Research Article

Constitutive expression of the Period1 gene impairs behavioral and molecular circadian rhythms

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PNAS March 7, 2006 103 (10) 3716-3721; https://doi.org/10.1073/pnas.0600060103

  1. Communicated by Joseph S. Takahashi, Northwestern University, Evanston, IL, January 4, 2006

  2. ↵ †R.N., S.Y., and N.U. contributed equally to this work. (received for review March 25, 2005)

Abstract

Three mammalian Period (Per) genes, termed Per1, Per2, and Per3, have been identified as structural homologues of the Drosophila circadian clock gene, period (per). The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo, we generated two transgenic rat lines in which the mouse Per1 (mPer1) transcript was constitutively expressed under the control of either the human elongation factor-1α (EF-1α) or the rat neuron-specific enolase (NSE) promoter. The transgenic rats exhibited an ≈0.6–1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes.

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus (1–4). Circadian rhythms persist under constant conditions and are entrained by environmental light cycles (5). Removal of the SCN causes arrhythmicity of locomotor activity and transplant of fetal SCN tissue restores circadian rhythmicity with the period of the donor SCN (4, 6, 7). Mammalian circadian photoreceptors are located in the retina, and specialized retinal ganglion cells carrying light information project directly to the SCN (8, 9).

The mammalian Per1 gene has been identified as a structural homologue of the Drosophila circadian clock gene period (10, 11). Two other homologues, Per2 and Per3, have also been found in mammals (12–15). Both transcripts and proteins of all three Per genes exhibit circadian rhythms in the SCN. Per1 transcription is activated by the CLOCK–BMAL1 complex through its binding to E-box sequences located in the Per1 promoter region (16–18) and is repressed by Cryptochrome (Cry1 or Cry2) (19). Loss of function of the Clock (20, 21), Bmal1 (22), Cry1, or Cry2 (23) genes affects not only the molecular oscillation of Per1 and Per2 but also circadian rhythmicities in behavior and the neural activity of the SCN (24–26). Per1 and Per2 expression is induced immediately after light exposure (27–30), and both light-induced behavioral phase shifts and glutamate-induced phase shifts in SCN slice preparations are inhibited or attenuated by pretreatment with an antisense oligonucleotide against Per1 (31, 32).

To determine the role of the rhythmic expression of Per1 and its induction by light, we produced transgenic rat lines in which the transgene mPer1 was constitutively expressed under the control of the human elongation factor-1α (EF-1α) or rat neuron-specific enolase (NSE) promoter. Constitutive expression of the mPer1 transgene disrupted endogenous rPer1 and rPer2 oscillations, lengthened circadian period, and impaired light entrainment.

Results

Generation and Characterization of Transgenic Rats Overexpressing mPer1.

Transgenic rat lines in which the expression of mPer1 was driven by the promoter of either EF-1α or NSE developed normally and showed no obvious defects.

We examined the distribution of Per1 in the brains of E12(Per1) and N3(Per1) rats by in situ hybridization. The Per1 probe used in this experiment did not distinguish between the transcripts of the endogenous rPer1 and the mPer1 transgene. Although a clear circadian fluctuation of rPer1 was observed in the SCN from the wild-type siblings, no circadian oscillation of the Per1 signal (sum of the signals of both mPer1 and rPer1) was detected in the SCNs of E12(Per1) or N3(Per1) rats (Fig. 1 B). Furthermore, in N3(Per1) rats strong signals, which we assumed to be N3(Per1) transcripts, were also detected in the whole brain as well as in the SCN (Fig. 1 B). As expected, fluctuation of the PER protein was observed in the SCN of wild-type rats but not in the SCNs of E12(Per1) or N3(Per1) rats (Fig. 1 C).

Fig. 1.
Fig. 1.

Structures of EF-1α::mPer1 and NSE::mPer1 transgenes and histological analysis of SCN of transgenic rat lines. (A) mPer1 cDNA was sited between the human EF-1α or rat NSE::Per1 promoters and SV40 poly(A) signals. Primers (arrows) and probes (white boxes) used for the screening of the transgenic rat lines are indicated below each construct. (B) Per1 mRNA levels in coronal sections of the brain of the wild-type, E12(Per1), and N3(Per1) rat lines were analyzed by in situ hybridization. Per1 transcripts were constitutively expressed in the SCN of the E12(Per1) and the N3(Per1) transgenic rats lines, whereas clear circadian cycling of rPer1 expression was observed in the SCN of the wild-type rat lines. (C) Immunohistochemistry of PER1 protein in SCN of transgenic rat lines at CT 0 and CT 12. Photomicrographs of SCN and other brain regions after staining with the PER1 antibody. (D) Light induction of Per1 mRNA in the SCN of wild-type, E12(Per1), and N3(Per1) rats was compared by in situ hybridization analysis. Per1 mRNA was not induced in the SCN of the E12(Per1) and N3(Per1) transgenic rat lines after a 30-min light exposure at CT 16. Results are quantified in the histogram. (E) Light induction of c-fos mRNA in the SCN of wild-type, E12(Per1), and N3(Per1) rat lines. (Lower and Upper) Illustrated is the SCN with and without a light pulse at CT 16, respectively.

We then asked whether light exposure increased the amount of Per1 mRNA in the SCN. Light exposure [30 min at circadian time (CT) 16] induced a 3-fold increase of Per1 transcript in the SCN in the wild-type rats (Fig. 1 D). In contrast, no Per1 induction was observed in the SCNs of E12(Per1) and N3(Per1) transgenic rats after light exposure (Fig. 1 D). We also measured the light induction of c-fos mRNA in the SCN by in situ hybridization and observed that a light pulse at CT 16 rapidly increased c-fos mRNA in both wild-type and transgenic rats (Fig. 1 E).

Behavioral and Physiological Phenotypes of mPer1 Transgenic Rats.

We found altered circadian features in both the E12(Per1) and N3(Per1) lines. Representative body temperature rhythms from both transgenic lines and their wild-type siblings are shown in Fig. 2 A and B. Both transgenic rat lines failed to entrain to laboratory light/dark (LD) cycles (LD 12:12; ≈100–200 lux). Some masking was shown by both transgenic rat lines (the body temperature is low during the light period and becomes high immediately after switching off the light), and “relative coordination” (i.e., the period of the rhythm is modified as it crosses the LD cycle) also occurred in both lines. As can be seen in Fig. 2 A and B, progression of the high-temperature phase relative to the LD cycle was slower when a high temperature coincided with darkness and faster when it coincided with light. As a consequence, the high temperature tended to remain in the dark portion of the LD cycle before transiting quickly through the light portion. This observation suggests that the environmental light signal still reached the pacemaker of the transgenic rats, although it was too weak to fully entrain the rhythms. Relative coordination in locomotor activity rhythm was not as clear as in body temperature rhythms because of the stronger masking effect of light on the wheel running (see Fig. 4, which is published as supporting information on the PNAS web site). The free-running periods of body temperature rhythms in constant darkness (DD) from the male F1 E12(Per1) and N3(Per1) transgenic rats were 0.82 h and 0.95 h longer than from their wild-type siblings, respectively. Periods in individual rats were: 25.3, 25.3, and 25.2 h for E12(Per1); 24.4 and 24.5 h for wild-type siblings of E12(Per1); 25.3 h for N3(Per1); and 24.3 and 24.4 h for wild-type siblings of N3(Per1). Two other F1 transgenic rat lines similarly showed longer periods in wheel-running rhythms than their wild-type siblings (data not shown).

Fig. 2.
Fig. 2.

Body temperature and wheel-running activity rhythms of transgenic rats. Body temperature rhythms of E12(Per1) (A) and N3(Per1) (B) rats were reduced to actogram form (33) (shown double plotted) and compared with those of wild-type siblings. White and black bars at the top of figures indicate the LD cycle when present. Arrows indicate transitions from LD to DD. Both transgenic rat lines failed to entrain completely and, in DD, exhibited 0.82 h [E12(Per1)] and 0.95 h [N3(Per1)] longer circadian periods of body temperature than their wild-type siblings. General activity rhythms monitored by movement of the transmitter in these rats are shown in Fig. 4. (C) The period distribution of wheel-running rhythms in the offspring of crosses between F1 heterozygous transgenic rats of both lines and wild-type rats are plotted. ■, wild-type rats; □, transgenic rats.

The period distributions of wheel-running rhythms in the offspring of F1 heterozygous and wild-type rats are shown in Fig. 2 C. Approximately 50% of pups from these crosses carried the transgene. The distribution of the free-running periods of the transgenic rats and of their wild-type siblings did not overlap (Fig. 2 C). The average circadian periods of the transgenic rats were 24.92 ± 0.03 (SEM) [E12(Per1)] and 24.99 ± 0.02 [N3(Per1)] and were significantly longer than those of their wild-type siblings [24.29 ± 0.01 for wild-type siblings of E12(Per1); 24.35 ± 0.03 for wild-type siblings of N3(Per1)] (Fig. 2 C).

Expression of mPer1, rPer1, and rPer2 in SCN and the Eyes of mPer1 Transgenic Rats.

The rPer1 mRNA in the SCN of the wild-type rats was rhythmic, with a peak between CT 4–8 (Fig. 3 and Table 1). In the SCN of E12(Per1) rats, the mPer1 transgene was constitutively expressed at approximately twice the peak level of the rPer1 transcript in the wild-type rats. In the SCN of N3(Per1) rats, the mPer1 transgene was constitutively expressed at ≈7 times the peak level of the rPer1 transcript in the SCN of the wild-type rats (Fig. 3 A and E). The robustness of endogenous rPer1 mRNA cycling in the SCN of E12(Per1) and N3(Per1) rats was significantly attenuated compared with those in the wild-type rats (Fig. 3 A and E and Table 1).

Fig. 3.
Fig. 3.

Expression of Per1 and Per2 in SCN and eyes (whole eyes) of transgenic rat lines. The amounts of the rPer1, rPer2, and mPer1 mRNAs were quantified with TaqMan RT-PCR analysis and are plotted as relative mRNA abundance. Each point represents mean ± SEM values of four independent assays (n = 4). (A) Expression of mPer1 and rPer1 in the SCN of E12(Per1) and wild-type rats. (B) Expression of mPer1 and rPer1 in the eyes of E12(Per1) and wild-type rats. Expression of rPer2 mRNA in the SCN (C) and eyes (D) of E12(Per1) and wild-type rats. (E) Expression of mPer1 and rPer1 in the SCN of N3(Per1) and the wild-type rats. (F) Expression of mPer1 and rPer1 in the eyes of N3(Per1) and wild-type rats. Expression of rPer2 mRNA in the SCN (G) and eyes (H) of N3(Per1) and wild-type rats. ▵, mPer1; □, rPer1 in transgenic rats; ♦, rPer1 in wild-type rats (A, B, E, and F); □, rPer2 in transgenic rats; ♦, rPer2 in wild-type rats (C, D, G, and H).

View this table:
Table 1.

Cosinor summary of rhythm variations in transgenic rats

We also measured the levels of the rPer2 transcripts in the same samples and found that rPer2 had expression profiles similar to those of rPer1. In the SCN of the wild-type rats, rPer2 mRNA showed a robust circadian pattern with a peak at CT 12, but the robustness of the oscillation was attenuated in the SCN of E12(Per1) and N3(Per1) rats [with the exception of the residual rhythmicity in the SCN of E12(Per1) rats] (Fig. 3 C and G and Table 1).

Similar effects were observed in the mRNA extracted from eyes (Fig. 3 B, D, F, and H). The mPer1 transgene was constitutively expressed in the eyes of E12(Per1) and N3(Per1) rats at ≈3 and 4 times the peak level of the rPer1 transcript in wild-type rats. The amplitudes of oscillation of rPer1 and rPer2 mRNA levels in the eyes of both E12(Per1) and N3(Per1) rats were much lower than that of wild-type rats, and residual rhythmicities of the rPer1 and rPer2 transcripts were observed only in E12(Per1) rats (Fig. 3 B, D, F, and H and Table 1).

Discussion

Two transgenic rat lines in which the mPer1 transgene was constitutively expressed under the control of either the EF-1α or NSE promoter showed significantly lengthened circadian periods and abnormal entrainment of the rhythms of locomotor activity and body temperature. The amplitude of the circadian rhythms of endogenous rPer1 and rPer2 was severely attenuated in these transgenic rats. That the phenotypes of both transgenic rat lines were almost identical can be explained if Per1 overexpression is assumed to be saturating despite the different copy numbers of the mPer1 transgene and the different levels of mPer1 expression in the two lines. From these results, we conclude that the similar phenotypes in the two transgenic rat lines are due to the constitutive expression of mPer1 rather than due to disruption of the genes surrounding the insertion sites of the transgenes, and furthermore, we conclude that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes. The data indicate that the light induction and rhythmic oscillation of Per1 expression in the SCN are strongly correlated with the entrainment and molecular oscillation.

In Drosophila, constitutive expression of per does not eliminate cyclic PER expression and behavioral rhythms, although the mechanism that maintains PER cycling in the absence of rhythmic per transcription is still not completely understood (34). A similar posttranscriptional regulation of PER expression might be expected in mammals because the sequences in the 3′UTR of mPer1 inhibit the translation of mPer1 mRNA (35). Despite the differences between rats and mice, cross-species comparisons of the phenotypes resulting from overexpression or absence of expression of Per genes are of significant interest (36–40). Per1 −/− and Per2 −/− mice exhibit shortened periods of wheel-running activity. The double knockout (Per1 −/− Per2 −/−) is arrhythmic (36, 37). These results indicate that loss of expression of either Per1 or Per2 does not abolish the circadian oscillation but does influence the length of its period. Because the Per1 −/− Per2 −/− mice are completely arrhythmic, it has been suggested that there is partial redundancy in the function of the Per1 and Per2 genes (36, 37). Therefore, it seems that posttranscriptional regulation of Per genes and functional redundancy of the Per1 and Per2 genes may explain the residual behavioral rhythm observed in our transgenic animals. In addition, because the Per1 −/− mouse has a shortened period (36, 38) and because we observed lengthening in the period in our Per1 transgenic rats, the period of the rhythm may be positively correlated with the gene dosage of Per1. Thus, future construction of transgenic animals in which the expression of Per1 is clamped at low, middle, and high levels and those in which both Per1 and Per2 are overexpressed may clarify the roles of these genes in the determination and generation of circadian rhythms.

More dramatic than the period phenotype of our transgenic rats is the defect in entrainment. There are three possible explanations for this effect of mPer1 overexpression: (i) The transgenic rats may be less sensitive to the entraining light stimulus. This hypothesis seems unlikely because our data demonstrate rapid c-fos induction in the SCN of the transgenic rats after a light pulse (Fig. 1 E). Thus, the light induction of rPer1 must be obscured by the mPer1 overexpression in the transgenic rats (Fig. 1 D). (ii) The lengthened period may place the circadian pacemaker outside the limits of entrainment without affecting light entrainment mechanisms. To test this possibility, we subjected wild-type rats (n = 5) to 23-h light cycles (LD 11.5:11.5; 100 lux) and found that they could entrain (S.Y. and N. Schneider, unpublished data). Because entrainment to this cycle requires a larger phase shift each day (≈1.5-h advance) than the ≈1-h advance that would be required to entrain the transgenic rats to the 24-h cycle (to which they failed to entrain), we conclude that the lengthened period of the transgenic rats cannot by itself account for the entrainment defect. (iii) A direct effect of excess mPer1 on the oscillator or a combination of such a direct effect with reduced light sensitivity and/or lengthened period could cause the circadian oscillator of Per1-overexpressing rats to be unable to advance ≈1 h/day as would be required for entrainment. Although Albrecht et al. (39) reported that Per1 mutant mice fail to phase advance in response to a 15-min light exposure at Zeitgeber time 22 on the first night in DD and Per2 mutant mice fail to phase delay at Zeitgeber time 14, Cermakian et al. (38) found that Per1 mutant mice show a normal phase response to a 30-min light exposure at CT 14 or 20 after 3–5 days in DD. Bae and Weaver (40) in a T-cycle photoperiod experiment concluded that both Per1 and Per2 mutant mice have the ability to shift in both directions. Moreover, antisense studies suggest that Per1 is involved in light-induced phase delays (31, 32, 41). Because Per1 and Per2 interact with each other, overexpression of mPer1 in transgenic rats could affect the mechanism involved in both phase advances and delays.

The present study shows that oscillations of both endogenous rPer1 and rPer2 expression in the SCN are impaired in mPer1-overexpressing transgenic rats. Per1 and Per2 transcription is induced by the CLOCK–BMAL1 complex (16–18) and repressed by CRY1 or CRY2 (19). Although each of the PER proteins interacts with the CRY1 and CRY2 proteins, overexpression of PERs in mouse NIH 3T3 cells has almost no critical effect on the transcription of Per1 and Per2 (18, 19). This discrepancy may be due to the difference in methods used in our study (transgenic) and others (transient expression). An extensive elucidation of the molecular function of PER is necessary for further understanding of the regulation of Per1 transcription.

Materials and Methods

Animals.

All of the transgenic rat lines used in this study were generated and maintained by using the Wistar rat strain (Charles River Laboratories Japan). To overexpress mPer1, we made two different DNA constructs. The promoter regions of the human EF-1α and rat NSE genes were directly ligated to the mPer1 ORF flanked by transcriptional termination signals derived from the SV40 early gene and were designated as EF-1α::mPer1 and NSE::mPer1, respectively (Fig. 1 A). We confirmed the expression of the mPer1 mRNA and mPer1 protein in Chinese hamster ovary cells (JCRB9018, Japanese Collection of Research Bioresources Cell Bank, Osaka) and PC12 cells (rat adrenal pheochromocytoma cells; IFO50015, Health Science Research Resources Bank, Osaka) 24 h after the transfection with these two plasmids (data not shown). The two transgenic vectors were linearized by using either SmaI/NotI (for EF-1α::mPer1) or HindIII/SacII (for NSE::mPer1) and injected into fertilized Wistar rat zygotes. Transgenic rats were screened by PCR and Southern blot analyses by using their tail DNA. We obtained 17 and 10 founder rats in the EF-1α::mPer1 and NSE::mPer1 transgenic experiments, respectively. Two founder rats of EF-1α::mPer1 transgenic lines were mosaic and four founder rats of NSE::mPer1 transgenic lines were either mosaic (three lines) or sterile (one line). In the initial screening, we measured the wheel-running activity of the F1 heterozygous transgenic rats and compared it with that of their wild-type siblings. We found that two lines of the EF-1α::mPer1 transgenic rats and one line of the NSE::mPer1 transgenic rats showed free-running periods significantly longer than those of wild-type rats in DD. In this study, we used a line designated E12(Per1) from the EF-1α::mPer1 transgenic rats and a line designated N3(Per1) from the NSE::mPer1 transgenic rats to analyze molecular and physiological circadian rhythms. The copy numbers of the mPer1 transgenes in the rat genomes in these two lines were estimated by Southern blot and TaqMan RT-PCR (Applied Biosystems) analyses. We found that approximately five copies of the transgene were inserted in the genome of E12(Per1) rats and 40 copies in the genome of N3(Per1) rats. All of the animal studies conducted were in accordance with the guidelines of the Committee on Animal Care and Use of University Tokyo, University of Virginia, Yamaguchi University, and Y. S. New Technology Institute, Inc.

Behavioral Analysis.

F1 heterozygous transgenic rats and their wild-type siblings (8 weeks old) were individually housed in cages with running wheels (8 cm wide with a 17-cm diameter). Wheel revolutions were monitored with wheel-activated micro switches every 6 min for 8 days in LD (12 h of light and 12 h of dark) and 19 days in DD by using the Data Quest system (Data Sciences International, Arden Hills, MN). Using this behavioral screen, we found that F1 heterozygous E12(Per1) and N3(Per1) rats showed free-running periods ≈45 min longer than those of their wild-type siblings. Therefore, we investigated more detailed circadian characteristics in these rats [three E12(Per1) heterozygous males and two wild-type male siblings; one N3(Per1) heterozygous male and two wild-type male siblings]. Body temperature was measured by telemetry by using implanted transmitters (model VM-FH; Mini-Mitter, Sunriver, OR). A radio receiver (model RA-1010; Mini-Mitter) coupled to a data acquisition system (Dataquest III; Data Sciences International) was placed under the cage. Body temperature was recorded in 6-min bins and analyzed by actiview (Mini-Mitter).

F1 female heterozygous transgenic rats [five females in E12(Per1) and two females in N3(Per1)] were crossed with wild-type rats. We obtained 25 E12(Per1) heterozygous (14 males and 11 females) and 30 wild-type (17 males and 13 females) siblings and 15 N3(Per1) heterozygous (9 males and 6 females) and 15 wild-type (9 males and 6 females) siblings. When the rats reached 4 weeks of age, they were individually housed in running cages, and their wheel-running activities were monitored for 14 days in LD and 10 days in DD. Free-running periods were obtained on the basis of the eye fitted line, which marked the onset of wheel-running activity during 10 days in DD. Tissues for mRNA analysis were sampled from these rats after 10 days in DD.

Cages were changed every 2 weeks and water bottles every week. Half of the bedding was replaced between each cage change. All animal care in DD was performed under infrared illumination by using an infrared viewer (FJW Optical Systems, Palatine, IL). Ambient temperature in the cage was ≈21°C.

Quantification of mPer1, rPer1, rPer2, and GAPDH Transcripts.

The animals produced by crossing the F1 heterozygous and wild-type rats were raised under the LD conditions as described above. For the N3(Per1) group, we did additional sampling (12 transgenic and 12 wild type), and these samples are included in the data analysis in Fig. 3. After 10 days in DD, activity onset (CT 12) was predicted on the basis of the eye fitted line that marked the onset of wheel-running activity during the 10-day period. Brains and eyes (entire eye) were sampled at 4 h intervals starting at CT 0 of the 11th cycle in DD. By using an infrared viewer, the rats were killed with CO2. Both eyes were removed with scissors without visible light exposure and frozen in liquid nitrogen. The brain was removed under light and frozen immediately on dry ice. Paired SCNs were punched out from a 1-mm-thick coronal slice by using a microdissecting needle (inner diameter, 600 μm). Total RNA from the SCN and eyes was extracted by using TRIzol solution (Invitrogen), treated with DNase I (Stratagene), and further purified with TRIzol LS solution (Invitrogen). The mPer1, rPer1, rPer2, and GAPDH transcripts were quantified by using the TaqMan RT-PCR kit (Applied Biosystems) and a 7700 Sequence Detection System (Applied Biosystems). Serial dilutions of the rat genomic DNA solutions (from 0.64 to 10,000 copies per μl) (Promega) were used as standards of the copy numbers for the rPer1, rPer2, and GAPDH cDNAs. To quantify the mPer1 cDNA, the EF1α::mPer1 DNA solution was mixed with the rat genomic DNA solution to obtain standard solutions containing equal numbers of copies of each DNA (from 0.64 to 10,000 copies per μl). The transcripts of the endogenous rPer1 gene and mPer1 transgene were quantified by using specially designed primer sets; the forward and reverse primers for rPer1 were designed within a unique 3′-untranslated region of rPer1; the forward primer for mPer1 was designed in the 3′ terminal of the mPer1 ORF and the reverse primer in poly(A) signals of SV40 (Fig. 1 A). The specific primer pairs used in this study were: rPer1 forward, 5′-AGCAGAGTGGAAGTTTTCAGCC-3′; rPer1 reverse, 5′-ACCACTTCAGCAGCTTGTCAGC-3′; mPer1 forward, 5′-ACTCTGCCATGGAGGAAGAAGA-3′; mPer1 reverse, 5′-TGTGAAATTTGTGATGCTATTGCTT-3′; rPer2 forward, 5′-GGTGTGGCAGCTTTTGCTTC-3′; rPer2 reverse, 5′-CGGCACAGAAACGTACAGTGTG-3′.

The GAPDH transcript was used for normalizing the expression of each transcript. The amplitudes of the rhythms were calculated by cosinor analysis (time series single cosinor 6.2; Laboratory of Applied Statistics and BioMedical Computing Expert Soft Technologie, Richelieu, France), and the results are presented in Table 1.

In Situ Hybridization.

The preparation and sampling of the animals (n = 3) were described above. After 14 days in DD, activity onset was predicted on the basis of the eye fitted line that marked the onset of wheel-running activity during the 14-day period. Brains were sampled 60 min after a 30-min light exposure at CT 16. Light was produced by a 40 W fluorescent lamp [60 μW/cm2 (≈200 lux) at cage level]. In situ hybridization analysis of Per1 and c-fos transcripts was performed as described in ref. 28. Brains were fixed in 4% paraformaldehyde/PBS solution for 24 h, followed by immersion in 20% sucrose/PBS solution for 48 h. Brains were then sliced into 30-μm-thick sections with a Cryostat (Leitz CM-1510). The sections were hybridized with the 33P-labeled antisense cRNA probe complementary to the sequence from nucleotides 436 to 931 of mPer1 or complementary to the sequence from nucleotides 513 to 1021 of c-fos. Briefly, tissue sections were incubated with proteinase K (2 μg/ml in 10 mM Tris·HCl, pH 7.5/0.5 M EDTA) for 10 min at 37°C and acetic anhydride in 0.1 M triethanolamine for 10 min. Then they were incubated in hybridization buffer (60% formamide/10% dextran sulfate/1 M Tris·HCl, pH 7.4/5 M NaCl/0.5 M EDTA/500 μg/ml tRNA/10% SDS/1 × Denhardt’s solution) containing mPer1 or c-fos antisense cRNA probes for 17–18 h at 60°C. After the posthybridization wash and RNaseA treatment, hybridized sections and 14C-acrylic standards (Amersham Pharmacia Biosciences) were exposed to BioMax film (Kodak) for 7 days.

Immunohistochemistry.

The preparation and sampling of the animals were described above. Rats were transferred to DD conditions 2 days before the experiment and killed at CT 0 and CT 12. Anti-mouse PER1 antibody was purchased from Affinity BioReagents (catalog no. PA1-524). After postfixation in paraformaldehyde and dehydration, brains were sectioned into 20-μm-thick slices with a Cryostat microtome (Leica CM1510). The sections were incubated with a polyclonal rabbit antibody against PER1 diluted 1:1,000 in PBS containing 0.3% Triton X-100 for 72 h at 4°C after the inactivation of endogenous peroxidase with 0.3% H2O2/50% methanol. Then they were incubated with anti-rabbit IgG diluted 1:1,000 in PBS for 24 h at 4°C and with avidin–biotin peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories), and peroxidase was visualized by using 0.01% diaminobenzidine as chromogen and 0.01% H2O2 in PBS as substrate for 10 min.

Acknowledgments

We thank Drs. K. Hanaoka, K. Sakimura, and Y. Kimura for the gift of the plasmid containing EF-1α and NSE promoters and poly(A) signals of the SV40 early gene, respectively; Dr. J. Eguchi for excellent technical assistance in the TaqMan RT-PCR analysis; Drs. K. Nakao and M. Katsuki for helpful discussions; and Dr. C. Green for the critical reading of the manuscript. This work was partially supported by research grants from the Japan Society for the Promotion of Science, the Japanese Ministry of Education, Science, Sports and Culture, and the Japanese Ministry of Health and Welfare, and National Institute of Mental Health Grant MH56647 (to M.M.).

Footnotes

  • §§To whom correspondence should be sent at the present address:
    Research Group of Chronogenomics, Mitsubishi Kagaku Institute of Life Sciences, 11, Minami-Oya, Mahida, Tokyo 194-8511, Japan.
    E-mail: tei{at}libra.ls.m-kagaku.co.jp

  • ↵ ‡Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200.

  • ↵ Present address: Department of Biological Sciences, Vanderbilt University, VU Station B, Box 35-1634, Nashville, TN 37235-1634.

  • Author contributions: H.T. designed research; R.N., S.Y., N.U., T.S., M.S., R.-i.T., M.U., A.M., K.Y., and H.T. performed research; R.N., Y.S., and H.T. contributed new reagents/analytic tools; R.N., S.Y., N.U., T.S., M.S., Y.S., S.-I.T.I., M.M., and H.T. analyzed data; and R.N., S.Y., S.-I.T.I., M.M., and H.T. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • Abbreviations:
    SCN,
    suprachiasmatic nucleus;
    NSE,
    neuron-specific enolase;
    EF-1α,
    elongation factor-1α;
    CT,
    circadian time;
    LD,
    light/dark;
    DD,
    constant darkness.
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Constitutive expression of the Period1 gene impairs behavioral and molecular circadian rhythms
Rika Numano, Shin Yamazaki, Nanae Umeda, Tomonori Samura, Mitsugu Sujino, Ri-ichi Takahashi, Masatsugu Ueda, Akiko Mori, Kazunori Yamada, Yoshiyuki Sakaki, Shin-Ichi T. Inouye, Michael Menaker, Hajime Tei
Proceedings of the National Academy of Sciences Mar 2006, 103 (10) 3716-3721; DOI: 10.1073/pnas.0600060103
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Proceedings of the National Academy of Sciences of the United States of America: 103 (10)
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