Abstract
Mouse period1 (mPer1) and mPer2 are mammalian homologs of the Drosophila clock gene period that show robust oscillation in the suprachiasmatic nucleus, the mammalian master clock, and have been implicated as essential components of the core clock mechanism. Gene-targeting studies have demonstrated that mPer2 plays a dominant function in behavioral rhythm generation, although the role of mPer1 has not been fully clarified. Here, we report that prolongation of the lighting period (4-16 h) induces a larger-delay phase shift of the behavioral rhythm in mPer1-deficient (mPer1-/-) mice. During the light-elongation task, mPER2 protein decay in mPer1-/- mice is slower (∼4 h) than in wild-type mice, which thereby causes larger behavioral phase delay. mPer1-/- mice could not adapt to environmental light/dark cycles in long complete photoperiods with dim light or in long skeleton photoperiods. These photoperiodic conditions mimic natural environmental changes present at high latitudes, indicating that mPer1 could operate in the adaptation of the circadian clock of nocturnal mice to large seasonal changes of environmental light/dark cycles.
Introduction
Mouse period1 (mPer1) and mPer2 are mammalian homologs of the Drosophila clock gene period (for review, see Dunlap, 1999) that show robust oscillation in the suprachiasmatic nucleus (SCN), the mammalian master clock (Klein et al., 1991), and have been implicated as essential components of the core clock mechanism. Gene-targeting studies have demonstrated that mPer2 knock-out mice are arrhythmic in constant darkness (DD) (Zheng et al., 1999; Bae et al., 2001), whereas mPer1 knock-out (mPer1-/-) mice elicit a persistent rhythmicity with a slightly shorter period length (Bae et al., 2001; Cermakian et al., 2001; Zheng et al., 2001). Although it is demonstrated that mPer1 has a crucial role for rhythm generation in peripheral clocks (Cermakian et al., 2001; Pando et al., 2002), its function in the central oscillator remains unclear.
Phase shifting by light is an essential feature of circadian rhythms (Daan and Pittendrigh, 1976a). The phase-shift profiles arising from short exposure to light do not show a difference between mPer1-/- and wild-type (mPer1+/+) mice (Cermakian et al., 2001; Spoelstra et al., 2004), although a decrease in phase advances was noted just after the move to DD (Albrecht et al., 2001; Spoelstra et al., 2004). Because it is considered that the larger the duration of light exposure, the greater the phase shift that occurs (Daan and Pittendrigh, 1976b), in the present study, we adopted long light exposure as a task for revealing the role of mPer1 to detect the difference between mPer1-/- and mPer1+/+ mice. Here, we found that the magnitude of phase delays arising from long light in mPer1-/- mice was larger than that of wild-type mice, accompanying the delay of mPER2 protein decay without effecting a change at the mRNA level in the initial phase during the light exposure. The altered core clock machinery in these mice was evident in long complete photoperiods with dim light or in long skeleton photoperiods in which animals show free-running rhythms not adapting to environmental cycles.
Materials and Methods
Animals and behavioral rhythm monitoring. mPer1+/+, heterozygous mutant (mPer1+/-), and mPer1-/- mice (Cermakian et al., 2001) were bred and housed under light/dark (LD) 12 h cycles (fluorescent light, 200-300 lux). Locomotor activity was detected by passive (pyroelectric) infrared sensors (FA-05 F5B; Omron, Kyoto, Japan) (Shigeyoshi et al., 1997). Data were monitored and analyzed as described previously (Masubuchi et al., 2000) by Chronobiology kit (Stanford Software Systems, Stanford, CA). The experimental protocol of the current research was approved by the Committee for Animal Research at Kobe University.
In situ hybridization. In situ hybridization histochemistry using the free-floating sections was performed according to the method detailed previously (Shigeyoshi et al., 1997). We used 33P-radiolabeled cRNA probes for mPer2 (Takumi et al., 1998) and albumin gene D-site-binding protein (dbp) (Yamaguchi et al., 2000) for the in situ hybridization studies. The peak value of mPer1+/+ mice was adjusted to 100, and relative RNA abundance was used.
Immunocytochemistry. Immunocytochemistry was performed with the avidin-biotin-peroxidase method applied to free-floating sections (Ban et al., 1997). Serial frontal sections (30 μm thick) from the rostral end to the caudal end of the SCN were incubated for immunostaining with 1 μg/ml anti-mPER2 (affinity-purified rabbit antisera; Alpha Diagnostic International, San Antonio, TX) (Matsuo et al., 2003), which was finally visualized brown with 3,3′-diaminobenzidine tetrahydrochloride. We counted the mean number of immunoreactive cells in three sections in the middle portion of the SCN. For each point, we used four animals.
Statistical analysis. The 24 h mRNA and protein variations were statistically tested by one-way ANOVA. The effects of genotypes on the behavioral rhythm onsets were tested by two-way ANOVA. A post hoc Bonferroni/Dunn test was used for the comparison between the values of Per1+/+, Per1+/-, and Per1-/- groups at the same time points. The effects of genotypes on the entrainment daily lighting tasks (complete and skeleton photoperiod) were tested by Fisher's exact probability test.
Results
Extended light exposure induces larger phase delay of behavioral rhythm in mPer1-/- mice
mPer1+/+, mPer1+/-, and mPer1-/- mice (Cermakian et al., 2001) entrained to an LD cycle (12 h; fluorescent light, 200-300 lux) were exposed to 0, 4, 8, 12, and 16 h of light prolongation (LP) (task LP00, LP04, LP08, LP12, and LP16, respectively) from lights off [Zeitgeber time 12 (ZT12)] at the last day of LD. Afterward, mice were kept in DD. We defined the lights-off points of each task as L00 (corresponding to ZT12), L04 (ZT16), L08 (ZT20), L12 (ZT24), and L16 (ZT28, ZT4 of the next cycle), respectively. The phase-delay effect of the prolongation of light of the last day on the behavioral rhythm was evaluated using extrapolated eye-fitted lines made by >10 d of activity onsets. Only the switch from LD to DD does not shift the onset of the extrapolated rhythm in both in mPer1+/+ and mPer1-/- mice, when there was no light prolongation (LP00) (Fig. 1A, left). The onset of the behavioral rhythm was delayed in proportion to the increase of the duration of the light exposure (LP04, LP08, LP12) in all three groups (mPer1+/+, mPer1+/-, and mPer1-/- mice; one-way ANOVA; p < 0.0001) (Fig. 1B). The phase-delay effect peaked at 12 h (LP12) and then decreased at 16 h (LP16). Interestingly, depending on the genotype, the elongation of light exposure differentially affects the magnitude of the phase delay. As shown in Figure 1A, when mice were exposed to light prolongation for 12 h (LP12) for 1 d, the onset of activity rhythm in mPer1-/- mice was shifted by ∼12 h, strikingly different from mPer1+/+ mice (∼5 h). The effects of elongation of light exposure on the phase delay were significantly different between genotypes (two-way ANOVA; mPer1-/- vs mPer1+/+, p < 0.0001; mPer1-/- vs mPer1+/-, p < 0.0001; mPer1+/+ vs mPer1+/-, p < 0.01): the behavioral phase delay is much larger in mPer1-/- than mPer1+/+ (p < 0.0001 in LP04, LP08, LP12, and LP16 tasks) and than mPer1+/- (p < 0.0001 in LP08, LP12, and LP16 tasks) and slightly larger in mPer1+/- than mPer1+/+ (p < 0.0001 in LP16 task) (Fig. 1B). The prolongation of light exposure (4-16 h) produces larger phase delay of behavioral rhythm in mPer1-/- mice.
Extended light exposure does not yield differences in mPer2 gene expression in the SCN of mPer1-/- mice
Because the SCN is the master clock that times rhythmicity at the systemic level (Schibler and Sassone-Corsi, 2002), the behavioral changes observed here must be a reflection of the alteration of the circadian core transcription/translation feedback loop within the SCN. Indeed, rapid resetting by photic cues was confirmed in the SCN in vivo (Best et al., 1999) and in vitro (Asai et al., 2001). Because mPer2 is essential for the timing of the core feedback loop in the mammalian circadian clock (Hastings et al., 2003), we examined mPer2 expression profiles in the SCN during the light-prolongation task. In the usual 12 h LD regimen (ZT0, ZT4, ZT8, ZT12, ZT16, ZT20), in both mPer1-/- and mPer1+/+ mice, mPer2 mRNA began to increase before dawn, peaked at ZT12, and then steadily decreased by ZT16-20 (two-way ANOVA; mPer1-/- vs mPer1+/+; p = 0.094) (Fig. 2A). In 16 h of elongated light exposure (from L00 to L16) from lights off at ZT12 (L00), which induces a larger phase delay in mPer1-/- mice, mPer2 transcript levels increased with 4 h of light prolongation (L04); thereafter, expression decreased during extended light exposure in both mPer1-/- and mPer1+/+ mice in a time-dependent manner (one-way ANOVA; p < 0.0001) (Fig. 2B). Because mPer1-/- and mPer1+/+ mice display analogous profiles of mPer2 expression (two-way ANOVA; p = 0.128) (Fig. 2B), light-induced transient increase of mPer2 transcription may occur similarly in these mice. These results indicated that the cause of the enhanced delay of behavioral onset by light prolongation in mPer1-/- mice was not attributable to changes at the mPer2 transcription level.
Core clock oscillation and its output show a larger phase delay in the second cycle after extended light exposure in mPer1-/- mice
We then examined mPer2 expression profiles after the end of a 12 h light prolongation (LP12 task) with 4 h intervals (D04, D08, D12, D16, D20, and D24). A LP12 task (12 h of light prolongation) was chosen because the magnitude of the behavioral phase shift was largest in both genetic backgrounds (Fig. 1B). mPer1+/+ mice displayed an increase at D08 and a peak at D16 followed by a decrease (Fig. 2C). The increase of mPer2 expression in mPer1-/- mice began at D12 and peaked at D20. Thus, in contrast to similar expression profiles observed during the long light prolongation in mPer1-/- and mPer1+/+ mice (Fig. 2B), mPer2 mRNA peaks 4 h later in mPer1-/- mice (two-way ANOVA; p < 0.0001) (Fig. 2C). The magnitude of the behavioral phase shift (4-6 h) did not correlate with the first mPer2 mRNA expression profiles in light but correlated with the phases of mPer2 expression in the next cycle. As an indicator of clock output levels, we next examined the expression profiles of a clock-controlled gene, dbp, the transcription of which is directly regulated by clock genes (Ripperger et al., 2000; Yamaguchi et al., 2000). As shown in Figure 2D, dbp expression was equivalent in mPer1-/- and mPer1+/+ mice during the extended-light task (from L00 to L12; two-way ANOVA; p = 0.2696). However, after the task, dbp expression in mPer1-/- mice displayed a 4 h delay when compared with mPer1+/+ mice (from D04 to D24; two-way ANOVA; p < 0.0001). These findings suggest that expression of core clock genes and clock-controlled genes did not reflect the behavioral phase shift seen during extended light exposure without mPer1. But after the task, the expression of these genes does reflect the behavioral rhythms; this occurs after the cessation of the lighting task.
Delayed disappearance of mPER2 protein in the SCN of mPer1-/- mice after the extended light exposure
Because there is a dissociation between clock gene expression during the extended-light task and the observed behavioral phase shifts, we examined the expression profile of clock protein levels to know whether there is a dissociation between protein expression patterns and behavioral rhythms. In the usual 12 h LD regimen, in both mPer1-/- and mPer1+/+ mice, mPER2 protein began to increase at ZT8, peaked at ZT16, and then steadily decreased by ZT0 (Fig. 3A). We examined the expression of mPER2 protein in the SCN during (L00, L04, L08, and L12) and after light prolongation (D04, D08, and D12) (Fig. 3B, left). At L00 (ZT12), mPER2 protein levels were similarly high in both mPer1-/- and mPer1+/+ mice. In mPer1+/+ mice, mPER2 levels began to decrease at L08 and reached minimal expression at D08, to increase thereafter (D12) (Fig. 3B, left). In contrast, the high expression of mPER2 did not decrease until L08 in mPer1-/- mice, to reach minimal levels at L12 (4 h later than mPer1+/+ mice) and then to steadily decrease until D12. From L04 to D08, mPER2 levels are clearly higher in mPer1-/- mice than that in mPer1+/+ mice (two-way ANOVA; p < 0.01). We then examined mPER2 protein expression profiles in the next cycle at 4 h intervals (D16, D20, D24, D28, D32, and D36). In mPer1+/+ mice, mPER2 at D16 steadily decreased until D32. In mPer1-/- mice, however, mPER2 protein increased until D24 and then decreased thereafter (Fig. 3B, right). Clear phase difference (4-8 h) of mPER2 expressions between mPer1-/- mice and mPer1+/+ mice was observed in the second cycle (two-way ANOVA; p < 0.0001) (Fig. 3B, right). These findings reveal that mPer1 regulates mPER2 protein levels without affecting mPer2 transcription and that the alteration is likely to induce profound changes within transcription of core clock components and clock-controlled genes for the next cycle, which is then reflected at behavioral level.
mPer1-deficient mice cannot adapt to environmental light/dark cycles consisting of long complete photoperiods of dim light or long skeleton photoperiods
The larger phase resetting that we observed after several hours of light prolongation raised a possibility that mPer1-/- mice have a difficulty in entraining to a long-day environment. It is known that expression profiles of Per genes alter in response to photoperiodic change (Steinlechner et al., 2002). First, we put animals in a long-day schedule: we exposed mPer1-/- and mPer1+/+ mice to LD 20/4 h (light on from ZT0 to ZT20; mPer1-/-, n = 14; mPer1+/+, n = 12; 200 lux illumination) (Fig. 4A-C) and LD 17/7 h (mPer1-/-, n = 8; mPer1+/+, n = 8; 200 lux illumination) (Fig. 4D-F). During the period of observation (3-10 weeks), all mice entrained to the both long-day tasks. Because this entrain-ability is also influenced by the intensity of light, we decrease the intensity of luminescence to ∼0.3 lux, adding the long-day schedules in the first group (LD 20/4 h group). It is possible that low light intensity (0.3 lux) (Fig 4 A-C,G) conditions are more natural for nocturnal rodents than the classically high light intensity (200-300 lux) in which usual laboratory animals are housed, because nocturnal rodents live in dim light or darkness in day-time. Nine of 14 mPer1-/- mice could not entrain to the dim long-day photoperiod in 35 d (Fisher's exact probability test; p < 0.005) (Fig. 4G) by phase delaying (Fig. 4A) and advancing (Fig. 4B). In contrast, all mPer1+/+ mice kept entrained to the long complete photoperiod for >35 d (Fig. 4C,G). However, low light conditions per se are not enough to entrain the rhythm, because all control mice (mPer1-/-, n = 8; mPer1+/+, n = 6) of the first group kept entrained to LD 12 h lighting cycle in 0.3 lux for >35 d (data not shown; Fisher's exact probability test; p = 0.65).
Next, a second group (LD 17/7 h group; light on, ZT19; lights off, ZT12) was exposed to skeleton photoperiods (Fig. 4D-F,H). Johnson et al. (2003) speculated that in the case of nocturnal animals, a skeleton photoperiod mimics natural seasonal lighting changes better than complete photoperiods. DeCoursey (1986) suggests that nocturnal rodents in nature reset their clocks by short light exposures. In her simulation experiments, nocturnal rodents (flying squirrels) housed in cages containing dark nest boxes returned to their nest during day-time, and therefore they reset their clocks by short periods (only several minutes per day) of daily light sampling. After entrainment to a LD 17/7 h lighting cycle for 3 weeks, mice were moved to skeleton photoperiods [L/D/L/D, 0.5/16/0.5/7 h; light on from ZT19 to ZT19.5 and from ZT11.5 to ZT12 (the beginning of the skeleton photoperiod being ZT19)]. As shown in Figure 4, D and E, all eight mPer1-/- mice could not entrain to the skeleton photoperiod during 30 d. In contrast, as shown in Figure 4F, seven of eight mPer1+/+ mice kept entrained to the skeleton photoperiod for 44 d (Fisher's exact probability test; p < 0.005) (Fig. 4H). In this experimental condition, all desynchronized mice moved to another light interval by delaying the clock. In contrast, after entrainment to a LD 12 h lighting cycle for >3 weeks, control mice (mPer1-/-, n = 8; mPer1+/+, n = 8) of the second group were moved to a skeleton photoperiod [L/D/L/D, 0.5/11/0.5/12 h; light on from ZT0 to ZT0.5 and from ZT11.5 to ZT12 (the beginning of skeleton photoperiod being ZT0]. In this skeleton photoperiod, seven of eight mPer1-/- mice and all eight mPer1+/+ mice remained entrained for 44 d (data not shown; Fisher's exact probability test; p = 0.5). Thus, whether synchronization occurs is a result of the difficulty of the task, and we speculate that mPer1-/- mice have weaker adaptation ability to environmental light/dark cycle.
Discussion
In the present study, we adopted a strong-lighting task to test the role of mPer1 in clock resetting. The prolongation of the lighting period clearly induces a larger-delay phase shift of the behavioral rhythm in mPer1-/- mice. Compared with wild-type mice, in mPer1-/- mice, long light exposure initially changed mPER2 protein levels, although the expression profiles of mPer2 at mRNA level was not altered. This result suggests that mPer1 suppresses synthesis or enhances decay of light induced mPER2 in the SCN. In mPer1-/- mice, this increased mPER2 may cause larger behavioral phase delay, because mPer2 mutant show attenuated phase delay by light pulse (Spoelstra et al., 2004). It is possible that desynchronization from the photoperiod (Fig. 4) is caused by the stability of mPER2 in mPer1-/- mice.
These findings raise the attractive possibility that mPER1 represses light-induced mPER2 at the protein level and attenuates phase resetting by light. Because this process is abolished in mPer1 knock-out mice, environmental light information readily changes the phases of these animals. Although the molecular process involved is unknown at present, the dimerization of mPER1 and mPER2 (Zylka et al., 1998; Field et al., 2000; Yagita et al., 2000) may provide a likely explanation, because it could indeed influence the phosphorylation and/or ubiquitine proteasome-dependent mPER2 degradation.
mPer1-/- mice could not entrain to experimental long photoperiod with dim light or to skeleton photoperiods. These conditions (dim light, skeleton photoperiod) may mimic the natural lighting schedule for nocturnal rodents. From the present investigation, therefore, it is possible that a nocturnal animal that has mPer1 has an advantage for entrainment to environmental light/dark cycles.
One essential issue is whether the duplication of clock genes in mammals has happened only to ensure functional redundancy or whether it has a physiological significance in the context of evolution. Our study underscores the importance of mPer1 in entrainment to dim long photoperiods and long skeleton photoperiods, conditions that mimic seasonal changes of day/night (long-day condition) for nocturnal rodents living in nature. Our results demonstrate that mPer1 appears to perform a function completely distinct from mPer2, because it is involved in the plasticity of the circadian system, allowing it to adapt to changing photoperiodic cycles.
Footnotes
This work was supported by the Scientific Research Fund from the Ministry of Health, Labor and Welfare of Japan (H.O.), by SRF (H.O.), by grants from the Special Coordination Funds on Priority Areas of the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.O.), by the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Université Louis Pasteur, Human Frontier Science Program (RG-240), and Ligue contre le Cancer (P.S.-C.), and by a grant-in-aid for Encouragement of Young Scientists from the Japan Society for the Promotion of Science (S.M.). We thank W. Schwartz for discussions and H. Isejima for assistance.
Correspondence should be addressed to Dr. Hitoshi Okamura, Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: okamurah{at}kobe-u.ac.jp.
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