Photic and nonphotic circadian phase resetting in a diurnal primate, the common marmoset

Despite the considerable literature on circadian entrainment, there is little information on this subject in diurnal mammals. Contributing to this lack of understanding is the problem of separating photic from nonphotic (behavioral) phase-resetting events in diurnal species. In the present study, photic phase resetting was obtained in diurnal common marmosets held under constant dim light (DimDim; <0.5 lx) by using a 20-s pulse of bright light to minimize time available for behavioral arousal. This stimulus elicited phase advances at circadian time (CT) 18-22 and phase delays at CT9-12. Daily presentation of these 20-s pulses produced entrainment with a phase angle of approximately 11 h (0 h = activity onset). Nonphotic phase resetting was obtained under DimDim with the use of a 1-h-induced activity pulse, consisting of intermittent cage agitation and water sprinkling, delivered in total darkness to minimize photic effects. This stimulus caused phase delays at CT20-24, and entrainment to a scheduled daily regimen of these pulses occurred with a phase angle of approximately 0 h. These results indicate that photic and nonphotic phase-response curves (PRCs) of marmosets are similar to those of nocturnal rodents and that nonphotic PRCs are keyed to the phase of the suprachiasmatic nucleus pacemaker, not to the phase of the activity-rest cycle.     

Nonphotic entrainment in a diurnal mammal, the European ground squirrel (Spermophilus citellus).

Entrainment by nonphotic, activity-inducing stimuli has been investigated in detail in nocturnal rodents, but little is known about nonphotic entrainment in diurnal animals. Comparative studies would offer the opportunity to distinguish between two possibilities. (1) If nonphotic phase shifts depend on the phase of the activity cycle, the phase response curve (PRC) should be about 180 degrees out of phase in nocturnal and diurnal mammals. (2) If nonphotic phase shifts depend on the phase of the pacemaker, the two PRCs should be in phase. We used the diurnal European ground squirrel (Spermophilus citellus) in a nonphotic entrainment experiment to distinguish between the two possibilities. Ten European ground squirrels were kept under dim red light (<1 lux) and 20 +/- 1 degrees C. During the entrainment phase of the experiment, the animals were confined every 23.5 h (T) to a running wheel for 3 h. The circadian rhythms of 6 squirrels entrained, 2 continued to free run, and 2 possibly entrained but displayed arrhythmicity during the experiment. In a second experiment, a photic pulse was used in a similar protocol. Five out of 9 squirrels entrained, 1 did not entrain, and 3 yielded ambiguous results. During stable entrainment, the phase-advancing nonphotic pulses coincided with the end of the subjective day, while phase-advancing light pulses coincided with the start of the subjective day: mean psi(nonphotic) = 11.4 h; mean psi(photic) = 0.9 h (psi defined as the difference between the onset of activity and the start of the pulse). The data for nonphotic entrainment correspond well with those from similar experiments with nocturnal Syrian hamsters where psi(nonphotic) varied from 8.09 to 11.34 h. This indicates that the circadian phase response to a nonphotic activity-inducing stimulus depends on the phase of the pacemaker rather than on the phase of the activity cycle.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

Circadian and acamprosate modulation of elevated ethanol drinking in mPer2 clock gene mutant mice

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12?h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2?h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ～60 vs. ～25?mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300?mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout.     

Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes

In mammals, sleep is regulated by circadian and homeostatic mechanisms. The circadian component, residing in the suprachiasmatic nucleus (SCN), regulates the timing of sleep, whereas homeostatic factors determine the amount of sleep. It is believed that these two processes regulating sleep are independent because sleep amount is unchanged after SCN lesions. However, because such lesions necessarily damage neuronal connectivity, it is preferable to investigate this question in a genetic model that overcomes the confounding influence of circadian rhythmicity. Mice with disruption of both mouse Period genes (mPer)1 and mPer2 have a robust diurnal sleep-wake rhythm in an entrained light-dark cycle but lose rhythmicity in a free-run condition. Here, we examine the role of the mPer genes on the rhythmic and homeostatic regulation of sleep. In entrained conditions, when averaged over the 24-h period, there were no significant differences in waking, slow-wave sleep (SWS), or rapid eye movement (REM) sleep between mPer1, mPer2, mPer3, mPer1-mPer2 double-mutant, and wild-type mice. The mice were then kept awake for 6 h (light period 6-12), and the mPer mutants exhibited increased sleep drive, indicating an intact sleep homeostatic response in the absence of the mPer genes. In free-run conditions (constant darkness), the mPer1-mPer2 double mutants became arrhythmic, but they continued to maintain their sleep levels even after 36 days in free-running conditions. Although mPer1 and mPer2 represent key elements of the molecular clock in the SCN, they are not required for homeostatic regulation of the daily amounts of waking, SWS, or REM sleep.     

Phase responses to light pulses in mice lacking functional per or cry genes

The phase-resetting properties of the circadian system in mice with a functional deletion in mCry1, mCry2, mPer1, or mPer2 were studied in 2 experiments. In experiment 1, mCry1(-/-) and mCry2(-/-) mice as well as mPer1(Brdm1) and mPer2(Brdm1) mutant mice were exposed to 15-min light pulses during the 1st cycle following entrainment, either early (external time [ExT] 20) or late (ExT 4) in the subjective night. In experiment 2, a full PRC was measured for all these strains by exposure to light pulses of the same duration and intensity in free-running conditions in constant darkness. Directly after entrainment (experiment 1), mPer1(Brdm1) animals did not show significant phase advances by a light pulse in the late subjective night (ExT 4), as in the study by Albrecht et al. In the same experiment, mPer2(Brdm1) mice became arrhythmic too frequently to reliably measure their phase responses. Mice with a targeted gene disruption in mCry1 or mCry2 showed increased phase delays compared to wild type after exposure to a light pulse in the early subjective night (ExT 20). Otherwise, phase shifts were not significantly affected. In free run (experiment 2), all genotypes did show phase advances and phase delays. The mPer2(Brdm1) mutant PRC was above the mPer1(Brdm1) mutant and wild-type PRC (i.e., less delayed and more advanced) at most circadian phases. The mPer1(Brdm1) mutant PRC was not distinguishable from the wildtype PRC. The mCry2(-/-) mice showed much smaller phase delays than did mCry1(-/-) mice in the subjective evening (delay phase). In general, mPer2(Brdm1) mutant mice were more accelerated by light compared to mPer1(Brdm1) and wildtype control mice, whereas mCry1(-/-) mice were more delayed by light than were mCry2(-/-) mice.     

Period gene expression in mouse endocrine tissues

Circadian rhythms are generated by the oscillating expression of the Per1 and Per2 genes, which are expressed not only in the central brain pacemaker but also in peripheral tissues. Hormones are likely to coordinate physiological function in time. We performed in situ hybridization to localize mPer1 and mPer2 mRNA to particular cell types and tissue compartments in adrenal, thyroid, and testis. BALB/c mice maintained in a 12:12-h light-dark cycle expressed mPer1 in adrenal medulla, particularly in late afternoon and early night. mPer2 mRNA was more intensely expressed in adrenal cortex, especially in afternoon and evening. mPer1 mRNA was detected in thyroid. mPer1 was found in some but not all seminiferous tubules of each mouse at all times of day. Quantitation in C57BL/6 mice revealed a significant increase in the number of heavily labeled seminiferous tubules early in the night. Consistent with in situ hybridization, immunocytochemistry showed PER1 protein in spermatocytes and spermatids (spermatogenic stages VII-XII). Staining in spermatogonia and interstitial cells was inconsistent. Double labeling with 5'-bromodeoxyuridine showed PER1 expression first occurring 5 days after DNA replication. We conclude that mPeriod genes are expressed in peripheral endocrine glands. Central regulation, adenohypophyseal control, and functional importance of expression and phase remain to be elucidated.     

Light-induced phase shifts in mice lacking mPER1 or mPER2

Three homologs of the Drosophila Period gene have been identified in mammals. In mice, these three genes (mPer1, mPer2, and mPer3) have distinct roles in the circadian clockwork. While products of mPer1 and mPer2 play important roles in the maintenance of circadian rhythmicity, mPer3 gene products are dispensable for rhythmicity. Several studies also implicate mPER1 and mPER2 in transduction of photic information to the core circadian clockwork. The phase-shifting effects of light were examined in mPER1-deficient and mPER2-deficient mice using T cycle paradigms, in which mice received 1 h of light per day at an interval of T hours. To assess phase delays, repeated exposure to 1 h of light per day at T = 24 was used. To assess phase advances, exposure to 1-h light pulses at T = 22-h intervals was used. The degeneration of rhythmicity in the mutant mice prevented assessment of a response in most cases. Nevertheless, clear examples of phase delays and phase advances were observed in both mPer1 and mPer2 mutant mice. These results are not consistent with the hypothesis that mPER1 and mPER2 play necessary and nonoverlapping roles in mediating the effects of light on the circadian dock.     

Scheduled voluntary wheel running activity modulates free-running circadian body temperature rhythms in Octodon degus

Entrainment of the circadian pacemaker to nonphotic stimuli, such as scheduled wheel-running activity, is well characterized in nocturnal rodents, but little is known about activity-dependent entrainment in diurnal or crepuscular species. In the present study, effects of scheduled voluntary wheel-running activity on circadian timekeeping were investigated in Octodon degus, a hystricomorph rodent that exhibits robust crepuscular patterns of wakefulness. When housed in constant darkness, O. degus exhibited circadian rhythms in wheel-running activity and body temperature (Tb) with an average period length (tau) of 23.39 +/- 0.11 h. When wheel running was restricted to a fixed 2-h schedule every 24 h, tau increased on average 0.39 +/- 0.09 h but did not result in steady-state entrainment. Instead, relative coordination between the fixed running schedule and circadian timing was observed. Tau was greatest when scheduled wheel running occurred at CT 20.5 (0.4 h greater than DD baseline tau). Scheduled running activity also influenced Tb waveform symmetry, reflecting concomitant changes in the circadian activity-rest ratio (alpha:rho). Aftereffects of the scheduled wheel-running paradigm were also observed. In 2 animals, tau lengthened from 23.20 and 23.80 h to 24.14 and 24.15 h, respectively, and remained relatively stable for approximately 1 month during the wheel schedule. Although behavioral activity appears to be a weak zeitgeber in this species, these data suggest that nonphotic stimuli can phase delay the circadian pacemaker in O. degus at similar times of the day as in nocturnal hamsters and mice, and in humans.     

Loss of dexras1 alters nonphotic circadian phase shifts and reveals a role for the intergeniculate leaflet (IGL) in gene-targeted mice

Loss of Dexras1 in gene-targeted mice impairs circadian entrainment to light cycles and produces complex changes to phase-dependent resetting responses (phase shifts) to light. The authors now describe greatly enhanced and phase-specific nonphotic responses induced by arousal in dexras1(-/-) mice. In constant conditions, mutant mice exhibited significant arousal-induced phase shifts throughout the subjective day. Unusual phase advances in the late subjective night were also produced when arousal has little effect in mice. Bilateral lesions of the intergeniculate leaflet (IGL) completely eliminated both the nonphotic as well as the light-induced phase shifts of circadian locomotor rhythms during the subjective day, but had no effect on nighttime phase shifts. The expression of FOS-like protein in the suprachiasmatic nucleus (SCN) was not affected by either photic or nonphotic stimulation in the subjective day in either genotype. Therefore, the loss of Dexras1 (1) enhances nonphotic phase shifts in a phase-dependent manner, and (2) demonstrates that the IGL in mice is a primary mediator of circadian phase-resetting responses to both photic and nonphotic events during the subjective day, but plays a different functional role in the subjective night. Furthermore, (3) the change in FOS level does not appear to be a critical step in the entrainment pathways for either light or arousal during the subjective day. The cumulative evidence suggests that Dexras1 regulates multiple photic and nonphotic signal-transduction pathways, thereby playing an essential role modulating species-specific characteristics of circadian entrainment.     

IMPAIRED EXPRESSION OF THE mPer2 CIRCADIAN CLOCK GENE IN THE SUPRACHIASMATIC NUCLEI OF AGING MICE

The expression of circadian clock genes was investigated inthe suprachiasmatic nuclei (SCN) of young adult and old laboratory mice. Sampleswere taken at two time points, which corresponded to the expected maximum(circadian time 7 [CT7]) or minimum (CT21) of mPermRNA expression. Whereas the young mice had a stable and well-synchronizedcircadian activity/rest cycle, the rhythms of old animals were less stableand were phase advanced. The expression of mPer1mRNA and mPer2 mRNA was rhythmic in bothgroups, with peak values at CT7. The levels of mClockand mCry1 mRNA were not different dependingon the time of day and did not vary with age. In contrast, an age-dependentdifference was found in the case of mPer2(but not mPer1) mRNA expression, with themaximum at CT7 significantly lower in old mice. The decreased expression of mPer2 may be relevant for the observed differencesin the overt activity rhythm of aged mice. (ChronobiologyInternational, 18(3), 559–565, 2001)     

Patterns of wheel running are related to Fos expression in neuropeptide-Y-containing neurons in the intergeniculate leaflet of Arvicanthis niloticus

A variety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGL of Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.     

Photic phase response curve in Octodon degus: assessment as a function of activity phase preference

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.     

MPer1 and mper2 are essential for normal resetting of the circadian clock

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. A light pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.     

Serine 714 might be implicated in the regulation of the phosphorylation in other areas of mPer1 protein

The phosphorylation of mPer proteins may play important roles in the mechanism of the circadian clock via changes in subcellular localization and degradation. However, the mechanism has remained unclear. Previously, we identified three putative casein kinase (CK)1epsilon phosphorylation motif clusters in mPer1. In this work, we examined the role of the phosphorylation of serine residue, Ser(S)714, in mPer1. mPer1 S[714-726]A mutant, in which potential phosphorylation serine residues replaced by alanine residues, is rapidly phosphorylated compared with wild-type mPer1 by CK1epsilon. Coexpression with S[714]G mutant of mPer1 advanced phase of circadian expression of mPer2-luc expression, which was monitored by in vitro bioluminescence system. This result showed that the mPER1 S[714]G mutation affects circadian core oscillator. Considering these, it seems that Ser 714 might be involved in the regulation of the phosphorylation of other sites in mPer1 by CK1epsilon.     

Bimodal clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues under a 7-hour light and 5-hour dark schedule

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.