Wheel-running Activity Rhythms Research Articles

NOCTURNALISM INDUCED BY SCHEDULED FEEDING IN DIURNAL OCTODON DEGUS

Octodon degus, a mainly diurnal rodent, characterized by its ability to shift to a nocturnal locomotor pattern under laboratory conditions, was studied to determine whether restricted food access during the scotophase could induce nocturnalism. To address this question, wheel running activity, feeding, and body temperature rhythms were analyzed for diurnal degus housed with a wheel and subjected to either long (12 h) or short (2 h) food availability periods, in the latter case with random or scheduled food access times. The results show that allowing nocturnal feeding for 2 h, but not 12 h, can shift a previous diurnal phase preference for wheel running activity and body temperature to the scotophase, with random feeding being more effective than scheduled food availability. However, this behavioral inversion proved to be unstable, as the degus returned to the diurnal phase within only a few days after the restricted feeding was discontinued. In addition, the negative masking effect induced by light, which is characteristic of the degus' nocturnal chronotype, was not observed when the animals were forced to feed at night. Thus, neither long, short, random, nor scheduled food-availability during the scotophase was able to induce all the characteristics of the nocturnal chronotype in Octodon degus. (Author correspondence: jamadrid@um.es)

Endogenous Rhythms inPeriod1Mutant Suprachiasmatic NucleiIn VitroDo Not Represent Circadian Behavior

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) controls daily rhythms of behavior and physiology. Lesions of the SCN cause arrhythmicity of locomotor activity, and transplants of fetal SCN tissue restore rhythmic behavior that is consistent with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. While several studies have demonstrated that the circadian characteristics of in vitro SCN rhythms represent circadian behavior, others have shown that the periods of explanted SCN are not always congruent with locomotor activity. We find that the aberrant rhythms of ex vivo SCN lacking functional Period1 (Per1(-/-)) do not represent the behavioral rhythms of the mutant animals. Surprisingly, in C57BL/6J Per1(-/-) mice, the real-time circadian gene promoter activity rhythm is weak or absent in adult SCN slices in vitro even though the free-running wheel-running activity rhythm is indistinguishable from wild-type (Per1(+/+)) mice. While some neurons in Per1(-/-) SCN explants exhibit robust circadian rhythms, others have irregular and/or low-amplitude rhythms. Together, these data suggest that either a small population of rhythmic neurons in the Per1(-/-) SCN is sufficient to control wheel-running activity or that in vivo physiological factors can compensate for the aberrant endogenous rhythms of Per1(-/-) SCN.

High‐fat feeding alters the clock synchronization to light

High-fat feeding in rodents leads to metabolic abnormalities mimicking the human metabolic syndrome, including obesity and insulin resistance. These metabolic diseases are associated with altered temporal organization of many physiological functions. The master circadian clock located in the suprachiasmatic nuclei controls most physiological functions and metabolic processes. Furthermore, under certain conditions of feeding (hypocaloric diet), metabolic cues are capable of altering the suprachiasmatic clock's responses to light. To determine whether high-fat feeding (hypercaloric diet) can also affect resetting properties of the suprachiasmatic clock, we investigated photic synchronization in mice fed a high-fat or chow (low-fat) diet for 3 months, using wheel-running activity and body temperature rhythms as daily phase markers (i.e. suprachiasmatic clock's hands). Compared with the control diet, mice fed with the high-fat diet exhibited increased body mass index, hyperleptinaemia, higher blood glucose, and increased insulinaemia. Concomitantly, high-fat feeding led to impaired adjustment to local time by photic resetting. At the behavioural and physiological levels, these alterations include slower rate of re-entrainment of behavioural and body temperature rhythms after 'jet-lag' test (6 h advanced light-dark cycle) and reduced phase-advancing responses to light. At a molecular level, light-induced phase shifts have been correlated, within suprachiasmatic cells, with a high induction of c-FOS, the protein product of immediate early gene c-fos, and phosphorylation of the extracellular signal-regulated kinases I/II (P-ERK). In mice fed a high-fat diet, photic induction of both c-FOS and P-ERK in the suprachiasmatic nuclei was markedly reduced. Taken together, the present data demonstrate that high-fat feeding modifies circadian synchronization to light.

From daily behavior to hormonal and neurotransmitters rhythms: Comparison between diurnal and nocturnal rat species

Mammalian species can be defined as diurnal or nocturnal, depending on the temporal niche during which they are active. Even if general activity occurs during nighttime in nocturnal rodents, there is a patchwork of general activity patterns in diurnal rodents, including frequent bimodality (so-called crepuscular pattern, i.e., dawn and dusk peaks of activity) and a switch to a nocturnal pattern under certain circumstances. This raises the question of whether crepuscular species have a bimodal or diurnal – as opposed to nocturnal – physiology. To this end, we investigated several daily behavioral, hormonal and neurochemical rhythms in the diurnal Sudanian grass rat ( Arvicanthis ansorgei) and the nocturnal Long–Evans rat ( Rattus norvegicus). Daily rhythms of general activity, wheel-running activity and body temperature, with or without blocked wheel, were diurnal and bimodal for A. ansorgei, and nocturnal and unimodal for Long–Evans rats. Moreover, A. ansorgei and Long–Evans rats exposed to light–dark cycles were respectively more and less active, compared to conditions of constant darkness. In contrast to other diurnal rodents, wheel availability in A. ansorgei did not switch their general activity pattern. Daily, unimodal rhythm of plasma leptin was in phase-opposition between the two rodent species. In the hippocampus, a daily, unimodal rhythm of serotonin in A. ansorgei occurred 7 h earlier than that in Long–Evans rats, whereas a daily, unimodal rhythm of dopamine was unexpectedly concomitant in both species. Multiparameter analysis demonstrates that in spite of bimodal rhythms linked with locomotor activity, A. ansorgei have a diurnally oriented physiology.

Entrainment and coupling of the hamster suprachiasmatic clock by daily dark pulses

The circadian rhythm of locomotor activity of hamsters kept in constant light (LL) can split into two distinct components that, in steady state, lie 180 degrees apart. The splitting phenomenon is the result of antiphase circadian oscillations between left and right sides of the suprachiasmatic nuclei (SCN), the master circadian clock in mammals. In unsplit hamsters housed in LL, a single dark pulse produces a phase-shift of the wheel-running activity rhythm, accompanied by a transient down-regulation of clock gene expression in the SCN. In the present study, we evaluated the effects of daily 1-hr dark pulses on wheel-running activity rhythm and on the expression of clock and nonclock proteins in the SCN of Syrian hamsters exposed to LL conditions. The results show that a daily 1-hr dark pulse entrained the rhythm of wheel-running activity of unsplit hamsters. In addition, in split animals, unimodal coupling of the two locomotor activity components was produced by daily 1-hr dark pulses. In the SCN, the effects of entrainment and unimodal coupling of the two separate components by dark observed in behavior were also evident in the bilateral expression of the proteins c-FOS, p-ERK, PERIOD 1, and calbindin. These results show that the bilaterally asymmetric SCN clock, underlying split circadian behavior, can be recoupled in phase and entrained by short daily dark exposure, indicating the synchronizing potency of darkness on the main circadian clock.

Serotonergic potentiation of dark pulse‐induced phase‐shifting effects at midday in hamsters

In mammals, resetting of the suprachiasmatic clock (SCN) by behavioral activation or serotonin (5-HT) agonists is mimicked by dark pulses, presented during subjective day in constant light (LL). Because behavioral resetting may be mediated in part by 5-HT inputs to the SCN, here we determined whether 5-HT system can modulate dark-induced phase-shifts in Syrian hamsters housed in LL. Two hours of darkness at mid-subjective day (circadian time 6; CT-6) resulted in increased concentrations of 5-HT in the SCN tissue and induction of c-FOS expression in the raphe nuclei. Injections of the 5-HT(1A/7) agonist +8-OH-DPAT or dark pulses at CT-6 induced phase-advances of the wheel-running activity rhythm and down-regulated the expression of the clock genes Per1-2 and c-FOS in the SCN in a similar way. The combination of both treatments [+8-OH-DPAT + dark pulses], however, resulted in larger phase-advances, while associated molecular changes were not significantly modified, except for the gene Dbp, in comparison to +8-OH-DPAT or dark pulses alone. Dark resetting was blocked by pre-treatment with a 5-HT(7) antagonist, but not with a 5-HT(1A) antagonist. The additive phase-shifts of two different cues to reset the SCN clock open wide the gateway for non-photic shifting, leading to new strategies in chronotherapy.

Behavioral and gene expression analyses of Wfs1 knockout mice as a possible animal model of mood disorder

Wolfram disease is a rare genetic disorder frequently accompanying depression and psychosis. Non-symptomatic mutation carriers also have higher rates of depression and suicide. Because WfS1, the causative gene of Wolfram disease, is located at 4p16, a linkage locus for bipolar disorder, mutations of WfS1 were suggested to be involved in the pathophysiology of bipolar disorder. In this study, we performed behavioral and gene expression analyses of Wfs1 knockout mice to assess the validity as an animal model of mood disorder. In addition, the distribution of Wfs1 protein was examined in mouse brain. Wfs1 knockout mice did not show abnormalities in circadian rhythm and periodic fluctuation of wheel-running activity. Behavioral analysis showed that Wfs1 knockout mice had retardation in emotionally triggered behavior, decreased social interaction, and altered behavioral despair depending on experimental conditions. Wfs1-like immunoreactivity in mouse brain showed a similar distribution pattern to that in rats, including several nuclei potentially relevant to the symptoms of mood disorders. Gene expression analysis showed down-regulation of Cdc42ep5 and Rnd1, both of which are related to Rho GTPase, which plays a role in dendrite development. These findings may be relevant to the mood disorder observed in patients with Wolfram disease.

Daily Meal Timing is Not Necessary for Resetting the Main Circadian Clock by Calorie Restriction

In rodents, entrainment and/or resetting by feeding of the central circadian clock, the suprachiasmatic nucleus (SCN), is more efficient when food cues arise from a timed calorie restriction. Because timed calorie restriction is associated with a single meal each day at the same time, its resetting properties on the SCN possibly depend on a combination of meal time-giving cues and hypocaloric conditions per se. To exclude any effect of daily meal timing in resetting by calorie restriction, the present study employed a model of ultradian feeding schedules, divided into six meals with different durations of food access (6 x 8-min versus 6 x 12-min meal schedule) every 4 h over the 24-h cycle. The effects of such an ultradian calorie restriction were evaluated on the rhythms of wheel-running activity (WRA) and body temperature (Tb) in rats. The results indicate that daily/circadian rhythms of WRA and Tb were shifted by a hypocaloric feeding distributed in six ultradian short meals (i.e. 6 x 8-min meal schedule), showing both phase advances and delays. The magnitude of phase shifts was positively correlated with body weight loss and level of day-time behavioural activity. By contrast, rats fed daily with six ultradian meals long enough (i.e. 6 x 12-min meal schedule) to prevent body weight loss, showed only small, if any, phase shifts in WRA and Tb rhythms. The results obtained reveal the potency of calorie restriction to reset the SCN clock without synchronisation to daily meal timing, highlighting functional links between metabolism, calorie restriction and the circadian timing system.

The experience in the water maze task can affect the circadian rhythm of locomotor activity

Wistar rats maintained in cages with running wheels and submitted to a skeleton photoperiod or to a light – dark cycle were tested in the Morris water maze. Half of the animals were exposed to the task during their active phase while the other half was exposed during their inactive phase. The effect of the experience in the water maze, a strong arousing event, on the rhythm of wheel-running activity was evaluated. In the first experiment, a group of animals submitted to a skeleton photoperiod was trained every day in the reference memory version of the task. The novel experience in the water maze had a strong phase-dependent masking effect: it produced an intense post-training bout of activity in the animals tested during their inactive phase. Another experiment was run using single working memory sessions in the water maze and with animals submitted to a light – dark cycle. The circadian rhythm of locomotor activity was evaluated on undisturbed days and compared with testing days. The experience in the water maze produced a significant increase in variability of activity onset during both circadian phases. Taken together, the data suggest that there is a modulating effect of the arousing experience in the pool on the overt circadian rhythm of locomotor activity.

Metabolic rhythm abnormalities in mice lacking VIP-VPAC2 signaling

The circadian pacemaker in the suprachiasmatic nuclei (SCN) controls endogenous near 24-h physiological and behavioral rhythms in metabolism, neuroendocrine function, and locomotor activity. Recently, we showed that vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2) are critical to the intercellular communication between individual SCN neurons, and appropriate synchronization and phasing of these oscillatory cells. Mice defective in VIP signaling manifest grossly impaired circadian rhythms of SCN neuronal firing activity and are typically unable to maintain rhythmic wheel-running behavior in the absence of external time cues. Here we report that daily rhythms of metabolism and feeding behavior are also overtly altered in these animals. Under diurnal conditions (12:12-h light-dark; LD), metabolic and feeding rhythms are advanced in mice lacking either VIP or VPAC(2) receptor expression, peaking in the late day, rather than early night, as observed in wild-type mice. When placed in constant light (LL), both VIP-deficient and VPAC(2) receptor-knockout mice exhibit dampening of metabolic and feeding rhythms, which deteriorate after a few days. In addition, overall metabolic rate is greatly reduced in VPAC(2)-knockout mice, when compared with wild-type mice, regardless of lighting condition. The advancement of metabolic and feeding rhythms in these mice under LD suggests that these rhythms are less sensitive to masking by light. These results demonstrate that altering SCN function not only affects neuronal and wheel-running activity rhythms but also dramatically impairs temporal regulation of metabolism and feeding.

Evaluation of serotonin, noradrenaline and dopamine reuptake inhibitors on light-induced phase advances in hamster circadian activity rhythms

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for the treatment of anxiodepressive states that are often associated with perturbed circadian rhythms including, in certain patients, phase advances. Surprisingly, the influence of SSRIs upon circadian activity rhythms has been little studied in experimental models. Accordingly, this study examined the ability of SSRIs to modulate the phase-setting properties of light on circadian activity rhythms in hamsters. Their actions were compared to those of the mixed serotonin/noradrenaline reuptake inhibitor (SNRI), venlafaxine, the selective noradrenaline reuptake inhibitor, reboxetine, and the dopamine reuptake inhibitor, bupropion. Wheel-running activity rhythms were recorded in male Syrian hamsters. Drugs were administered systemically before a light stimulus that was used to advance the timing of the hamster running rhythms. Four chemically diverse SSRIs, citalopram (1-10 mg/kg, intraperitoneally), fluvoxamine (1-10), paroxetine (1-10), and fluoxetine (10 and 20), all robustly and significantly inhibited the ability of light to phase advance hamster circadian wheel-running activity rhythms. Their actions were mimicked by venlafaxine (1-10) that likewise elicited a marked reduction in phase advances. Conversely, reboxetine (1-20) and bupropion (1-20) did not exert significant effects. These data suggest that suppression of serotonin (but not noradrenaline or dopamine) reuptake by SSRIs and SNRIs modifies circadian locomotor activity rhythms in hamsters. Further, they support the notion that an inhibitory influence upon the early-morning light-induced advance in circadian activity contributes to the therapeutic effects of serotonin uptake inhibitors in certain depressed patients.

Gastrin releasing peptide and neuropeptide Y exert opposing actions on circadian phase

Microinjection of gastrin releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other's effects on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other's effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells.

The After-Hours Mutant Reveals a Role for Fbxl3 in Determining Mammalian Circadian Period

By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.

Entrainment to Feeding but Not to Light: Circadian Phenotype of VPAC2Receptor-Null Mice

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2-/-) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2-/- mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2-/- mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

Behavioral characterization and modulation of circadian rhythms by light and melatonin in C3H/HeN mice homozygous for the RORβ knockout

This study reports for the first time the effects of retinoid-related orphan receptors [RORbeta; receptor gene deletion RORbeta(C3H)(-/-)] in C3H/HeN mice on behavioral and circadian phenotypes. Pineal melatonin levels showed a robust diurnal rhythm with high levels at night in wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. The RORbeta(C3H)(-/-) mice displayed motor ("duck gait," hind paw clasping reflex) and olfactory deficits, and reduced anxiety and learned helplessness-related behaviors. Circadian rhythms of wheel-running activity in all genotypes showed entrainment to the light-dark (LD) cycle, and free running in constant dark, with RORbeta(C3H)(-/-) mice showing a significant increase in circadian period (tau). Melatonin administration (90 microg/mouse sc for 3 days) at circadian time (CT) 10 induced phase advances, while exposure to a light pulse (300 lux) at CT 14 induced phase delays of circadian activity rhythms of the same magnitude in all genotypes. In RORbeta(C3H)(-/-) mice a light pulse at CT 22 elicited a larger phase advance in activity rhythms and a slower rate of reentrainment after a 6-h advance in the LD cycle compared with (+/+) mice. Yet, the rate of reentrainment was significantly advanced by melatonin administration at the new dark onset in both (+/+) and (-/-) mice. We conclude that the RORbeta nuclear receptor is not involved in either the rhythmic production of pineal melatonin or in mediating phase shifts of circadian rhythms by melatonin, but it may regulate clock responses to photic stimuli at certain time domains.

Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice.

The basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) domain transcription factor BMAL1 is an essential component of the mammalian circadian pacemaker. Bmal1-/- mice lose circadian rhythmicity but also display tendon calcification and decreased activity, body weight, and longevity. To investigate whether these diverse functions of BMAL1 are tissue-specific, we produced transgenic mice that constitutively express Bmal1 in brain or muscle and examined the effects of rescued gene expression in Bmal1-/- mice. Circadian rhythms of wheel-running activity were restored in brain-rescued Bmal1-/- mice in a conditional manner; however, activity levels and body weight were lower than those of wild-type mice. In contrast, muscle-rescued Bmal1-/- mice exhibited normal activity levels and body weight yet remained behaviorally arrhythmic. Thus, Bmal1 has distinct tissue-specific functions that regulate integrative physiology.

Enhanced circadian photoresponsiveness after prolonged dark adaptation in seven species of diurnal and nocturnal rodents

Previous studies in mice and Syrian hamsters have described an enhancement of circadian photoresponsiveness after exposure to darkness for several weeks. The present study investigated the generality of the phenomenon in 3 diurnal and 4 nocturnal rodent species. In four of the species tested, phase delays of the running-wheel activity rhythm evoked by 1-h light pulses were several-fold larger after 3 to 4 weeks of exposure to darkness than after a single day. This drastic change in photoresponsiveness has important implications for the understanding of the process of photic entrainment. Differences between species that showed a significant effect of dark adaptation and species that showed no effect were not accounted for by temporal niche (diurnal versus nocturnal) or photic sensitivity (albino versus pigmented). Further research is needed to elucidate the mechanisms responsible for inter-species differences in the occurrence of enhanced photoresponsiveness after dark adaptation and to identify the neural substrates of this phenomenon in species that exhibit it.

Glutamatergic activity modulates the phase‐shifting effects of gastrin‐releasing peptide and light

Previous studies have established that microinjection of gastrin-releasing peptide (GRP) into the suprachiasmatic nucleus (SCN) region or third ventricle causes circadian phase shifts similar to those produced by light pulses. Activation of N-methyl-d-aspartate (NMDA) receptors in the SCN region also produces light-like phase shifts. This study was designed to test the effects of (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist, and l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate reuptake inhibitor, on GRP-induced phase shifts. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the third ventricle were housed in constant darkness until stable free-running rhythms of wheel-running activity were apparent. Microinjection of GRP into the third ventricle at circadian time (CT)13 induced large phase delays. These GRP-induced phase delays were completely blocked by co-administration of AP5, suggesting that GRP-induced phase delays require concurrent activation of NMDA receptors. Microinjection of AP5 alone did not induce significant phase shifts. A second set of experiments was designed to test whether GRP-induced phase shifts would be enhanced by PDC. Co-administration of PDC and GRP elicited significantly larger phase delays at CT13 than GRP alone. However, administration of PDC alone did not induce a significant phase shift. Finally, when administered just prior to a light pulse, PDC elicited significantly larger phase delays than light pulse plus vehicle controls. These data suggest that the effects of GRP on the circadian clock phase are highly dependent on the level of excitation provided by activated NMDA receptors.

Illuminating serotonergic gateways for strong resetting of the mammalian circadian clock

circadian clocks regulate animal behavior and physiology by generating daily rhythms and coordinating these with local time. If you sleep well and abstain from flying around the globe or working rotating shift schedules, this pervasive influence is easy to take for granted, a background rhythm of

Gender differences in complaints

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.