The Role of Wheel Running in the Coupling of Two Simultaneous Circadian Rhythms of Motor Activity in the Rat

Motor activity is among the non-photic stimuli that act on the internal clock. We have tested the role of motor activity in the circadian pattern of rats under conditions near the lower limits of entrainment, that induce circadian rhythm dissociation. Three groups of 8 rats each were used: a) rats kept individually in 25×25×15 cm cages, b) rats in 50×25×15 cm cages, and c) rats in 50×25×15 cm cages with access to a running wheel. All the rats were kept under light-dark cycles of 22 hours (T22, 11L:11D) for 50 days, after which they were transferred to constant darkness. All the rats without a running wheel showed a motor activity pattern with two statistically significant circadian rhythms in the periodogram of Sokolove and Bushell: one circadian component entrained by the LD cycle, and another free-running. The rats with access to a running wheel showed several patterns: 5 rats showed only one rhythm entrained to the LD cycle, 2 rats showed circadian rhythm dissociation, and 1 showed only a free running rhythm. We believe that the simultaneous manifestation of two circadian components reflects the functional dissociation of the oscillators population that constitutes the circadian pacemaker, of the rat. Physical exercise acts on the pacemaker reinforcing the strongest group of oscillators, which, depending on the structure of the circadian system of the rat, is usually the one entrained to the LD cycle. This study supports the hypothesis that motor activity couples the oscillators that form the circadian system of the rat.     

Parametric and nonparametric entrainment of circadian locomotor rhythm in the Japanese honeybee Apis cerana japonica

The circadian rhythm of locomotor activity in the Japanese honeybee Apis cerana japonica was studied to determine the involvement of parametric and/or nonparametric entrainment. The rhythm was entrained to a skeleton photoperiod in which a 1-h first light pulse was imposed in the morning along with a second light pulse in the evening, as well as to a complete photoperiodic regime (LD 12:12). However, the timing of peak activity relative to the lights-off in the evening in the skeleton photoperiod was earlier than that in the complete photoperiod. A single daily light pulse in the evening entrained the rhythm, whereas a daily light pulse in the morning allowed free-running as in constant darkness. The free-running period (τ) of locomotor activity in constant light became longer as the light intensity increased. A Winfree's type I phase response curve of the locomotor activity rhythm was obtained using a single 1-h light pulse. The results suggest that both parametric and nonparametric entrainment are involved in the circadian rhythm of individual locomotor activity in this honeybee.     

Period-Dependent Oscillatory Free-Run in the Locomotor Activity Rhythm of the Field Mouse Mus booduga Under Skeleton Photoperiodic Regimes

The entrainment behaviour of the circadian rhythm of locomotor activity in the field mouse Mus booduga was studied in order to evaluate the role of the animals' free-running period (τ) and the duration of skeleton photoperiods in determining entrainment of animals with τ values beyond and close to the “limits of entrainment”. We predicted that animals with τ lesser than the lower “limit of entrainment” would entrain only to short skeleton photoperiods (≤ 6 h) and not to longer skeleton photoperiods. Experimental animals (n = 25) were entrained to light/dark (LD) 12:12 h schedule, and then subjected to various skeleton photoperiods in which the duration of one of the two intervals of darkness was successively reduced while holding the zeitgeber period (T) constant. Some animals (n = 9) entrained to long as well as short photoperiods, whereas others (n = 5) entrained only to extremely short skeleton photoperiods of 6 h or less. The mean τ of the animals entraining to all photoperiods (23.78 ± 0.22 h) was significantly greater than that of the animals that entrained only to very short skeleton photoperiods (22.43 ± 0.41 h) (t _{df 12} = 5.3, p < 0.001). We also selected a few animals (n = 11) with average τ value of 23.13 ± 0.38 h and studied them under several skeleton photoperiods. To our surprise the animals which were subjected to restricted dark intervals invariably underwent “phase-jump” assuming the longer dark interval as “subjective night”. We suggest that the observed variation in entrainment behaviour might be due to the variation seen among individual animals in τ and the shape of their PRC. These results support the view that the duration of the skeleton photoperiod and the τ of an individual animal interact to determine its entrainment, and underscore the relevance of inter-individual variation in circadian organisation to studies of circadian rhythms.     

Forced desynchronization model for a diurnal primate

The circadian system is organized in a hierarchy of multiple oscillators, with the suprachiasmatic nucleus (SCN) as the master oscillator in mammals. The SCN is formed by a group of coupled cell oscillators. Knowledge of this coupling mechanism is essential to understanding entrainment and the expression of circadian rhythms. Some authors suggest that light-dark (LD) cycles with periods near the limit of entrainment may be good models for promoting internal desynchronization, providing knowledge about the coupling mechanism. As such, we evaluated the circadian activity rhythm (CAR) pattern of marmosets in LD cycles at lower limits of entrainment in order to study induced internal dissociation. To that end, two experiments were conducted: (1) 6 adult females were under symmetrical LD cycles T21, T22 and T21.5 for 60, 35 and 48 days, respectively; and (2) 4 male and 4 female adults were under T21 for 24 days followed by 18 days of LL, back to T21 for 24 days, followed by 14 days of LL. The CAR of each animal was continuously recorded. In experiment 1, vocalizations were also recorded. Under Ts shorter than 24 days, a dissociation pattern was observed for CAR and vocalizations. Two simultaneous circadian components emerged, one with the same period as the LD cycle, called the light-entrained component, and the other in free-running, denominated the non-light-entrained component. Both components were displayed in the CAR for all the animals in T21, five animals (83.3%) in T21.5 and two animals (33.3%) in T22. Our results are in accordance with the multioscillatory nature of the circadian system. Dissociation is partial synchronization to the LD cycle, with at least one group of oscillators synchronized by relative coordination and masking, while another group of oscillators free runs, but is also masked by the LD cycle. Since only T21 promoted the emergence of both circadian components in the circadian rhythms of all marmosets, it was considered the promoter period of circadian rhythm dissociation in this species, and is proposed as a good animal model for forced desynchronization in non-human diurnal primates.     

The Role of Wheel Running in the Coupling of Two Simultaneous Circadian Rhythms of Motor Activity in the Rat

Motor activity is among the non-photic stimuli that act on the internal clock. We have tested the role of motor activity in the circadian pattern of rats under conditions near the lower limits of entrainment, that induce circadian rhythm dissociation. Three groups of 8 rats each were used: a) rats kept individually in 25×25×15 cm cages, b) rats in 50×25×15 cm cages, and c) rats in 50×25×15 cm cages with access to a running wheel. All the rats were kept under light-dark cycles of 22 hours (T22, 11L:11D) for 50 days, after which they were transferred to constant darkness. All the rats without a running wheel showed a motor activity pattern with two statistically significant circadian rhythms in the periodogram of Sokolove and Bushell: one circadian component entrained by the LD cycle, and another free-running. The rats with access to a running wheel showed several patterns: 5 rats showed only one rhythm entrained to the LD cycle, 2 rats showed circadian rhythm dissociation, and 1 showed only a free running rhythm. We believe that the simultaneous manifestation of two circadian components reflects the functional dissociation of the oscillators population that constitutes the circadian pacemaker, of the rat. Physical exercise acts on the pacemaker reinforcing the strongest group of oscillators, which, depending on the structure of the circadian system of the rat, is usually the one entrained to the LD cycle. This study supports the hypothesis that motor activity couples the oscillators that form the circadian system of the rat.     

Significance of Nonparametric Light Effects in Entrainment of Circadian Rhythms in Owl Monkeys (Aotus Lemurinus Griseimembra) by Light-Dark Cycles

In a total of 12 adult Colombian owl monkeys, Aotus lemurinus griseimembra, the significance of nonparametric light effects for the entrainment of the circadian system by light-dark (LD) cycles was studied by carrying out (a) phase-response experiments testing the phase-shifting effect of 30-min light pulses (LPs) of 250 lx applied at various phases of the free-running circadian activity rhythm (LL 0.2 lx) and (b) synchronization experiments testing the entraining effect of 24-h single LP photoperiods consisting of 30-min L of 80 lx and 23.5-h D of 0.5 lx (sP 0.5) and skeleton photoperiods consisting of two 30-min LPs of 80 lx, given against a background illuminance of 0.5 lx either symmetrically at 12-h intervals (PP 12:12) or asymmetrically at 9- and 15-h intervals (PP 9:15). The phase-response characteristics in Aotus, as evidenced by the phase-response curve, generally correspond to those of nocturnal rodents, proving that this neotropical simian primate chronobiologically is a genuine nocturnal species. When free-running with a spontaneous period close to 24 h (24.3 ± 0.1 h), the PP 12:12 produced entrainment in only two of five owl monkeys, whereas the sP 0.5 entrained four of them. The PP 9:15, however, brought about stable entrainment of the circadian rhythms of locomotor activity, feeding activity, and core temperature in all animals tested (n = 8). Changes in phase position of the activity time with the endogenous rhythm entrained by a PP 12:12, by an sP 0.5, or by a PP 9:15 give evidence that both LPs of a skeleton photoperiod contribute to the phase setting of the circadian system. When free-running with a considerably lengthened spontaneous period (τ ≥ 25.5 h), even the sP 0.5 and the PP 9:15 failed to entrain the owl monkeys' circadian rhythms, whereas a 24-h photoperiod with a very long LP of 3 h caused entrainment. The results indicate that in Aotus lemurinus griseimembra, in addition to the nonparametric light effects, parametric light effects play a significant role in the entrainment of circadian rhythms by LD cycles.     

Behavioral and neurochemical sources of variability of circadian period and phase: studies of circadian rhythms of npy-/- mice

The cycle length or period of the free-running rhythm is a key characteristic of circadian rhythms. In this study we verify prior reports that locomotor activity patterns and running wheel access can alter the circadian period, and we report that these treatments also increase variability of the circadian period between animals. We demonstrate that the loss of a neurochemical, neuropeptide Y (NPY), abolishes these influences and reduces the interindividual variability in clock period. These behavioral and environmental influences, from daily distribution of peak locomotor activity and from access to a running wheel, both act to push the mean circadian period to a value < 24 h. Magnitude of light-induced resetting is altered as well. When photoperiod was abruptly changed from a 18:6-h light-dark cycle (LD18:6) to LD6:18, mice deficient in NPY were slower to respond to the change in photoperiod by redistribution of their activity within the prolonged dark and eventually adopted a delayed phase angle of entrainment compared with controls. These results support the hypothesis that nonphotic influences on circadian period serve a useful function when animals must respond to abruptly changing photoperiods and point to the NPYergic pathway from the intergeniculate leaflet innervating the suprachiasmatic nucleus as a circuit mediating these effects.     

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.     

Circadian Rhythms and time Measurement in Locomotor Activity of the Scorpion Leiurus Quinquestriatus (Buthidae)

The circadian rhythms of locomotor activity of the scorpion Leiurus quinqueslriatus were examined under different light-dark cycles and in free-running conditions. The circadian rhythm is bimodal in LD 12:12 with alternating cycles of temperature (35°-25°C) with high intensity (1300 lux) or in LD 12: 12 with constant temperature 35° C with 300 lux. In LD 12:12 (1300 lux), in long or in short light spans with constant temperature, the bimodal pattern is slightly changed with the appearance of a third minor peak of activity. In free-running conditions, the bimodal rhythm of locomotor activity persists in DD with T about 24 hr, but in LL the rhythm becomes unimodal with T about 24 hr. Cosinor and power spectrum analysis showed the presence of more than one periodic component. It seems that there is a correlation between the range of light regimens, temperature, light intensity and the coincidence of these components. These components are independently entrained by the environmental light cycle. The mechanism of entrainment of components is discussed.     

Adult locomotor rhythmicity as "hands" of the maternal photoperiodic clock regulating larval diapause in the blowfly, Calliphora vicina.

Some basic properties of the adult locomotor activity rhythm and of the maternal induction of larval diapause in Calliphora vicina are described. Diapause responses in Nanda-Hamner experiments indicate that circadian rhythmicity is involved in photoperiodic time measurement (PPTM). However, although the locomotor rhythm shows long-lasting changes in free-running period (aftereffects of photoperiod and constant light) and occasional "splitting," thereby indicating a structural complexity to the circadian system, the overt rhythm may be used as an indicator of phase relationships (or "hands") of the covert system involved in PPTM, within the framework of a simple external-coincidence model for the diapause clock. Thus, in light-dark (LD) cycles close to "resonance" with the circadian pacemaker(s) (T 24, LD 12:12; T 48, LD 12:36; and T 72, LD 12:60), light is restricted to the subjective day and diapause incidence is high. In T 36 (LD 12:24) and T 60 (LD 12:48), light falls into the subjective night and illuminates the postulated light-sensitive phase (phi i), and diapause incidence is low. Within the primary range of entrainment, light invades the late subjective night in T 20 (LD 12:8), illuminates phi i, and causes low incidence of diapause; however, it invades the early subjective night in T 30 (LD 12:18) and diapause remains high.     

Tau differences between short-day responsive and short-day nonresponsive white-footed mice (Peromyscus leucopus) do not affect reproductive photoresponsiveness

In laboratory-bred rodent populations, intraspecific variation in circadian system organization is a known cause of individual variation in reproductive photoresponsiveness. The authors sought to determine whether circadian system variation accounted for individual variation in reproductive photoresponsiveness in a single, highly genetically variable population of Peromyscus leucopus recently derived from the wild. Running-wheel activity patterns of male and female mice, aged 70 to 90 days, from artificially selected lines of reproductively photoresponsive (R) and nonresponsive (NR) lines were monitored under short-day photoperiod (8 h light, 16 h dark), long-day photoperiod (16 h light, 8 h dark), and constant darkness (DD). NR mice displayed a significantly longer mean free-running period (24.08 h) in DD compared with R mice (23.75 h), due in large part to a difference between NR and R females (24.25 h vs. 23.74 h, respectively). All other entrainment characteristics (alpha, phase angle of activity) under short days, long days, and DD were similar between R and NR mice. Variation in free-running period and entrainment characteristics has been shown to affect photoresponsiveness in other rodent species by altering the manner in which the circadian system interprets short days. To determine whether variation in photoresponsiveness in P. leucopus is due to differences in free-running period instead of variation downstream from the central circadian clock in the pathway controlling photoresponsiveness, the authors exposed young R and NR mice to DD and measured the effect on reproductive organ development. If variation in free-running period affected how the circadian system of mice interpreted short days, then both R and NR mice exposed to DD should have exhibited a delay in gonadal development. Only R mice exhibited pubertal delay in DD. NR mice exhibited large paired testes, paired seminal vesicles, paired ovaries, and uterine weight typical of mice nonresponsive to short days, whereas R mice exhibited reproductive organ weight typical of mice responsive to short days. These data suggest that despite significant differences in free-running period between R and NR mice, individual variation in photoresponsiveness is not due to differences in how the circadian systems of R and NR mice interpret the LD cycle.     

The comparison between circadian oscillators in mouse liver and pituitary gland reveals different integration of feeding and light schedules

The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues.     

Latitudinal clines in the properties of a circadian pacemaker

The circadian rhythm of eclosion activity and its pacemaker were analyzed in a series of latitudinal races of Drosophila auraria ranging from 34.2 degrees to 42.9 degrees N in Japan. The phase of the rhythm (psi EL) to the daily photoperiod (PP) changes as daylength is increased, and the slope of psi EL (PP) changes with latitude. Is is sufficiently greater in the north to cause a phase reversal of northern and southern races on long versus short photoperiods. This reversal is found in assays of the pacemaker's phase (psi PL) as well as that of the rhythm (psi EL). Assay of the pacemaker shows that its period (tau) is longer in northern than in southern races, and that the amplitude of its phase response curve (PRC) is lower in the north. The period of the rhythm in all latitudinal races is longer than 24 hr in short photoperiods (LD 1:23), but is probably less than 24 hr (as an aftereffect of photoperiod) in longer days such as LD 14:10. The observed north-south differences in the phase relation of both pacemaker and rhythm to the light cycle are explained by the latitudinal clines in pacemaker properties and a postulated aftereffect of photoperiod on tau. It is suggested that the latitudinal cline in PRC amplitude has functional significance in conserving the amplitude of the pacemaker's signal to the rest of the system it times. Computer simulation shows that without such a reduction in the perceived light intensity, pacemaker amplitude will be lowered by the increase in duration of the daily light at higher latitudes.     

Photoperiod influences endogenous rhythm of ambient temperature selection by the honeybee Apis mellifera

The aim of this study was to evaluate whether the day–night cycle phase is a critical factor modulating diurnal rhythm of isolated honeybee's thermal preference or other factors are involved. The insects were exposed to standard (LD 12:12) and reversed (DL 12:12) photoperiods as well as to constant light and constant darkness conditions. Thermal preference and motor activity of honeybees were recorded for 3–5 days in a thermal gradient system. Under the standard (control) photoperiod conditions mean values of temperature selected by honeybees changed rhythmically within the period of about 24 h. Honeybees, exposed to the modified light–darkness cycle distinctly modified their rhythm of thermal preference. Under the reversed photoperiod conditions period of selected ambient temperature was much longer than before, until a complete reversal of the circadian oscillation was established at the end of the experiment. Experiments performed under constant light and constant darkness yielded undisturbed 24 h rhythms of both ambient temperature selection and locomotor activity. Under these conditions only a slight, nonsignificant flattening of the temperature selection curves was noticed. Both lack of substantial changes in the amplitude and occurring phase shifts of the rhythm, recorded in our experiments suggest its endogenous character. Our results prove that diurnal rhythm of ambient temperature selection by bee workers may be entrained by light–dark cycles. This implies a critical role of photoperiod in the modulation of nychthemeral oscillations of thermal preference in honeybees.     

Relationship between infant and mother circadian rest-activity rhythm pre- and postpartum, in comparison to an infant with free-running rhythm

Some infants show a free-running rhythm in their rest-activity. We do not know why, nor do we know exactly what the entrainment factors are for the development of the normal 24-h rest-activity rhythm. Actigraphic recordings on 10 primiparae during late pregnancy and these mothers and their infants during the 2nd, 6th, and 12th wks after birth were made over 3-5 continuous days to investigate maternal and infant entrainment. One infant showed a free-running rest-activity circadian rhythm. In late pregnancy, the period in the autocorrelogram of the mother with the free-running infant was longer than the significant period of the mean autocorrelogram of the mothers with non-free-running infants. The finding of this study indicates the free-running rhythm of infant is not reset by maternal entrainment factors.     

Exposure to T-cycles of 22 and 23 h during lactation modifies the later dissociation of motor activity and temperature circadian rhythms in rats

Early environmental conditions may affect the development and manifestation of circadian rhythms. This study sought to determine whether the maintenance of rats under different T-cycles during lactation influences the subsequent degree of dissociation of the circadian rhythms of motor activity and core body temperature. Two groups of 22 day-old Wistar rats were kept after weaning under T-cycles of 22 h (T22) or 23 h (T23) for 70 days. Subsequently, they were kept in constant darkness (DD). Half of the animals in each group were born and reared under these experimental conditions, while the other half were reared until weaning under 24 h LD cycles (T24). Rats transferred from T24 to T22 or T23 showed two circadian components in motor activity and temperature, one entrained by light and the other free-running. In T22, there was also desynchronization between temperature and motor activity. Rats submitted to T23 from birth showed higher stability of the 23 h component than rats transferred from T24 to T23 after weaning. However, in comparison to rats born under T24 and subsequently changed to T22, animals submitted to T22 from birth showed shorter values of the period of the non-light-dependent component during T22, more aftereffects when transferred to DD, and a lack of desynchronization between motor activity and temperature. The results suggest that T-cycles in the early environment may modify overt rhythms by altering the internal coupling of the circadian pacemaker.     

Effects of constant darkness and constant light on circadian organization and reproductive responses in the ram

The relationship between circadian rhythms in the blood plasma concentrations of melatonin and rhythms in locomotor activity was studied in adult male sheep (Soay rams) exposed to 16-week periods of short days (8 hr of light and 16 hr of darkness; LD 8:16) or long days (LD 16:8) followed by 16-week periods of constant darkness (dim red light; DD) or constant light (LL). Under both LD 8:16 and LD 16:8, there was a clearly defined 24-hr rhythm in plasma concentrations of melatonin, with high levels throughout the dark phase. Periodogram analysis revealed a 24-hr rhythm in locomotor activity under LD 8:16 and LD 16:8. The main bouts of activity occurred during the light phase. A change from LD 8:16 to LD 16:8 resulted in a decrease in the duration of elevated melatonin secretion (melatonin peak) and an increase in the duration of activity corresponding to the changes in the ratio of light to darkness. In all rams, a significant circadian rhythm of activity persisted over the first 2 weeks following transfer from an entraining photoperiod to DD, with a mean period of 23.77 hr. However, the activity rhythms subsequently became disorganized, as did the 24-hr melatonin rhythms. The introduction of a 1-hr light pulse every 24 hr (LD 1:23) for 2 weeks after 8 weeks under DD reinduced a rhythm in both melatonin secretion and activity: the end of the 1-hr light period acted as the dusk signal, producing a normal temporal association of the two rhythms. Under LL, the 24-hr melatonin rhythms were disrupted, though several rams still showed periods of elevated melatonin secretion. Significant activity rhythms were either absent or a weak component occurred with a period of 24 hr. The introduction of a 1-hr dark period every 24 hr for 2 weeks after 8 weeks under LL (LD 23:1) failed to induce or entrain rhythms in either of the parameters. The occurrence of 24-hr activity rhythm in some rams under LL may indicate nonphotoperiodic entrainment signals in our experimental facility. Reproductive responses to the changes in photoperiod were also monitored. After pretreatment with LD 8:16, the rams were sexually active; exposure to LD 16:8, DD, or LL resulted in a decline in all measures of reproductive function. The decline was slower under DD than LD 16:8 or LL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Non-parametric photic entrainment of Djungarian hamsters with different rhythmic phenotypes

To investigate the role of non-parametric light effects in entrainment, Djungarian hamsters of two different circadian phenotypes were exposed to skeleton photoperiods, or to light pulses at different circadian times, to compile phase response curves (PRCs). Wild-type (WT) hamsters show daily rhythms of locomotor activity in accord with the ambient light/dark conditions, with activity onset and offset strongly coupled to light-off and light-on, respectively. Hamsters of the delayed activity onset (DAO) phenotype, in contrast, progressively delay their activity onset, whereas activity offset remains coupled to light-on. The present study was performed to better understand the underlying mechanisms of this phenomenon. Hamsters of DAO and WT phenotypes were kept first under standard housing conditions with a 14:10 h light–dark cycle, and then exposed to skeleton photoperiods (one or two 15-min light pulses of 100 lx at the times of the former light–dark and/or dark–light transitions). In a second experiment, hamsters of both phenotypes were transferred to constant darkness and allowed to free-run until the lengths of the active (α) and resting (ρ) periods were equal (α:ρ = 1). At this point, animals were then exposed to light pulses (100 lx, 15 min) at different circadian times (CTs). Phase and period changes were estimated separately for activity onset and offset. When exposed to skeleton-photoperiods with one or two light pulses, the daily activity patterns of DAO and WT hamsters were similar to those obtained under conditions of a complete 14:10 h light–dark cycle. However, in the case of giving only one light pulse at the time of the former light–dark transition, animals temporarily free-ran until activity offset coincided with the light pulse. These results show that photic entrainment of the circadian activity rhythm is attained primarily via non-parametric mechanisms, with the “morning” light pulse being the essential cue. In the second experiment, typical photic PRCs were obtained with phase delays in the first half of the subjective night, phase advances in the second half, and a dead zone during the subjective day. ANOVA indicated no significant differences between WT and DAO animals despite a significantly longer free-running period (tau) in DAO hamsters. Considering the phase shifts induced around CT0 and the different period lengths, it was possible to model the entrainment patterns of both phenotypes. It was shown that light-induced phase shifts of activity offset were sufficient to compensate for the long tau in WT and DAO hamsters, thus enabling a stable entrainment of their activity offsets to be achieved. With respect to activity onsets, phase shifts were sufficient only in WT animals; in DAO hamsters, activity onset showed increasing delays. The results of the present paper clearly demonstrate that, under laboratory conditions, the non-parametric component of light and dark leads to circadian entrainment in Djungarian hamsters. However, a stable entrainment of activity onset can be achieved only if the free-running period does not exceed a certain value. With longer tau values, hamsters reveal a DAO phenotype. Under field conditions, therefore, non-photic cues/zeitgebers must obviously be involved to enable a proper circadian entrainment.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.