rPer1 and rPer2 induction during phases of the circadian cycle critical for light resetting of the circadian clock

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

rPer1 and rPer2 induction during phases of the circadian cycle critical for light resetting of the circadian clock Mamoru Nagano a , Akihito Adachi a , Koh-hei Masumoto b , Elizabeth Meyer-Bernstein c , Yasufumi Shigeyoshi a,⁎ a

Department of Anatomy and Neurobiology, Kinki University School of Medicine 377-2 Ohno-Higashi, Osakasayama City, Osaka 589-8511, Japan b Department of Physics, Informatics and Biology, Yamaguchi University, Yoshida, Yamaguchi 753-8512, Japan c Department of Biology, College of Charleston, Charleston, SC 29424, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Photic resetting of a biological clock is one of the fundamental characteristics of circadian

Accepted 17 June 2009

systems and allows living organisms to adjust to a particular environment. Nocturnal light

Available online 24 June 2009

induces the Per1 and Per2 genes, which leads to a resetting of the circadian clock in the suprachiasmatic nucleus (SCN), the mammalian circadian center. In our present study, we

Keywords:

investigated whether light differentially induces the rat Per1 (rPer1) and Per2 (rPer2) genes to

SCN

enable resetting of their circadian clocks. In a 24-hour LD cycle (12 h light:12 h dark), which is

Entrainment

shorter than the normal free-running period for rats, Per1 alone showed strong induction in

Per1

the ventrolateral region of the SCN (VLSCN) during the early day. In contrast, in a 25 hour LD

Per2

cycle (12.5 h light:12.5 h dark), which is longer than the free running period for these

In situ hybridization

animals, rPer2 alone was strongly induced in the VLSCN, at the end of the light phase and

Circadian rhythm

during the early dark periods. Our current findings therefore suggest that Per1 and Per2 are differentially regulated for daily entrainment to the LD cycle. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Circadian rhythms are generated by endogenous timekeeping mechanisms that have been conserved throughout evolution. In mammals, the suprachiasmatic nucleus (SCN) is the center of these circadian rhythms and within the SCN, individual neurons are autonomous oscillators that generate circadian firing rhythms (Herzog et al., 1998; Liu et al., 1997; Welsh et al., 1995). Molecular dissection of these processes has further demonstrated that the generation of the circadian rhythm is produced by a transcription/translation feedback loop that regulates clock genes (Chang and Reppert, 2001; Dunlap, 1999; Ripperger and Schibler, 2001; Wager-Smith and Kay, 2000).

The endogenous circadian clock of the mammalian SCN can be adjusted by light exposure, which can in turn synchronize the biological clock in the SCN with the external environment. Although the molecular mechanisms underlying this are not yet fully understood, a number of recent findings have now suggested that the induction of Per1/Per2 expression by light during the night is the principal event that produces long term state changes, i.e. the phase shift of the circadian rhythm. Per1 and Per2 are induced in the SCN after light exposure during the night (Albrecht et al., 1997; Shearman et al., 1997; Shigeyoshi et al., 1997; Takumi et al., 1998; Yan and Silver, 2002; Zylka et al., 1998) and the PER1 protein has also been shown to respond to light during the night,

⁎ Corresponding author. Fax: +81 72 368 1031. E-mail address: [email protected] (Y. Shigeyoshi). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.06.051

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suggesting that it plays a role in photic entrainment (Field et al., 2000; Yan and Silver, 2004). Furthermore, antisense oligonucleotides against either mPer1 or mPer2 have been shown to attenuate the extent of the phase shift produced by a light exposure event (Akiyama et al., 1999; Tischkau and Gillette, 2005; Wakamatsu et al., 2001). These findings together suggest that Per1 and Per2 are fundamentally involved in the resetting of the circadian clock. Since the period of the endogenous circadian clock is not an even 24 h, biological organisms have to adjust their internal clock to the environmental LD cycle on a daily basis. Morning and evening light is particularly important for the resetting of the central circadian clock. Living organisms with circadian periods of less than 24 h require exposure to clock-delaying light at dusk and organisms with periods that are greater than 24 h need to be exposed to clock-advancing light at dawn (Refinetti, 2006). Hence, and Assuming that Per1 and Per2 activation is fundamental to phase resetting, these genes will need to be induced during the critical period in which the central clock is reset. In our present study, we examined whether Per1 and Per2 in the rat are transactivated both at dawn and at dusk during the daily resetting of the SCN clock to steady LD conditions. We utilized a T-cycle experimental system that compels the animals to advance or delay their entrainment to environmental LD cycles and then determined whether Per1 and Per2 are differentially induced.

2.

Results

2.1. Time course analysis of rPer1/rPer2 expression during a 24 hour period under LD and DD conditions We examined the expression of the rPer1/rPer2 genes at around dawn and dusk. These timepoints corresponded to periods from 30 min before (−30 min: ZT23.5) to 120 min after (+120 min: ZT2) the onset of the light phase and 90 min before (−90 min: ZT10.5) to 60 min after (+60 min: ZT13) the end of the light phase (Figs. 1 and 2), respectively. A cluster of intenselylabeled rPer1-expressing neurons was detectable in the VLSCN at 30 min after the onset of the light phase (+30 min: ZT0.5; Fig. 1B) under LD conditions and increased in number over the course of the following 2 h (Figs. 1A, C). In the DMSCN, a similar linear increase was found in the rPer1-positive cell number at around the equivalent of dawn under both LD and DD conditions (Fig. 1C). Under LD conditions, we observed a significant increase in the number of rPer1 mRNA-positive cells (two way ANOVA; p < 0.01) in the VLSCN, but not in the DMSCN (two way ANOVA; p = 0.27), from 30 min before to 120 min after the onset of the light phase (Figs. 1A, C). In the VLSCN, a Tukey's multiple comparison test revealed signifi-

cant differences from 30 min (+30 min) to 120 min (+120 min) after lights on (p < 0.01; Fig. 1C). In the LD experiment, rPer2 mRNA-positive neurons were also found to increase shortly after light onset. Weakly labeled rPer2-expressing neurons appeared in the VLSCN by 30 min after the onset of the light phase (+30 min: ZT0.5; Fig. 1E) and increased in number over the course of the next 2 h (Figs. 1D, F). In the DMSCN, there was also a similar linear increase in the rPer2 cell number at lights on under both LD and DD conditions (Figs. 1D, F). The morning light in the LD experiment significantly increased the number of rPer2 mRNA-positive cells (two-way ANOVA; p < 0.01) in the VLSCN, but not in the DMSCN (two-way ANOVA; p = 0.15; Figs. 1D, F). By analysis with Tukey's multiple comparison test, significant differences were again revealed from 30 min (+30 min) to 120 min (+120 min) after lights on (p < 0.01; Fig. 1F). At around dusk, both rPer1- and rPer2-expressing cells were found to increase in the VLSCN but more modestly when compared with the dawn conditions (Fig. 2). The number of rPer1 mRNA-positive neurons under LD conditions increased and peaked at 30 min before the end of the light exposure (− 30 min: ZT11.5; Fig. 2B), and these numbers gradually decreased until 60 min after the end of the light phase (+60 min: ZT13; Figs. 2A–C). However, under DD conditions, no significant differences were observed (Figs. 2A, C). Light exposure at around dusk significantly increased the number of rPer1 mRNA-positive cells (two-way ANOVA; p < 0.01) in the VLSCN but not in the DMSCN (two-way ANOVA; p = 0.36) (Figs. 2A, C). In the VLSCN also, a Tukey's multiple comparison test revealed significant differences at 30 min before lights off (− 30 min) and also at 0 min (0 min) (p < 0.01; Fig. 2C). rPer2 was found to be induced in the VLSCN also during the late day (Figs. 2D, F). From CT and ZT0 before the end of the light phase (0 min: ZT12), rPer2-positive neurons had increased in the VLSCN (Figs. 2D, F) in LD compared with DD, apparently as a result of light exposure. Light exposure at around dusk significantly increased the number of rPer2 mRNA-positive cells in the VLSCN (two-way ANOVA; p < 0.01) and DMSCN (two-way ANOVA; p = 0.045; Figs. 2D–F). In the VLSCN, analysis using Tukey's test revealed significant differences from 0 to 60 min after lights off (p < 0.05 at 0 and 30, p < 0.01 at 60; Fig. 2F).

2.2. Time course analysis of rPer1/rPer2 expression over a 25 hour period under LD and DD conditions To examine the expression of rPer1 and rPer2 at times when animals need to delay their circadian clock to enable entrainment to environmental lighting conditions, we utilized the T-cycle paradigm. The rats were housed under LD 12:12 hour conditions for 2 weeks, and then exposed to LD 12.5:12.5 hour conditions. Under both of these sets of conditions, the rats were entrained to the LD cycles (Fig. 3). As the rat is known to

Fig. 1 – Time course analysis of rPer1 and rPer2 expression at around dawn under 24 h LD and DD conditions. Dashed lines indicate the SCN boundaries and denote the dorsomedial and ventrolateral subdivisions within the SCN. Scale bar, 200 μm. (A, D) Representative coronal sections of the SCN at around dawn subjected to rPer1 (A) or rPer2 (D) in situ hybridization. The top black–white bar indicates an LD cycle and the black bar indicates a DD cycle. (B, E) Higher magnification images of the panels shown in A and D, respectively. (C, F) Graphs showing the number of SCN cells expressing rPer1 (C) or rPer2 (F) mRNA in the VLSCN or DMSCN under entrained conditions or in the constant dark. *p < 0.05, **p < 0.01, Tukey's test. Each data series represents the mean ± SEM.

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Fig. 3 – Representative locomotor activity records from a Wistar rat. Representative activities of the rats were double-plotted. The 24 h LD cycle (indicated by the bar at the top) was shifted as indicated by the arrow (25 h LD cycle indicated by black and white bar). After the photoperiod shift, the rats appeared to be entrained to the 25 hour LD cycle. The top and bottom bars indicate the schedules of the LD cycles on the first and last day, respectively.

have a free-running rhythm of longer than 24 h, but shorter than 25 h (Honma et al., 1985), our subject animals were observed to entrain their rhythms by delaying their circadian clocks to adjust to the environmental 25 hour T-cycle. At around the onset of the light phase, the induction of the rPer1 and rPer2 genes was found to occur in the VLSCN. Twoway ANOVA analysis revealed that light exposure at around dawn in LD 12.5:12.5 hour conditions significantly increased the number of rPer1 mRNA-positive cells (p < 0.01) in the VLSCN but not in the DMSCN (p = 0.11) (Figs. 4A–C). In the VLSCN, the Tukey's test further revealed significant differences from 60 min (60 min) to 120 min after lights off (120 min; p < 0.01) (Fig. 4C). In addition, rPer2 showed significant increases in the VLSCN (two way ANOVA p < 0.01) but not in the DMSCN (two-way ANOVA; p = 0.20). We performed further post hoc analysis but found significant differences only at 120 min after light onset (Fig. 4F). In an LD 12.5:12.5 hour environment, the differential induction of rPer2 in the VLSCN during the late day was the most prominent observation. During the late day, a few rPer1expressing neurons were observed to be scattered within the VLSCN from about 90 min before (−90 min: ZT11) the end of the light exposure to 60 min after lights off (+60 min: ZT13.5) (Figs. 5A–C). Under DD conditions, however, practically no

labeled cells were evident. Two-way ANOVA revealed that light exposure at around dusk under LD 12.5:12.5 hour conditions significantly increased the number of rPer1 mRNA-positive cells (p < 0.01) in the VLSCN but not in the DMSCN (p = 0.11; Figs. 5A–C). By post hoc analysis we found a significant difference from 90 min (90 min) before to 60 min after lights off (p < 0.01; Fig. 4C). In contrast to the above mentioned results, light exposure at dusk significantly increased the number of rPer2 mRNApositive cells (two-way ANOVA; p < 0.01) in both the VLSCN and DMSCN (two-way ANOVA; p < 0.01; Fig. 5D–F). A cluster of strongly-labeled rPer2 neurons was also detectable in the VLSCN by 90 min before the end of the light phase (− 90 min: ZT11) and these cells continued to increase in number in LD conditions even at 60 min after lights off (+60 min: ZT13.5; Figs. 5D–F). At that time, the effects of light exposure upon the expression of rPer2 mRNA in the DMSCN were also evident (two-way ANOVA; p < 0.01), and a constant number of positive cells were detectable until 60 min after the offset of the light phase (+60 min: ZT13.5). This is in contrast to the gradual decrease observed for rPer2 positive-neurons in the DMSCN in DD conditions (Figs. 5D, F). Post hoc analysis showed a significant difference from 90 min (90 min) before lights off to 60 min after lights off (p < 0.01; Fig. 5F) but not in the DMSCN.

Fig. 2 – Time course analysis of rPer1 and rPer2 expression around dusk under 24 h LD and DD conditions. Dashed lines indicate the SCN boundaries and denote dorsomedial and ventrolateral subdivisions within the SCN. Scale bar, 200 μm. (A, D) Representative coronal sections of the SCN at around dusk subjected to rPer1 (A) or rPer2 (D) in situ hybridization. The top black–white bar indicates an LD cycle and the black bar indicates a DD cycle. (B, E) Higher magnification images of the panels shown in A and D, respectively. (C, F) Graphs showing the number of SCN cells expressing rPer1 (C) or rPer2 (F) mRNA in the VLSCN and DMSCN under entrained conditions or in the constant dark. *p < 0.05, **p < 0.01, Tukey's test. Each data series represents the mean ± SEM.

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2.3. Comparison of the light induction of rPer1 and rPer2 mRNA between 24 and 25 hour LD cycles To elucidate the effects of our T-cycle experiments on Per1 and Per2 expression during dawn and dusk conditions, we compared the rPer1 and rPer2 expression profiles between the 12 h light:12 h dark and 12.5 h light:12.5 hour dark schedules using ANOVA. In the VLSCN, but not in the DMSCN, the 12:12 hour LD cycle increased the number of cells expressing rPer1 following light exposure during the dawn period to a greater extent than in the 12.5:12.5 hour LD cycle (two way ANOVA; p < 0.01 versus p = 0.8; Fig. 6A). The Tukey's post hoc test revealed significant differences from 30 min after lights on (+30 min) to 120 min (+120 min) (p < 0.01; Fig. 6A). The 24 h LD cycle also increased the number of rPer2-positive cells in the VLSCN in the light during the dawn to a higher level than under LD 25 h conditions (ANOVA; p < 0.01) but not in the DMSCN (ANOVA; p = 0.84; Fig. 6B). The post hoc test revealed that a greater number of Per1-positive cells were present under LD 24 h conditions from 30 min (+30 min) to 120 min after lights on (+120 min; Fig. 6B). With regard to rPer1 expression at around lights off, twoway ANOVA revealed significant differences between the LD and DD conditions, but by Tukey's multiple comparison, neither the DMSCN nor the VLSCN showed any significant differences over the time course we examined (Fig. 6C). In terms of rPer2, ANOVA revealed significant differences between LD 24 h and LD 25 h with regard to the number of rPer2 mRNA-positive neurons both in the DMSCN (ANOVA; p < 0.01) and the VLSCN (ANOVA; p < 0.01; Fig. 6D). Multiple comparisons revealed significant differences in the VLSCN at the time points examined (Tukey HSD; p < 0.01) but we did not detect any significant differences in the DMSCN using this analysis (p > 0.05; Fig. 6D).

3.

Discussion

In the present study, we show that strong induction of rPer1 and rPer2 occurs in the VLSCN during the early day when the animals are housed under LD 12:12 hour cycles and need to advance their circadian clock. In contrast, the LD 12.5:12.5 hour cycle that compels the rats to delay their circadian clock was found to be associated with a strong induction of rPer1 during the dawn and of rPer2 at dusk in the VLSCN. A comparison of the number of rPer1 or rPer2 mRNA-containing cells at dawn and dusk demonstrated that Per1 is more strongly induced in a 24 h LD cycle during the dawn and that Per2 is more strongly transactivated at dusk in a 25 h LD cycle. It is possible that the expression profiles of the rPer1 and rPer2 genes are affected by the nature of the light exposure during the photosensitive

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period. The very start of the light onset induces both of these genes during the dawn period. In contrast, at dusk, the phase of the photosensitive periods for Per1 and Per2 decides the onset time of their expression. In the latter case, after the circadian clock enters the photosensitive phase, rPer1 and rPer2 expression in the VLSCN is continuously upregulated as long as the animals are exposed to light. Our current findings thus suggest that there is a phase difference between the Per1 and Per2 photosensitive periods. Under LD 24 h conditions (12 h:12 h) when τ, or the free running period, is longer than the T cycle, the phase angles of the Per1 and Per2 photosensitive periods are fixed to promote early entrainment (Fig. 7A). Under LD 25 h conditions (12.5 h:12.5 h) however, when τ is shorter than the T cycle, the phase angles of Per1 and Per2 photosensitive periods are fixed to delay entrainment (Fig. 7B). Although both the Per1 and Per2 genes are induced at the beginning and at the end of the light phase, strong levels of expression of these genes were also found to be limited to specific periods of the light phase. The phase response curve (PRC) of individual animals (Honma et al., 1985; Johnson et al., 2004) shows that exposure to light early in the night delays the internal clock but that exposure to light late in the night advances the circadian clock. The timing of Per1 and Per2 induction is thus very consistent with the hypothesis that the transactivation of these genes underpins the daily resetting of the central pacemaker in the SCN. In LD 12:12 hour cycles, when the rats are receiving a daily phase advancing light pulse, strong rPer1 and weaker rPer2 expression was observed during the early light phase. On the other hand, in LD 12.5:12.5 h conditions, when the rats are receiving a daily phase-delaying light pulse, strong rPer2 and very weak rPer1 induction was observed during the late light phase. In mice, for which the free running period is shorter than 24 h requiring a delay of their circadian clock each day in LD 12:12 h conditions, strong Per2 induction was observed during the late light phase (Albrecht et al., 1997; Takumi et al., 1998; Zylka et al., 1998). This observation suggests that the basic framework for the differential induction of rPer1 and rPer2 genes by light is common among animals regardless of the length of their circadian periods. Moreover, these differences are decided by the phase angles of the photosensitive period during Per1 and Per2 gene induction. The observed differential induction of rPer1 and rPer2 during the different T-cycles we imposed in our present experiments suggests their differential usage for daily resetting. A possible hypothesis is that the induction of Per1 and Per2 in the SCN functions in the unidirectional shift of the circadian clock, i.e. an advance and delay, respectively. However, the profile of the PRC (phase response curve) militates against this possibility, since in rats it shows a maximum

Fig. 4 – Time course analysis of rPer1 and rPer2 expression at around dawn under 25 h LD and DD conditions. Dashed lines indicate the SCN boundaries and denote the dorsomedial and ventrolateral subdivisions within the SCN. Scale bar, 200 μm. (A, D) Representative coronal sections of the SCN at around dawn subjected to rPer1 (A) or rPer2 (D) in situ hybridization. The top black–white bar indicates an LD cycle and the black bar indicates a DD cycle. (B, E) Higher magnification images from the panels in A and D, respectively. (C, F) Graphs showing the number of SCN cells expressing rPer1 (C) or rPer2 (F) mRNAs in the VLSCN or DMSCN under entrained conditions or in the constant dark. *p < 0.05, **p < 0.01, Tukey's test. Each data series represents the mean ± SEM.

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delay at around CT16 (Honma et al., 1985; Pittendrigh and Daan, 1976), in which both Per1 and Per2 genes are strongly induced by light (Miyake et al., 2000; Yan et al., 1999). If rPer1 and rPer2 shift the circadian clock in opposite directions, the maximum length of the phase shift should occur when they are induced separately. However, Per1 and Per2 seem to work synergistically to initiate a phase shift in the same direction and the maximum length of this shift occurs when both of these genes are strongly induced by light in the SCN of both mice and rats. Therefore, it is more likely that the direction of the circadian phase shift is regulated by the timing of Per gene activation but not by the particular Per gene that is induced. Per1 is dominantly activated by light during the late subjective night and this gene therefore appears to produce an advance in the circadian clock at this time. On the other hand, Per2 is preferentially induced during the early subjective night and appears to produce a delay in the clock. Many studies have described the light response phase shift of the circadian rhythm in Per1 or Per2 mutant mice (Albrecht et al., 2001; Bae and Weaver, 2003; Cermakian et al., 2001; Spoelstra et al., 2004). Spoelstra et al. (2004) also reported the phase response curves of the mutant mice which showed that both Per1 and Per2 mutant mice can delay and advance their circadian rhythms by light. This finding suggests that rPer1 and rPer2 function redundantly and cooperatively with regard to the light-induced phase shift. However, differences in the phase angle of the photosensitive period results in differential induction of Per1 and Per2 genes under LD conditions as demonstrated in our present study. Our present analysis demonstrates that rPer1 and rPer2 genes are differentially activated by light. An important question that arises therefore is the nature of the mechanism that causes this. This difference might be attributable to the differentially activated intracellular signal transduction system and to specific responsive elements for activation of the rPer1 and rPer2 genes. Various intracellular signals are known to activate Per1 gene expression. Activation of protein kinase A, Protein kinase C and glucocorticoids, and an increase in intracellular calcium concentrations, can upregulate Per1 transcription (Gillette and Mitchell, 2002; Motzkus et al., 2000; Oh-hashi et al., 2002; Yokota et al., 2001). Furthermore, prolonged activation of MAP kinase also induces Per1 (Akashi and Nishida, 2000; Butcher et al., 2002; Coogan and Piggins, 2004; McArthur et al., 2000). On the other hand, the Per2 gene pathway includes activation of Gq protein-coupled receptors, phospholipases, production of inositol 1,4,5-trisphosphate (IP3), and release of Ca2+ from the intracellular Ca2+ store (Takashima et al., 2006). Therefore, it is possible that the differential induction of the rPer1 and rPer2 genes is due to the distinct signal transduction pathways that activate these genes. In conclusion, in our present study we describe the rPer1 and rPer2 expression profiles in the SCN in rats entrained by

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24 h and 25 h LD cycles. We have observed the induction of both genes during the early and late day, but found that Per1 is dominantly induced by light in the morning whereas Per2 is dominantly induced in the evening. Our findings suggest that Per1 and Per2 are induced by different signaling pathways and reveal a differential usage of rPer1 and rPer2 for the advance and delay of the circadian clock.

4.

Experimental procedures

4.1.

Animals and light conditions

Five week old male Wistar rats (JACJO, Osaka, Japan) were initially housed on a 24 h light (400 lx)–dark (LD 12:12) cycle with lights on at 7:00 and lights off at 19:00. One half of the animals were transferred to constant darkness (DD). At the first DD cycle, rats in DD and LD were (n = 3 at each time point) sacrificed simultaneously. At the designated time point, each rat was deeply anesthetized with ether and intracardially perfused with 50 ml of saline followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and processed for in situ hybridization to determine the expression levels of rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs in the SCN under these light conditions. Rats were sacrificed every 30 min around both dawn and dusk. Specifically, gene expression was analyzed in the SCN every 30 min, starting at 30 min prior until 120 min after lights on. We also analyzed rPer1, and rPer2 gene expression at 30 minute intervals from 90 min before to 60 min after lights off (n = 3 at each time point). To compare the mRNA expression from rats held on a 24 h T-cycle versus those held on a 25 h T-cycle, an additional group of rats initially entrained to LD12:12 were subsequently exposed to a 25 h T-cycle (LD12.5:12.5) for 2 weeks prior to being sacrificed, and were then perfused as described above (n = 3 at each time point). Similarly, a subgroup of the LD12.5:12.5 rats was moved to DD conditions and sacrificed on day one of this cycle as described above (n = 3 at each time point). This study was performed in compliance with the Rules and Regulations of the Animal Care and Use Committee, Kinki University School of Medicine, and followed the Guide for the Care and Use of Laboratory Animals, Kinki University School of Medicine.

4.2.

Probe synthesis

Digoxigenin-labeled probes for ratPer1 (rPer1) (bases: 736-1720; GenBank accession number XM340822), rPer2 (bases: 1390– 2915; NM031678), rat VIP (nucleotides 119–808 of rVIP, BC158798), were synthesized for in situ hybridization as previously described (Yan et al., 1999). Briefly, cDNA fragments

Fig. 5 – Time course analysis of rPer1 and rPer2 expression at around dusk under 25 h LD and DD conditions. Dashed lines indicate the SCN boundaries and denote dorsomedial and ventrolateral subdivisions within the SCN. Scale bar, 200 μm. (A, D) Representative coronal sections of the SCN at around dusk subjected to rPer1 (A) or rPer2 (D) in situ hybridization. Scale bar, 200 μm. The top black–white bar indicates an LD cycle and the black bar indicates a DD cycle. (B, E) Higher magnification images from the panels in A and D, respectively. (C, F) Graphs showing the number of SCN cells expressing rPer1 (C) or rPer2 (F) mRNA in the DMSCN or the VLSCN under LD and DD conditions.

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Fig. 7 – Representative schema for daily photic entrainment of the SCN circadian clock using LD cycles of different periods. The findings in this study are consistent with a model in which distinct timing of Per1 and Per2 photo-sensitive periods occurs in the SCN. When the LD cycle (LD 12:12 h) is shorter than the rat free running period, the photo-sensitive and photo-insensitive periods will be delayed so that the induction of Per1 alone can advance the internal clock in the SCN during the early day (A). On the other hand, in the case of the LD cycle (LD 12.5:12.5 h), since the advance of the photo-sensitive and photo-insensitive periods are caused by it being longer than the free running period of the animal, the photo-sensitive periods may be presented in the late day. Hence, light during the late day dominantly transactivates Per2 and causes a delay in the daily entrainment (B).

were obtained by PCR and cloned into the pGEM-T easy vector. The rPer1 and rPer2 cRNA sense and antisense probes were labeled with digoxigenin-UTP (Roche, Mannheim, Germany) through in vitro transcription using T7 or SP6 RNA polymerase.

4.3.

Fig. 6 – rPer1 and rPer2 induction in the SCN at dawn and dusk under 24 LD and 25 LD conditions. Graphs showing the number of SCN cells in the VLSCN and DMSCN expressing rPer1 and rPer2 at around dawn (A, B) and dusk (C, D). Data from Figs. 1, 2, 4, and 5 were used to show the effects of the T-cycle on the expression of rPer1 and rPer2. The top black–white bar indicates an LD cycle. Dashed lines and solid lines indicate the number of labeled cells in the SCN under 24 h and 25 h LD conditions, respectively. *p < 0.05, **p < 0.01, Tukey's test. Each data series represents the mean ± SEM.

In situ hybridization

Coronal brain sections (30 μm) containing the entire rostral to caudal extent of the SCN were cut using a cryostat and processed using the free floating in situ hybridization method as previously described (Nagano et al., 2003). Tissue sections were sequentially transferred through 2 × SSC (1 × SSC = 0.15 M NaCl and 0.0015 M sodium citrate) for 10 min, proteinase K (2 μg/ml in 10 mM Tris buffer (pH 7.4) including 10 mM EDTA) for 10 min at 37°C, and 4% paraformaldehyde in 0.1 M phosphate buffer for 5 min. Sections were then treated with 0.5% acetic anhydride in 0.1 M triethanolamine for 10 min, and 2 × SSC for 10 min. This was followed by incubation in hybridization buffer (60% formamide, 10% dextran sulfate, 10 mM Tris–HCl (pH 7.4), 200 μg/ml transfer RNA, 1 × Denhardt's, 1 mM EDTA (pH 8.0), 0.6 M NaCl, and 0.25% sodium dodecyl sulphate) containing rPer1, rPer2 or rc-fos antisense RNA probe for mRNA detection or sense RNA probe as a control (approximately 20 ng/ml) for 16 h at 60°C. Following two rinses in 2 × SSC/50% formamide at 60°C, the sections were treated with a solution containing 20 μg/ml RNase A,

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10 mM Tris–HCl (pH 8.0), 1 mM EDTA and 0.5 M NaCl for 30 min at 37°C. The sections were further washed twice in 2 × SSC/ 50% formamide and then twice in 0.4 × SSC at 60°C (15 min for each wash). They were then transferred into buffer 1 (100 mM Tris–HCl (pH 7.4), 150 mM NaCl) (room temperature, 5 min) followed by buffer 2 (buffer 1 containing 1.5% blocking reagent) for 1 h and incubated with alkaline phosphataselabeled anti-digoxigenin serum (Roche; 1:2000 diluted in buffer 1) for 16 h. The sections were again washed first in buffer 1 and then in buffer 3 (100 mM Tris–HCl (pH 9.5) containing 100 mM NaCl, 50 mM MgCl2). Finally, the hybridization staining was visualized as a blue signal by treatment with nitroblue tetrazolium salt (0.34 mg/ml) and 5-bromo-4chloro-3-indolyl phosphate toluidinium salt (0.18 mg/ml) in buffer 3 at room temperature for 6 h. The sections were then analyzed under a bright-field microscope to examine for positive staining.

4.4.

Histological analysis

We selected the most rostral to the most caudal sections of the SCN with a 30 μm thickness. In each experiment, we stained every section containing the SCN which produced 24 sections showing the rPer1 and rPer2 mRNA-expressing neurons as shown in the supplementary figure (Supplementary Fig. 1). We then selected the middle sections (generally, 12th to 15th levels of sections). In the figure, the sections of a rat SCN show the localization of rPer1- and VIP-expressing neurons by DIG in situ hybridization. The number of positively stained cells was manually determined in an observer blind fashion in the 4 serial sections in the middle portion of the SCN (middle in all of the 24 serial SCN sections) and averaged for each animal. Cells were counted only when the cytoplasm was clearly labeled against the background. This was repeated and the two counts were found to be highly correlated (r = 0.994). Values were generated separately for the DMSCN and VLSCN which were defined by the boundary of the VIP subregions, and corresponded to the criteria described previously (Van den Pol, 1980). As expected, no significant signals were detected using sense probes or following prior RNase treatment of the sections (data not shown). All values are expressed as mean ± standard error of the mean (SEM). For statistical analysis, one-way analysis of variance (ANOVA) followed by Tukey's test was performed.

4.5.

Behavior analysis

For the assessment of locomotor activity, rats were housed individually and their locomotor activity rhythm was continuously measured by area sensors (model FA-05 F5B; Omron, Tokyo, Japan) with a thermal radiation detector system. The obtained data were electronically stored.

Acknowledgments This research was supported by the “High Tech Research Center” project for Private Universities, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture of Japan.

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