRC but also parametric responses to light.

Central control of circadian phase in arousal-promoting neurons.

Cells of the dorsomedial/lateral hypothalamus (DMH/LH) that produce hypocretin (HCRT) promote arousal in part by activation of cells of the locus coeruleus (LC) which express tyrosine hydroxylase (TH). The suprachiasmatic nucleus (SCN) drives endogenous daily rhythms, including those of sleep and wakefulness. These circadian oscillations are generated by a transcriptional-translational feedback loop in which the Period (Per) genes constitute critical components. This cell-autonomous molecular clock operates not only within the SCN but also in neurons of other brain regions. However, the phenotype of such neurons and the nature of the phase controlling signal from the pacemaker are largely unknown. We used dual fluorescent in situ hybridization to assess clock function in vasopressin, HCRT and TH cells of the SCN, DMH/LH and LC, respectively, of male Syrian hamsters. In the first experiment, we found that Per1 expression in HCRT and TH oscillated in animals held in constant darkness with a peak phase that lagged that in AVP cells of the SCN by several hours. In the second experiment, hamsters induced to split their locomotor rhythms by exposure to constant light had asymmetric Per1 expression within cells of the middle SCN at 6 h before activity onset (AO) and in HCRT cells 9 h before and at AO. We did not observe evidence of lateralization of Per1 expression in the LC. We conclude that the SCN communicates circadian phase to HCRT cells via lateralized neural projections, and suggests that Per1 expression in the LC may be regulated by signals of a global or bilateral nature.

Animal care practices in experiments on biological rhythms and sleep: report of the Joint Task Force of the Society for Research on Biological Rhythms and the Sleep Research Society.

Many physiological and molecular processes are strongly rhythmic and profoundly influenced by sleep. The continuing effort of biological, medical, and veterinary science to understand the temporal organization of cellular, physiological, behavioral and cognitive function holds great promise for the improvement of the welfare of animals and human beings. As a result, attending veterinarians and IACUC are often charged with the responsibility of evaluating experiments on such rhythms or the effects of sleep (or its deprivation) in vertebrate animals. To produce interpretable data, animals used in such research must often be maintained in carefully controlled (often constant) conditions with minimal disruption. The lighting environment must be strictly controlled, frequent changes of cages and bedding are undesirable, and daily visual checks are often not possible. Thus deviations from the standard housing procedures specified in the Guide for the Care and Use of Laboratory Animals are often necessary. This report reviews requirements for experiments on biological rhythms and sleep and discusses how scientific considerations can be reconciled with the recommendations of the Guide.

Does the precision of a biological clock depend upon its period? Effects of the duper and tau mutations in Syrian hamsters.

Mutations which alter the feedback loops that generate circadian rhythms may provide insight into their insensitivity to perturbation robustness) and their consistency of period (precision). I examined relationships between endogenous period, activity and rest (τ(DD), α and ρ) in Syrian hamsters using two different mutations, duper and tau, both of which speed up the circadian clock. I generated 8 strains of hamsters that are homozygous or heterozygous for the tau, duper, and wild type alleles in all combinations. The endogenous period of activity onsets among these strains ranged from 17.94+0.04 to 24.13 ± 0.04 h. Contrary to predictions, the variability of period was unrelated to its absolute value: all strains showed similar variability of τ(DD) when activity onsets and acrophase were used as phase markers. The τ(DD) of activity offsets was more variable than onsets but also differed little between genotypes. Cycle variation and precision were not correlated with τ(DD) within any strain, and only weakly correlated when all strains are considered together. Only in animals homozygous for both mutations (super duper hamsters) were cycle variation and precision reduced. Rhythm amplitude differed between strains and was positively correlated with τ(DD) and precision. All genotypes showed negative correlations between α and ρ. This confirms the expectation that deviations in the duration of subjective day and night should offset one another in order to conserve circadian period, even though homeostatic maintenance of energy reserves predicts that longer intervals of activity or rest would be followed by longer durations of rest or activity. Females consistently showed greater variability of the period of activity onset and acrophase, and of α, but variability of the period of offset differed between sexes only in super duper hamsters. Despite the differences between genotypes in τ(DD), ρ was consistently more strongly correlated with the preceding than the succeeding α.

Effects of the duper mutation on circadian responses to light.

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.

Duper: a mutation that shortens hamster circadian period.

Three animals born to homozygous tau mutant (τ(ss), "super short") Syrian hamsters showed extremely short free-running periods of locomotor activity (τ(DD) of approximately 17.8 hours). Inbreeding produced 33 such "super duper" animals, which had a τ(DD) of 18.09 ± 0.05 hours, which was shorter than that of τ(ss) hamsters (20.66 ± 0.07 hours, p < 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of τ(DD), we backcrossed super duper hamsters to unrelated τ(ss) animals. The F(1) pups uniformly had a τ(DD) similar to that of τ(ss) hamsters (19.89 ± 0.15 hours), but F(2) animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F(1) of a cross between super duper hamsters and τ(ss) animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F(1) offspring uniformly produced F(2) pups with extremely short τ(DD) (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background through crossing of super duper with wild-type animals. Restriction digests identified short-period F(2) pups that lack the mutant CK1ε allele, and these animals had a mean τ(DD) of 23.11 ± 0.04 hours. τ(DD) of duper hamsters born and raised in DD was significantly shorter than in hamsters raised in 14L:10D (21.92 ± 0.12 hours, p < 0.0001). τ(DD) shortened twice as much in τ(s) and τ(ss) hamsters than in wild-type animals that were homozygous for duper, indicating the presence of epistatic interactions. Assortment of phenotypes in the F(2) generation fit the expected distribution for expression of duper as recessive (χ(2) = 6.41, p > 0.1). Neither CK1ε nor CK1δ coding region base sequences differed between super duper and τ(ss) hamsters. The growth rate of super duper mutants is similar to that of τ(ss) animals but slightly but significantly reduced at particular postweaning time points. We conclude that duper represents a new mutation that substantially reduces τ(DD) and has significant effects on physiology and metabolism.

Lateralization of the central circadian pacemaker output: a test of neural control of peripheral oscillator phase.

To evaluate the contribution of neural pathways to the determination of the circadian oscillator phase in peripheral organs, we assessed lateralization of clock gene expression in Syrian hamsters induced to split rhythms of locomotor activity by exposure to constant light. We measured the ratio of haPer1, haPer2, and haBmal1 mRNA on the high vs. low (H/L) side at 3-h intervals prior to the predicted activity onset (pAO). We also calculated expression on the sides ipsilateral vs. contralateral (I/C) to the side of the suprachiasmatic nucleus (SCN) expressing higher haPer1. The extent of asymmetry in split hamsters varied between specific genes, phases, and organs. Although the magnitude of asymmetry in peripheral organs was never as great as that in the SCN, we observed significantly greater lateralization of clock gene expression in the adrenal medulla and cortex, lung, and skeletal muscle, but not in liver or kidney, of split hamsters than of unsplit controls. We observed fivefold lateralization of expression of the clock-controlled gene, albumin site D-element binding protein (Dbp), in skeletal muscle (H/L: 10.7 +/- 3.7 at 3 h vs. 2.2 +/- 0.3 at 0 h pAO; P = 0.03). Furthermore, tyrosine hydroxylase expression was asymmetrical in the adrenal medulla of split (H/L: 1.9 +/- 0.5 at 0 h) vs. unsplit hamsters (1.2 +/- 0.04; P < 0.05). Consistent with a model of neurally controlled gene expression, we found significant correlations between the phase angle between morning and evening components (psi(me)) and the level of asymmetry (H/L or I/C). Our results indicate that neural pathways contribute to, but cannot completely account for, SCN regulation of the phase of peripheral oscillators.