Acta Physiologica Sinica, August 25, 2006, 58 (4): 359-364

Circadian rhythms and different photoresponses of Clock gene transcription in the rat suprachiasmatic nucleus and pineal gland

WANG Guo-Qing^1,2, FU Chun-Ling², LI Jian-Xiang², DU Yu-Zhen², TONG Jian^2,*
1Department of Physiology, Medical School; ²Chronobiology Laboratory, Department of Toxicology, School of Radiation Medicine and Public Health, Soochow University, Suzhou 215123, China

Abstract: The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light: 12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6 rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (<0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P<0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P>0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG) and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P<0.05), and the rhythmic profile was anti-phasic to that under DD condition (P<0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P<0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P<0.05). (3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P<0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.

Key words: Clock gene; circadian rhythm; light; suprachiasmatic nucleus; pineal gland

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

王国卿^{1, 2}，傅春玲²，李建祥²，杜玉珍²，童 建^2,*
苏州大学¹医学院基础医学系生理学教研室；²放射医学与公共卫生学院卫生毒理学教研室，苏州215123

摘 要 ：本研究旨在观察和比较视交叉上核(suprachiasmatic nucleus, SCN)与松果体(pineal gland, PG)中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗(constant darkness, DD)和12 h光照: 12 h黑暗交替(12 hour-light : 12 hour-dark cycle, LD)光制下分别被饲养8周(n=36)和4周(n=36)后，在一昼夜内每隔4 h采集一组SCN和PG组织(n=6)，提取总RNA，用竞争性定量RT-PCR测定不同昼夜时点(circadian times, CT or zeitgeber times, ZT)各样品中Clock基因的mRNA相对表达量，通过余弦法和Clock Lab软件获取节律参数，并经振幅检验是否存在昼夜节律性转录变化。结果如下：(1) SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化(P<0.05)，PG中Clock基因的转录也显示相似的内源性节律外观，即峰值出现于主观夜晚(SCN为CT15，PG为CT18)，谷值位于主观白天(SCN为CT3，PG为CT6) (P>0.05)。 (2) LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡(P<0.05)，但与其DD光制下节律外观相比，呈现反时相节律变化(P<0.05)，且其表达的振幅及峰值的mRNA水平均增加(P<0.05)，而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反(P<0.05)。 (3) 在LD光制下，光照使PG中Clock基因转录的节律外观反时相于SCN (P<0.05)，即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17，谷值分别位于黑暗期ZT22和光照期ZT5。结果表明，Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质，而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。

关键词：Clock基因；昼夜节律；光照；视交叉上核；松果体
中图分类号：Q811.213；R322.55；R852.6；R322.81

Received 2006-03-09 Accepted 2006-06-08

This work was supported by the National Natural Science Foundation of China (No.30170295), Medical Developmental Foundation of Soochow University (No.EE134031) and Young Teacher's Research Foundation of Soochow University (No.Q3134044).

^*Corresponding author. Tel: +86-512-65880069; Fax: +86-512-65880069; E-mail: tongjian@suda.edu.cn

Circadian rhythms driven by an internal biological clock have been observed in almost all the living organisms. The mammalian clock is generally considered as comprising three major components: the hypothalamic suprachiasmatic nucleus (SCN), pineal gland (PG) and bilateral retinas. The exogenous lighting signals via retinal input can entrain the endogenous oscillator, e.g., the SCN, which then generates circadian rhythms including the synthesis of melatonin (MEL) in the PG^[1]. The core oscillator is regulated through a transcription ¾ (post)translation based autoregulatory feedback loop involving a set of clock genes^{[2, 3]}.

As an important mammalian clock gene, the Clock is mainly expressed in the SCN, PG and retina in the rat brain^[4]. The Clock gene encodes a base helix-loop-helix (bHLH)/PER-ASNT-SIM (PAS) type transcription factor to form a heterodimer with the product of BMAL1^[5]. The Clock/BMAL1 heterodimer in turn activates the transcription of period1 (Per1) gene which has been proved to be another clock gene^[6]. The Clock gene transcription in the mammalian SCN displays a circadian rhythm^[7], which is involved in phase-dependent induction by light^[8]. The rat pineal Clock gene transcription also shows an oscillatory rhythm with the peak at the circadian time (CT) 18 and the trough at CT6 under constant darkness (DD)^[9]. Based on these findings, the Clock gene is believed to play critical roles in the mechanisms underlying circadian rhythmicity^[10,11].

The circadian differences of Clock gene transcriptions between the SCN and PG in mammals, however, have not been thoroughly investigated so far. In the present study, we examined and compared the temporal profiles of Clock gene transcription in the rat SCN and PG in order to better understand the roles of Clock gene in regulation of the central temporal organizations.

1 MATERIALS AND METHODS

1.1 Animal preparation and sampling

Male Sprague-Dawley rats (SPF degree) weighing 70~100 g were used according to the Guideline for the Care and Use of Laboratory Animals in Soochow University Medical School. They were continuously reared in the University Animal Center equipped with light and temperature-controlled facilities. The room environment was set either under DD or under 12 hour-light : 12 hour-dark cycle (LD), illuminated with fluorescent light at the intensity of ca. 150 lux from 5:00 to 17:00, and maintained at (25±1) ºC. Four animals were in a plexiglass cage with free access to food and water which were supplied twice daily, at 7:00 and 19:00, respectively. Cages were exchanged once a day at 7:00. All animal treatments and sampling during the dark phases were conducted under the red dim light (<0.1 lux). Rats housed for 4 weeks in LD (n=36) or 8 weeks under DD (n=36) were decapitated under anesthesia using aether at 6 circadian timepoints (n=6 for each, zeitgeber time, ZT or CT2, 6, 10, 14, 18 and 22), where ZT12 or CT12 equals to the time of light off (17:00 h) under LD cycle. The brain samples were quickly removed and a mass of hypothalamic tissue (5 mm×5 mm×5 mm) which contains the SCN was selected at the upside of optical chiasm and promptly frozen on a glass plate on dry ice. Hypothalamic slice with the SCN, a thickness of 400~450 µm, was made by using a frozen slicer (Leica CM1900, Germany). The SCN tissue was sampled on the slice under the optical microscope, while the PG tissue was directly obtained from the underneath of confluent of sinus with naked eyes. All tissue samples were quickly stored at _85 ºC (MDF-U4086S refrigerator, SANYO, Japan) until use.

1.2 Extraction of total RNA and synthesis of cDNA

Total RNA was extracted from the respective samples with TRIzol reagent (Invitrogen) and 5810R frozen high-speed centrifuge (Eppendorf, Germany). Ultraviolet spectrophotometer (SANYO, Japan) was used to determine the value of absorbance A_{260 nm}/A_{280 nm}. Sample value more than 1.7 was diluted with 0.01% DEPC-treated water (Amresco) to get RNA concentration of 1 mg /ml. cDNA was synthesized using DEPC-treated water, Oligo(dT)₃₆ and dNTP mix (SANGON, Shanghai), the RevertAid^TM M-MuLV Reverse Transcriptase and 5×Reaction Buffer (MBI Fermentas). Extraction of total RNA and synthesis of cDNA were manipulated according to market specification manual.

1.3 Semi-quantitative polymerase chain reaction (PCR)

The semi-quantitative PCR was done with a PTC-100TM hot circulator (MJ Research, Inc., USA). Taq enzyme and 10×Reaction Buffer were obtained from SANGON in Shanghai. The primers for amplification of target gene were designed by a computer program on line (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3-www-results.cgi). The sequences of the primer pairs were as follows: Clock, 5'-TCACCACGTTCACTC AGGACA-3', 5'-AAGGATTCCCATGGAGCAA-3', amplification fragment length 375 bp; H3.3 (as reference^[12]), 5'-GCGTGCTAGCTGGATGTCTT-3', 5'-CCACTGAACTTCTGATTCGC-3', length 230 bp. According to our preliminary experiments seeking after various appropriate parameters relating to PCR, a total reactive volume of 50 ml contained the template 4 ml, dNTP 100 mmol, respective Clock primers 0.7 mmol, respective H3.3 primers 0.3 mmol, MgCl₂ 1.5 mmol, 10×Buffer 5 ml, Taq enzyme 2.5 U. Degeneration was done at 95 ºC for 5 min, followed by hot cycles at 94 ºC for 1 min, 55 ºC for 1 min, 72 ºC for 2 min, 32 cycles, and finally at 72 ºC for 8 min. Agarose (1.5%~2%, SANGON, Shanghai) gel electrophoresis of each PCR product was conducted using SCR-6 constant-voltage electrophoresis system (Radio Communication Manufactory, Danyang, China). The product sizes were estimated by comparison with standard DNA markers (0.1~1 kb DNA Ladder, MBI Fermentas). A microcomputer image analyzing system (Gel Doc 2000^TM, BIORAD, Italy) was used to convert band signals into numerical values. The grayness ratio of Clock/H3.3 bands was served as the relatively quantitative criterion of the Clock gene expression.

1.4 Data analysis

The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 timepoints and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The Cosine function equation^13] was as follows: F (t)=M+Acos (wt+f), where M, A, wt and f stand for mesor, amplitude, 360º/24 h and peak phase, respectively, as explained in our previous report^[9]. Data were presented as mean±SD. One-way ANOVA (software package of SPSS11.0 for Windows) was used for statistical analysis, and differences were considered significant at P<0.05.

2 RESULTS

2.1 Circadian rhythm and photoresponse of Clock gene transcription in the SCN

Changes of Clock mRNA levels in the SCN were observed throughout the day under LD or DD condition (Fig.1A, B). Its relative level was higher during the light phase than the dark time under LD cycle. In contrast, its relative amount was slightly higher during the subjective night than the subjective day under DD condition. Quantitative analysis revealed that the temporal profile of the gene transcription in the SCN showed a significantly circadian oscillation (F test, P<0.05), with the peak at ZT10 and the trough at ZT22 under LD, or with the peak at CT15 and the trough at CT3 under DD regime (Fig.2). The circadian parameters, such as peak phase, amplitude, mesor, mRNA levels at the peak and trough are listed in Table 1. In comparison with that under DD condition, Clock gene transcription in the SCN under LD showed a significantly increased amplitude, an increased mRNA level at the peak and a phase shift forward with the peak at the daytime (P<0.05) (Table 1), which corresponded to a previous report that a light pulse upregulated the expression of Clock gene in the SCN in a phase-dependent manner^[8].

2.2 Circadian rhythm and photoresponse of Clock gene transcription in the PG

In the PG, Clock gene transcription also displayed changes throughout the day under LD or DD regime (Fig.1C, D), but its relative amount was higher in the dark phase of LD cycle. The similar tendency was also observed under DD condition (Fig.2). In comparison with that under DD regime, the amplitude, mesor and the mRNA level at the peak of the pineal Clock gene transcription under LD showed a significant decrease (P<0.05) (Table 1), while the phase differences at the peak and trough were not obtained, suggesting that light could influence the pineal Clock gene circadian transcription but not the phase shift. The temporal profiles of gene expressions in the PG showed a robust circadian oscillation (F test, P<0.05), with the peak at ZT17 and the trough at ZT5 under LD cycle, or with the peak at CT18 and the trough at CT6 under DD condition (Table 1 and Fig.2), which was coincident with previous study^[9].

2.3 Comparison of circadian rhythms and photoresponses of Clock gene transcription between the SCN and PG

Under DD regime, except for the parameters such as amplitude, mRNA level at the peak and trough (Table 1), the temporal profiles of Clock gene transcription in the PG were similar in pattern to those in the SCN (Fig.1B, D and Fig.2) with each peak at the subjective night and trough during the subjective day (P>0.05) (Table 1). Under LD cycle, the circadian profiles of Clock gene transcription in the PG (peak at nighttime ZT17 and trough during the daytime ZT5), however, were quite different from those in the SCN (peak at daytime ZT10 and trough during the nighttime ZT22) in which the Clock transcription rhythms were anti-phasic to the case in the PG (P<0.05) (Fig.1A, C; Fig.2; Table 1).

3 DISCUSSION

The mammalian PG governed by the SCN not only shows neuroendocrine effect, but also serves as a central circadian oscillator^[10]. Both its photic-input and endocrine-output pathways keep in close touch with the central rhythmic oscillation^[10,14]. In the present study, we observed that the mRNA levels of Clock gene in both the SCN and PG displayed significant circadian oscillation under DD or LD condition. It was considered that the transcription levels of Clock gene in the SCN kept constant with unconspicuous rhythmic oscillation within 24 h^[5,15]. Compared with the methods used in other studies such as in situ hybridization with sampling at only two circadian timepoints^[8,16], our results benefit from multiple samplings in a circadian day as well as a more sensitive tool of the reverse transcription PCR in differentiation of circadian gene expressions^[17].

Data under DD regime of this study suggest a synchronous endogenous nature of the Clock gene circadian transcription in the SCN and PG, which further supports the evidence that the pineal endogenous rhythm is driven by the SCN^[1].

It is well known that the alternation of light and dark serves as the main zeitgeber or synchronizer of circadian rhythms^[18]. In order to determine the effects of light entrainment on Clock gene circadian transcriptions in the SCN and PG, the LD (12 h:12 h) regime was used to simulate the natural day and night. Under this LD regime, it was found that Clock gene mRNA expressions in the two central nuclei still displayed a daily rhythm. A light pulse could decrease the amplitude and mRNA level at the peak of Clock gene transcription in the PG without phase shift, but increase those in the SCN with an advanced phase shift. These findings propose a possibility that light may upregulate the Clock gene expression in the SCN and downregulate it in the PG. A previous study^[16] reported that the pineal Clock gene and its functional partner, BMAL1 gene, showed circadian transcriptions under DD and a 30-minute light pulse did not affect the transcription levels of Clock or BMAL1. The difference in our results might be due to insufficient light time and the experimental method used. By comparison of circadian rhythms and photoresponses of Clock gene transcriptions in the SCN and PG under LD cycle, it was revealed that the circadian profiles as well as photoresponses of Clock gene expressions in the PG were quite different from those in the SCN, in which the Clock transcription rhythms were anti-phasic to the case in the PG. This might reflect difference in transcriptional mechanism of Clock gene between the two central nuclei in terms of light-entrained rhythmic oscillation. Daily oscillatory rhythms in the PG, though controlled by the SCN, might be also partly independent of the SCN. Foulkes et al.^[19] have demonstrated that PG itself may have residual clock properties and affect its rhythmic MEL production via the cyclic AMP-responsive element modulator (CREM) feedback loop. On the other hand, independent of the SCN, the pineal MEL, as an important temporal hormone messenger, could be directly affected by light^[20], and its high sensitive receptors in the SCN might modulate the SCN rhythmic output^[21,22]. Therefore, with the ambient photic entrainment, by an innervation of the retino-hypothalamic tract (RHT)^[1], Clock gene circadian expressions in the SCN and PG are likely regulated by different transcriptional pathways, i.e., for the former, light signal input¾RHT¾SCN^[1,10], and for the latter, light signal input¾RHT¾SCN¾neurotransmission/neuroendocrine transmission ¾ PG or light signal input¾ neurotransmission/neuroendocrine transmission¾ PG. In addition to forming a common heterodimer with the product of BMAL1 gene, as positive regulators, which then binds to the E box in the Per1 or cryptochrome (Cry) gene promoter regions for transcriptional induction of Per1 or Cry gene^[5,6,10,14], this and other studies^[19-21] bring forward the possibility that Clock gene in the SCN and PG may play diverse roles in light-entrained circadian rhythms.

It has been shown that the daily rhythm of the pineal MEL production, peaking at nighttime^[23], largely depends on the rhythmic changes in the mRNA levels of arylalkylamine N-acetyltransferase (NAT) gene^[10], and light exposure reduces MEL synthesis^[10,23]. Similar rhythmic profiles of the mRNA levels of NAT, Per1 and Cry genes and effects of light on these gene transcriptions in the rat PG have been also reported in previous studies^{[9,10,14,23-25]}, consistent with the present study. This and other observations raise the possibility that Clock, NAT, Per1 and Cry genes in the PG may jointly function in circadian rhythmic regulation and cooperate with each other to control the rhythmic release of MEL.

Together, the findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, as well as different roles of light entrainment in modulating circadian transcriptions of Clock gene in the two central nuclei.

* * *

ACKNOWLEDGEMENTS: This work was finished in Key Laboratory of Radiation Medicine and Protection of Jiangsu Province, Soochow University, Suzhou, China.

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