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.     

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.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO's free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO's free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Evidence for time-of-day dependent effect of neurotoxic dorsomedial hypothalamic lesions on food anticipatory circadian rhythms in rats

The dorsomedial hypothalamus (DMH) is a site of circadian clock gene and immediate early gene expression inducible by daytime restricted feeding schedules that entrain food anticipatory circadian rhythms in rats and mice. The role of the DMH in the expression of anticipatory rhythms has been evaluated using different lesion methods. Partial lesions created with the neurotoxin ibotenic acid (IBO) have been reported to attenuate food anticipatory rhythms, while complete lesions made with radiofrequency current leave anticipatory rhythms largely intact. We tested a hypothesis that the DMH and fibers of passage spared by IBO lesions play a time-of-day dependent role in the expression of food anticipatory rhythms. Rats received intra-DMH microinjections of IBO and activity and body temperature (T(b)) rhythms were recorded by telemetry during ad-lib food access, total food deprivation and scheduled feeding, with food provided for 4-h/day for 20 days in the middle of the light period and then for 20 days late in the dark period. During ad-lib food access, rats with DMH lesions exhibited a lower amplitude and mean level of light-dark entrained activity and T(b) rhythms. During the daytime feeding schedule, all rats exhibited food anticipatory activity and T(b) rhythms that persisted during 2 days without food in constant dark. In some rats with partial or total DMH ablation, the magnitude of the anticipatory rhythm was weak relative to most intact rats. When mealtime was shifted to the late night, the magnitude of the food anticipatory activity rhythms in these cases was restored to levels characteristic of intact rats. These results confirm that rats can anticipate scheduled daytime or nighttime meals without the DMH. Improved anticipation at night suggests a modulatory role for the DMH in the expression of food anticipatory activity rhythms during the daily light period, when nocturnal rodents normally sleep.     

Forced dissociation of activity entrained to T cycles of food access in rats with suprachiasmatic lesions

The ability of rats with suprachiasmatic lesions to entrain anticipatory wheel running to two food access times per day was investigated. In the presence of meal schedules with periods of 23.75 hr and 24 hr, two of seven rats entrained activity to both for many consecutive days, while other rats repeatedly shifted activity from one schedule to the other. A second group of rats was maintained on 25-hr and 26-hr meal schedules. One of nine rats showed prolonged entrainment to both schedules (i.e., forced dissociation). In the other rats, anticipatory activity (AA) waxed and waned repeatedly on each schedule. In both experiments, AA to the leading schedule increased when the interval between meals was about 5 hr or less, and AA to the trailing schedule diminished or ceased. Changes in AA were also common when the interval between meals was between 11 and 16 hr. The results are consistent with the hypothesis that entrainment of AA to periodic food access is mediated by at least two mutually coupled circadian pacemakers. Interactions between these putative pacemakers appear to be strongest when the pacemakers are nearly in phase or in antiphase.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

Circadian Temperature and Activity Rhythms in Mice under Free-Running and Entrained Conditions; Assessment after Purification of the Temperature Rhythm

Six female mice were studied separately for six weeks, first in constant light (300 lx), and then on a 12 : 12 L : D schedule (light on 07:00–19:00–h). Food and water were available ad libitum. Abdominal temperature and spontaneous locomotor activity were measured every 10 min. In constant light, the animals free-ran with both temperature and activity records showing circadian rhythms that were significantly greater than 24 h; by contrast, in the LD schedule, the circadian rhythms had become entrained and showed a stable phase relation to this schedule. The direct masking effects upon raw temperatures caused by bursts of activity were clearly seen, and could be removed by a process of ‘purification’. A comparison of the activity profiles during the entrained and free-running phases showed that the imposed light-dark cycle resulted in decreased activity in the light, increased activity in the dark, and bursts of activity at the light-dark and dark-light transitions. Masking effects due to the activity profile were present in the raw temperature profile, and many could be removed by purification using the activity profile; however, there was evidence that other masking effects, independent of activity, were present also. The efficacy of thermoregulatory compensation, as assessed from the rise of core temperature produced by spontaneous locomotor activity, was, in comparison with the free-running condition, increased in the dark phase and decreased in the light phase; this would appear to be one way to limit the temperature rise that occurs in the active phase of the rest-activity cycle.     

Circadian Temperature and Activity Rhythms in Mice under Free-Running and Entrained Conditions; Assessment after Purification of the Temperature Rhythm

Six female mice were studied separately for six weeks, first in constant light (300 lx), and then on a 12 : 12 L : D schedule (light on 07:00-19:00-h). Food and water were available ad libitum. Abdominal temperature and spontaneous locomotor activity were measured every 10 min. In constant light, the animals free-ran with both temperature and activity records showing circadian rhythms that were significantly greater than 24 h; by contrast, in the LD schedule, the circadian rhythms had become entrained and showed a stable phase relation to this schedule. The direct masking effects upon raw temperatures caused by bursts of activity were clearly seen, and could be removed by a process of 'purification'. A comparison of the activity profiles during the entrained and free-running phases showed that the imposed light-dark cycle resulted in decreased activity in the light, increased activity in the dark, and bursts of activity at the light-dark and dark-light transitions. Masking effects due to the activity profile were present in the raw temperature profile, and many could be removed by purification using the activity profile; however, there was evidence that other masking effects, independent of activity, were present also. The efficacy of thermoregulatory compensation, as assessed from the rise of core temperature produced by spontaneous locomotor activity, was, in comparison with the free-running condition, increased in the dark phase and decreased in the light phase; this would appear to be one way to limit the temperature rise that occurs in the active phase of the rest-activity cycle.     

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 in honeybees: entrainment by feeding cycles

ABSTRACT. Colonies of the South African honeybee race Apis mellifera capensis (Escholtz) were maintained under constant conditions of illumination (200 lux), temperature (25±lC) and relative humidity (65±3%). Activity was measured at the hive entrance. After ad libitum feeding for at least 5 days, food was presented for only 2 h/day either for 1 week (series 1) or for 2 weeks (series 2). In the last part of each experiment, food was again available all the time. Colonies which showed free-running circadian activity rhythms (with periods ranging from 22.6 to 24.8 h) during ad libitum feeding were submitted to feeding cycles with inter-feeding intervals (T) of 22, 23, 24 and 25 h. In most of these experiments the rhythms were synchronized by the feeding schedule, resulting in a stable phase-angle difference between onset of activity and onset of food availability. The duration of this anticipatory activity was positively correlated with T. When ad libitum feeding was resumed, the period of the rhythm induced by the feeding schedule persisted for a few days. Thereafter, the rhythm was free-running again with a period close to that observed in the first part of the experiment. The conclusion is drawn that, under the influence of periodic feeding, the activity of honeybee colonies has the characteristics of an entrained circadian system.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO’s free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO’s free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Circadian photoentrainment: parameters of phase delaying

Experiments were carried out using simulated den cages to delineate specific characteristics of phase delaying in circadian photoentrainment of a nocturnal rodent, the flying squirrel. The principal experiments entailed presentation of one to five consecutive 15-min white-light pulses per activity cycle at activity onset to animals free-running in darkness, in order to determine the immediate and final phase-shifting effect. Auxiliary experiments recorded entrainment patterns on light-dark (LD) schedules in the den cages. Phase response curves (PRCs) based on 15-min white-light pulses in standard wheel cages were also constructed for these animals as background information for interpreting the phase-delaying experiments. Exposure of a den animal to light by light sampling at the time of initial arousal from the rest state at circadian time (CT) 12, either by an LD schedule or by a 15-min light pulse, resulted in a return to the nest box for a short rest period. The phase delay occurring after a single light exposure at activity onset was equal to the induced rest, thus suggesting an immediate phase shift. The maximum delay was about 1 1/2 hr/cycle, with the amount of delay related to the number of light exposures. During the photoentrained state on an LD schedule, the activity rhythm of a den-housed animal was essentially free-running on the days following a phase delay. The data are used to expand current models for photoentrainment of circadian activity rhythms in nocturnal rodents.     

Circadian discrimination of reward: evidence for simultaneous yet separable food- and drug-entrained rhythms in the rat

A unique extra-suprachiasmatic nucleus (SCN) oscillator, operating independently of the light-entrainable oscillator, has been hypothesized to generate feeding and drug-related rhythms. To test the validity of this hypothesis, sham-lesioned (Sham) and SCN-lesioned (SCNx) rats were housed in constant dim-red illumination (LL(red)) and received a daily cocaine injection every 24 h for 7 d (Experiment 1). In a second experiment, rats underwent 3-h daily restricted feeding (RF) followed 12 d later by the addition of daily cocaine injections given every 25 h in combination with RF until the two schedules were in antiphase. In both experiments, body temperature and total activity were monitored continuously. Results from Experiment 1 revealed that cocaine, but not saline, injections produced anticipatory increases in temperature and activity in SCNx and Sham rats. Following withdrawal from cocaine, free-running temperature rhythms persisted for 2-10 d in SCNx rats. In Experiment 2, robust anticipatory increases in temperature and activity were associated with RF and cocaine injections; however, the feeding periodicity (23.9 h) predominated over the cocaine periodicity. During drug withdrawal, the authors observed two free-running rhythms of temperature and activity that persisted for >14 d in both Sham and SCNx rats. The periods of the free-running rhythms were similar to the feeding entrainment (period = 23.7 and 24.0 h, respectively) and drug entrainment (period = 25.7 and 26.1 h, respectively). Also during withdrawal, the normally close correlation between activity and temperature was greatly disrupted in Sham and SCNx rats. Taken together, these results do not support the existence of a single oscillator mediating the rewarding properties of both food and cocaine. Rather, they suggest that these two highly rewarding behaviors can be temporally isolated, especially during drug withdrawal. Under stable dual-entrainment conditions, food reward appears to exhibit a slightly greater circadian influence than drug reward. The ability to generate free-running temperature rhythms of different frequencies following combined food and drug exposures could reflect a state of internal desynchrony that may contribute to the addiction process and drug relapse.     

Influence of the mode of daily melatonin administration on entrainment of rat circadian rhythms.

In previous entrainment studies, melatonin (MEL) was administered by handling the animal, but because such handling may act as a confounding variable, the results from these studies are equivocal. The authors used MEL administration techniques that do not involve direct handling of the animal. Long Evans rats were used, and core body temperature (CBT) and wheel-running activity were recorded. One group of rats received a daily 1-h time-fixed infusion of MEL or the vehicle via a subcutaneous catheter. Animals in a second group had timed access to drinking water involving daily presence of drinking water containing MEL or the vehicle for 2 h at a fixed time of the day. Following entrainment to LD 12:12, both groups were transferred to constant darkness to free-run under vehicle administration. MEL was then administered, and entrainment occurred when activity onset coincided with MEL onset. Under both regimens, entrainment of wheel-running and CBT rhythms showed equal phase-relation to the onset of MEL administration, and free-running reoccurred when MEL was withdrawn. The authors concluded that MEL administration via drinking water and via infusion represent efficient ways to synchronize free-running rhythms in rats.     

ACTIVITY RHYTHMS OF WILD AND LABORATORY GOLDEN HAMSTERS (MESOCRICETUS AURATUS) UNDER ENTRAINED AND FREE-RUNNING CONDITIONS

The golden hamster (Mesocricetus auratus) is one of the most frequently used laboratory animals, particularly in chronobiological studies. One reason is its very robust and predictable rhythms, although the question arises whether this is an inbreeding effect or rather is typical for the species. We compared the daily (circadian) activity rhythms of wild and laboratory golden hamsters. The laboratory hamsters were derived from our own outbred stock (Zoh:GOHA). The wild hamsters included animals captured in Syria and their descendants (F1). Experiments were performed under entrained (light: dark [LD] 14h:10h) and under free-running (constant darkness, DD) conditions. Locomotor activity was recorded using passive infrared detectors. Under entrained conditions, the animals had access to a running wheel for a certain time to induce additional activity. After 3 weeks in constant darkness, a light pulse (15 min, 100 lux) was applied at circadian time 14 (CT14). Both laboratory and wild hamsters showed well-pronounced and very similar activity rhythms. Under entrained conditions, all hamsters manifested about 80% of their total 24h activity during the dark portion of the LD cycle. The robustness of the daily rhythms was also similar. However, interindividual variability was higher in wild hamsters for both measures. All animals used the running wheels almost exclusively during the dark portion of the LD cycle, although the wild hamsters were three times more active. The period length, measured in constant darkness, was significantly shorter in wild (23.93h ± 0.10h) than in laboratory hamsters (24.06 ± 0.07h). The light-induced phase changes were not different (about 1.5h). In summary, these results indicate that the laboratory hamster is not much different from the wild type. (Chronobiology International, 18(6), 921-932, 2001)     

Nonphotic enhancement of adjustment to new light-dark cycles: masking interpretation discounted

The adjustment of hamsters to advanced light-dark (LD) cycles can be greatly accelerated by scheduling a single 3-hr bout of extra activity in a novel running wheel, starting about 7 hr before the time when the animals become active in the preceding LD cycle. The present experiments were designed to provide stronger evidence that this effect depends on a shift in the pacemaker rather than on masking. It was shown that when hamsters were put into continuous darkness (DD) 1 day after the exercise-accelerated phase shift, their free-running rhythms took off from a time nearer to the onset of darkness in the new LD cycle than in the preceding LD cycle. An incidental finding was that in DD the free-running period of the hamsters with the accelerated phase shifts was longer than that of the control animals. Further evidence that the 3-hr exercise pulse had produced a greater phase advance than that occurring in undisturbed control animals was obtained by giving a light pulse at the same clock time to all animals after they had been in DD for 8 days. The animals that had previously exercised for the additional 3-hr phase-advanced in response to the light pulse, while the undisturbed control animals phase-delayed.     

Daily hoarding opportunity entrains the pacemaker for hamster activity rhythms

Summary The effects on activity rhythms of a daily 30 min opportunity to leave the home cage and hoard seeds from an open field were assessed in Syrian hamsters housed in continuous dim illumination. Six of ten hamsters responded with clear entrainment of their activity rhythms to the hoarding opportunity, as demonstrated by responses to phase shifts and by the onset phase of subsequent freerunning rhythms. No entrainable component separate from the freerunning rhythm was ever observed. Two hamsters showed phase shifts in response to the hoarding opportunity, but they did not meet the criteria for stable entrainment, and two did not respond with noticeable changes in rhythmicity. Ablations of the suprachiasmatic nuclei (SCN) were attempted in three hamsters that had entrained stably to the hoarding time. The effects of partial lesions in two animals indicated that the entrained rhythm was controlled by the light-entrainable pacemaker represented by the SCN. The one animal with an apparently complete lesion, however, developed a clear, but irregular, increase in activity in anticipation of the daily hoarding time. SCN ablation apparently unmasked an oscillator system separate from the SCN and susceptible to entrainment by a nonphotic cue. The oscillator mechanism affected by daily hoarding opportunities in hamsters appears to be tightly coupled to the SCN pacemaker, in contrast to the system in rats that is synchronized by daily feeding schedules.Abbreviations SCN suprachiasmatic nuclei - FEO food-entrain-able oscillator Portions of these results were previously reported at the 1986 meeting of the Animal Behavior Society, Tucson, Arizona, and at the 1987 meeting of the Society for Neuroscience, New Or leans, LA (Rusak et al. 1987)     

Rats made cirrhotic by chronic CCl4 treatment still exhibit anticipatory activity to a restricted feeding schedule

Food entrainment of clock genes in the liver suggests that this organ may underlie a food entrained oscillator (FEO), which manifests under restricted feeding schedule (RFS). In order to test the importance of a functional liver for the expression of FEO, chronic CCl4-treated cirrhotic rats and oil-treated controls were entrained to RFS and drinking behavior was continuously monitored. After 20 d of free-running conditions, food access was restricted to 2 h, followed by a refeeding-fasting protocol to test persistence of anticipatory drinking. Present data show no differences between groups for the onset and intensity of anticipation during RFS. After RFS, however, cirrhotic rats exhibited a significantly longer free-running period and a delay and lower intensity of the persistence of activity under fasting conditions. Histology confirmed injury of the liver chronically treated with CCl4. Present data indicate that a dysfunctional liver due to chronic CCl4 does not prevent animals from exhibiting anticipatory activity but may promote metabolic derangement of the clock mechanisms of the suprachiasmatic nucleus and the FEO.     

Circadian rhythmicity in dopamine content of mammalian retina: role of the photoreceptors

Dopamine, the predominant retinal catecholamine, is a neurotransmitter and neuromodulator known to regulate light-adaptive retinal processes. Because dopamine influences several rhythmic events in the retina it is also a candidate for a retinal circadian signal. Using high performance liquid chromatography (HPLC), we have tested whether dopamine and its breakdown products are rhythmic in Royal College of Surgeons (RCS) rats with normal and dystrophic retinas. In both normal and mutant animals entrained to a 12-h light/12-h dark cycle, we found robust daily rhythms of dopamine and its two major metabolites. To address circadian rhythmicity of dopamine content, rats were entrained to light/dark cycles and released into constant darkness, using the circadian rhythm of wheel-running activity as a marker of each individual's circadian phase. Circadian rhythms of dopamine and metabolite content persisted in both wild type and retinally degenerate animals held for two weeks in constant darkness. Our results demonstrate for the first time clear circadian rhythms of dopamine content and turnover in a free-running mammal, and suggest that rods and cones are not required for dopamine rhythmicity.     

Changes in circadian rhythms during puberty in Rattus norvegicus: developmental time course and gonadal dependency

During puberty, humans develop a later chronotype, exhibiting a phase-delayed daily rest/activity rhythm. The purpose of this study was to determine: 1) whether similar changes in chronotype occur during puberty in a laboratory rodent species, 2) whether these changes are due to pubertal hormones affecting the circadian timekeeping system. We tracked the phasing and distribution of wheel-running activity rhythms during post-weaning development in rats that were gonadectomized before puberty or left intact. We found that intact peripubertal rats had activity rhythms that were phase-delayed relative to adults. Young rats also exhibited a bimodal nocturnal activity distribution. As puberty progressed, bimodality diminished and late-night activity phase-advanced until it consolidated with early-night activity. By late puberty, intact rats showed a strong, unimodal rhythm that peaked at the beginning of the night. These pubertal changes in circadian phase were more pronounced in males than females. Increases in gonadal hormones during puberty partially accounted for these changes, as rats that were gonadectomized before puberty demonstrated smaller phase changes than intact rats and maintained ultradian rhythms into adulthood. We investigated the role of photic entrainment by comparing circadian development under constant and entrained conditions. We found that the period (τ) of free-running rhythms developed sex differences during puberty. These changes in τ did not account for pubertal changes in entrained circadian phase, as the consolidation of activity at the beginning of the subjective night persisted under constant conditions in both sexes. We conclude that the circadian system continues to develop in a hormone-sensitive manner during puberty.