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.     

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 induced wheel running on the circadian activity rhythms of Syrian hamsters: entrainment and phase response curve

The goal of this study was to provide an example of nonsocial and nonphotic entrainment in Syrian hamsters, together with a corresponding phase response curve (PRC). Fourteen male hamsters were given 2-hr bouts of induced activity (mostly wheel running) at 23.83-hr intervals in constant darkness (DD). The activity onsets of 10 hamsters entrained to this manipulation, with no anticipatory activity present. After entrainment, the rhythms resumed free-running from a time 0.66-3.91 hr after the onset of the last bout of induced activity. Postentrainment free-running periods were shorter than pre-entrainment values. The PRC for 2-hr pulses of induced activity in DD revealed phase advances induced in some animals between circadian time (CT) 4 and CT 11 (approximately the last half of the hamsters' rest period), and delays between CT 23 and CT 3 and between CT 17 and CT 20. The CTs for phase advances are compatible with the phase angle differences observed between rhythm and zeitgeber at the end of entrainment. Many features of the results (not all animals entraining, PRC characteristics, lack of observable anticipation to the daily stimuli, phase relationship between zeitgeber and activity rhythms) are similar to those from a previous study on social entrainment in this species (Mrosovsky, 1988). These similarities reinforce the idea that induced activity and social zeitgebers act on activity rhythms via a common mechanism.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Circadian phototransduction: phase resetting and frequency of the circadian clock of Gonyaulax cells in red light

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic--rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16 degrees C and 25 degrees C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light.     

Conditioning in the Orcadian System

Light is the dominant environmental cue for entrainment of circadian rhythms. In mammals, light entrains rhythms by resetting the phase of a circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Until recently, the mechanism responsible for pacemaker resetting by light was thought to be exclusively sensitive to photic cues. New experiments indicate, however, that this mechanism is more plastic than once thought; is amenable to conditioned stimulus control; and is capable of acquiring, through conditioning, new response capabilities. These experiments showed that, in rats, a neutral stimulus paired with light in Pavlovian conditioning trials is capable of eliciting cellular and behavioral effects characteristic of circadian clock phase resetting by light, expression of Fos protein in the ventrolateral region of the SCN, and phase shifts of free-running rhythms. These novel results open up a previously unappreciated perspective on photic phase resetting and entrainment of circadian rhythms. Specifically, they suggest that the process normally initiated by light to reset the clock can be modified by learning and events in the environment that reliably precede the onset of light can assume the resetting function of light.     

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.     

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.     

Circadian rhythm of locomotor activity in the Japanese honeybee, Apis cerana japonica

Abstract.  To reveal circadian characteristics and entrainment mechanisms in the Japanese honeybee Apis cerana japonica , the locomotor-activity rhythm of foragers is investigated under programmed light and temperature conditions. After entrainment to an LD 12 : 12 h photoperiodic regime, free-running rhythms are released in constant dark (DD) or light (LL) conditions with different free-running periods. Under the LD 12 : 12 h regime, activity offset occurs approximately 0.4 h after lights-off transition, assigned to circadian time (Ct) 12.4 h. The phase of activity onset, peak and offset, and activity duration depends on the photoperiodic regimes. The circadian rhythm can be entrained to a 24-h period by exposure to submultiple cycles of LD 6 : 6 h, as if the locomotive rhythm is entrained to LD 18 : 6 h. Phase shifts of delay and advance are observed when perturbing single light pulses are presented during free-running under DD conditions. Temperature compensation of the free-running period is demonstrated under DD and LL conditions. Steady-state entrainment of the locomotor rhythm is achieved with square-wave temperature cycles of 10 °C amplitude, but a 5 °C amplitude fails to entrain.     

Unique food-entrained circadian rhythm in cysteine414-alanine mutant mCRY1 transgenic mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Daily scheduled restricted feeding (RF), in which the food available time is restricted for several hours each day, elicits anticipatory activity. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from the suprachiasmatic nucleus (SCN), the master pacemaker in mammals. In an earlier report, we described generation of transgenic (Tg) mice ubiquitously overexpressing cysteine414-alanine mutant mCRY1. The Tg mice displayed long locomotor free-running periods (approximately 28 h) with rhythm splitting. Furthermore, their locomotor activity immediately re-adjusted to the advance of light–dark cycles (LD), suggesting some disorder in the coupling of SCN neurons. The present study examined the restricted feeding cycle (RF)-induced entrainment of locomotor activity in Tg mice in various light conditions. In LD, wild-type controls showed both FAA and LD-entrained activities. In Tg mice, almost all activity was eventually consolidated to a single bout before the feeding time. The result suggests a possibility that in Tg mice the feeding cycle dominates the LD cycle as an entrainment agent. In constant darkness (DD), wild-type mice exhibited robust free-run activity and FAA during RF. For Tg mice, only the rhythm entrained to RF was observed in DD. Furthermore, after returning to free feeding, the free-run started from the RF-entrained phase. These results suggest that the SCN of Tg mice is entrainable to RF and that the mCRY1 mutation alters the sensitivity of SCN to the cycle of nonphotic zeitgebers.

     

Maternal entrainment of tau mutant hamsters.

Maternal entrainment of the circadian wheel-running activity rhythm was examined in Syrian hamsters heterozygous for a single gene mutation (tau) that affects the free-running period of circadian rhythms. Heterozygous tau pups were born to and raised by wild-type mothers under constant dim light. The pups' wheel-running activity was recorded after weaning on postnatal day 18 or 24. Pups weaned on day 18 had an average free-running period of 21.70 hr, demonstrating that the tau phenotype was fully expressed at this age. Using the activity onset of the postnatal free-running rhythms as a phase reference, we estimated the phase relationships between the pups and their mothers on days 18 and 24. In contrast to results with wild-type pups, the activity rhythms of tau pups were not in phase with the rhythms of their wild-type mothers; that is, activity onsets of mothers and pups did not coincide. The pups did, however, show synchrony among themselves, indicating that they had been exposed to a synchronizing signal sometime during development. It is likely that this synchronizing signal was provided by the mothers, since pups from different litters showed phase relationships similar to those of their mothers. Thus the mothers provided a signal that was sufficient to cause entrainment, despite the 2-hr difference in free-running period between the mothers and pups. Although the pups' activity rhythms appeared to have been entrained by the mothers, they were clearly free-running by postnatal day 18. The mechanism for entrainment is lost during the course of development, despite continued interaction between the mothers and pups.     

Resilience of circadian pacemaker development in hamsters

Disruptions of circadian rhythms have been linked to a wide range of pathologies from sleep disorders to cancer. The extent to which disruptions of circadian rhythms during development contribute to later conditions is not known. The present study tested the hypothesis that functional properties of the central circadian pacemaker, the suprachiasmatic nucleus (SCN), are affected by abnormal entrainment during development. The SCN is specialized for the generation of robust rhythms, for direct and indirect output to physiological and behavioral systems, and for entrainment to light/dark cycles via direct retinal input. It consists of thousands of neurons and glia with distinct phenotypes and has subdivisions delineated by both anatomical and functional criteria. In rodents, SCN rhythms develop within days after SCN cells are produced and before many other aspects of differentiation, such as synaptogenesis, are complete. We demonstrated that around the time of birth, the hamster SCN in vivo can undergo repeated phase shifts by a dopamine D(1) receptor agonist (SKF-38393). For 2 days before and 2 days after birth, one group of hamsters received regular exposure to the drug at the same time of day, while another group was exposed at varying times to induce repeated phase shifts. Free-running and entrained activity rhythms were compared between the groups at different ages after weaning. Repeated phase shifts during SCN development had a significant effect on free-running period measured immediately after weaning. This effect was eliminated by subsequent entrainment to a light/dark cycle, indicating that the effect was not permanent. These and other results suggest that SCN development required for functional properties such as free-running period is resilient to perturbation.     

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.     

Psi-Mutation Affects Phase Angle Difference, Free-Running Period and Phase Shifts in Aedes krombeini (Stegomyia)

A new mutation, designated as psi-mutant, affecting the timing of the circadian oviposition rhythm was discovered the in natural population of Aedes krombeini . This mutation advanced the phase of the oviposition median in an entraining light-dark cycle of 12:12 h by ca. 7.0 h and shortened the free running period t in constant darkness (DD) by ca. 4.0 h. Early oviposition in psi-mutants was also observed when while free-running in DD they were subjected to 24-h temperature cycles (29°C for 12 h and l8°C for l2 h). When the phase response curves (PRCs) for light pulses against DD as background were measured, the PRC for the psi-mutant had large delaying phase shifts, whereas, that of the wild strain had small delaying phase shifts.     

Psi-Mutation Affects Phase Angle Difference,Free-Running Period and Phase Shifts in Aedes krombeini (Stegomyia)

A new mutation, designated as psi-mutant, affecting the timing of the circadian oviposition rhythm was discovered the in natural population of Aedes krombeini. This mutation advanced the phase of the oviposition median in an entraining light-dark cycle of 12:12 h by ca. 7.0 h and shortened the free running period t in constant darkness (DD) by ca. 4.0 h. Early oviposition in psi-mutants was also observed when while free-running in DD they were subjected to 24-h temperature cycles (29°C for 12 h and l8°C for l2 h). When the phase response curves (PRCs) for light pulses against DD as background were measured, the PRC for the psi-mutant had large delaying phase shifts, whereas, that of the wild strain had small delaying phase shifts.     

Testicular hormones modulate circadian rhythms of the diurnal rodent, Octodon degus

Sex differences have been identified in a variety of circadian rhythms, including free-running rhythms, light-induced phase shifts, sleep patterns, hormonal fluctuations, and rates of reentrainment. In the precocial, diurnal rodent Octodon degus, sex differences have been found in length of free-running rhythm (tau), phase response curves, rates of reentrainment, and in the use of social cues to facilitate reentrainment. Although gonadal hormones primarily organize circadian rhythms during early development, adult gonadal hormones have activational properties on various aspects of circadian rhythms in a number of species examined. Gonadectomy of adult female O. degus did not influence tau, phase angle of entrainment, or activity patterns in previous experiments. The present experiment examined the role of gonadal hormones in adult male degus' circadian wheel-running rhythms. We predicted that male gonadal hormones would have an activational effect on some aspects of circadian rhythms, particularly those in which we see sex differences. Phase angles of entrainment, tau, length of the active period (alpha), maximum and mean activity levels, and activity amplitude were examined for intact and castrated males housed in LD 12:12. Responses to light pulses while housed in constant darkness (DD) were also compared. Castration had no significant effect on tau or light-induced phase shifts. However, castration significantly increased phase angle of entrainment and decreased activity levels. The data indicate that adult gonadal steroids are not responsible for the sex differences in endogenous circadian mechanisms of O. degus (tau, PRC), although they influence activity level and phase angle of entrainment. This is most likely due to masking properties of testosterone, similar to the activity-increasing effects of estrogen during estrus in O. degus females.     

Circadian organization of tau mutant hamsters: aftereffects and splitting

Homozygous tau mutant (tau(ss)) hamsters show an extremely short (20 h) circadian period (tau) that is attributable to altered enzymatic activity of casein kinase 1epsilon. It has been proposed that coupling of constituent circadian oscillators is strengthened in tau(ss) hamsters, explaining their tendency to show strong resetting after prolonged exposure to constant darkness. To evaluate further the circadian organization of tau(ss) hamsters, the authors assessed the extent of shortening of period as an aftereffect of exposure to light:dark cycles whose period (T) is 91% of tau and the ability of constant light to induce splitting. They find that tau(ss) hamsters show aftereffects comparable to wild types, indicating that normal CK1epsilon activity is not required for T cycles to shorten tau. This finding also contradicts the proposal that circadian period is homeostatically conserved. However, the authors find that tau(ss) hamsters rarely show splitting in constant light. Furthermore, LL does not induce lengthening of tau or reduction of activity duration (alpha) in these mutants. The authors' findings support the conclusion that the tau mutation alters the coupling between constituent circadian oscillators.     

Neural mechanisms for entrainment and generation of mammalian circadian rhythms.

The identification of a direct retinohypothalamic tract (RHT) terminating in the supra-chiasmatic nuclei (SCN) has focused attention on the role of these structures in the entrainment and generation of circadian rhythms in mammals. Light effects on circadian rhythms are mediated by both the RHT and portions of the classical visual system. The complex interactions of these systems are reflected both in their direct anatomical connections and in the functional changes in entrainment produced by interruption of either set of projections. Destruction of the RHT/SCN eliminated both normal entrainment and normal free-running circadian rhythms. No circadian rhythms has survived SCN ablation in rodents, but a variety of non-circadian cycles can be generated by lesioned animals. The complex behavioral patterns produced by SCN-lesioned hamsters suggest that circadian oscillators continue to function in these animals, but that their activity is no longer integrated into a single circadian framework. The available evidence indicates that the mammalian pacemaking system comprises a set of independent oscillators normally regulated by the SCN and by light information that is transmitted via several retinofugal pathways.