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Short communication
The pineal gland is not essential for circadian expression of rat period
homologue (rper
2
) mRNA in the suprachiasmatic nucleus and
peripheral tissues
a b a,c d d
Katsutaka Oishi , Ichiro Murai , Katsuhiko Sakamoto
, Hitoki Otsuka , Yoshiaki Miyake ,
e a,f ,
*
Takahiro Nagase , Norio Ishida
a
Ishida Group of Clock Gene, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Tsukuba,
Ibaraki305-8566, Japan
b
Department of Biochemistry, Nihon University School of Medicine, Itabashi, Tokyo 173-0032, Japan
c
Japan Science and Technology Corporation, Japan
d
Department of Obstetrics and Gynecology, Nihon University School of Medicine, Nerima, Tokyo 179-0072, Japan
e
Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan
f
Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501, Japan
Accepted 12 September 2000
Abstract
To investigate the functional involvement of the pineal gland in circadian expression of the rat period homolog gene (rPer2 ) in the suprachiasmatic nucleus (SCN) and peripheral tissues, we performed Northern blot analysis in tissues from pinealectomized rats. The ectomy did not have any significant effects on rPer2 mRNA expression patterns both in a daily light–dark condition and in a constant darkness. These results suggest that the rhythmic secretion of pineal melatonin is not essential for the circadian expression of clock genes in the SCN and other peripheral tissues of rats. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behaviour
Topic: Biological rhythms and sleep
Keywords: Circadian rhythm; Period homologue; Pinealectomy; Melatonin; Suprachiasmatic nucleus; Peripheral tissues
Mammalian homologs of Drosophila clock gene period mals [7]. These mRNAs are rhythmically expressed even ( per) are thought to be the essential elements that regulate in peripheral tissues such as eye, heart, lung, spleen, circadian rhythms in organisms [7]. Recently, Zheng et al. kidney and liver [17–19,23,28]. Previously, we showed [27] showed that a deletion mutation in the PAS domain of that the circadian expression of rPer2 mRNA in peripheral
mPer2 gene abolished a circadian rhythmicity of tissues is governed by the SCN, because the rhythmic locomotor activity in mice. This data define mPer2 as a expressions are abolished in SCN-lesioned rats [23]. We component of the mammalian circadian oscillators. Mam- also suggested that the humoral factors, driven by the malian per homologs ( per1, per2 and per3 ) and BMAL1 SCN, mediate the circadian expression of per genes in (a putative clock gene which encodes a basic helix-loop- peripheral tissues, because the rhythmic expression of per helix PAS transcription factor) mRNAs exhibit a robust genes are observed in circulating mononuclear leukocytes circadian expressions in the suprachiasmatic nucleus that have no neuronal connections [18]. In vitro experi-(SCN): the principal pacemaker that regulates various ments using rat-1 fibroblast culture systems have also physiological and behavioral circadian rhythms in mam- implied the existence of humoral factors that regulate the circadian expression of clock genes in peripheral tissues [2,25]. However, little is known about the factors mediat-*Corresponding author. Tel.: 181-298-61-6500; fax: 1
81-298-61-ing the rhythmic expression of mammalian clock genes. 6095.
E-mail address: [email protected] (N. Ishida). In mammals, the pineal hormone melatonin
methoxytryptamine) is involved in the regulation of numer- detection were performed as described [22]. Relative ous physiological and behavioral processes cued by the mRNA levels were normalized by determination of the environmental light–dark cycle [20]. Although pinealec- amount of b-actin mRNA (data not shown).
tomy has no significant effect on free-running locomotor At first, we measured blood melatonin concentrations of rhythms, exogenous melatonin modulates the function of rats under LD 12:12, to confirm that the operation for the circadian clock in the SCN, and entrains the activity pinealectomy had been achieved completely. The robust and sleep in mammals [1,4]. Circadian synthesis of pineal daily secretion of plasma melatonin was abolished by the melatonin is endogenously controlled by the SCN and is ectomy (Fig. 1). The small amount of circulating synchronized by environmental light [20]. While the SCN melatonin in the pinealectomized rats might have been is a major site of melatonin binding in the mammalian secreted by retina and other peripheral organs, since there brain, melatonin binding sites at the periphery have also is accumulating evidence showing that the mammalian been detected [3,5,16]. Thus, it is reasonable to speculate retina [9,21,22,26] and peripheral blood mononuclear that circulating melatonin from the pineal grand mediates leukocytes [10] can synthesize and even secrete melatonin. the circadian expression of mammalian clock genes in We examined the rPer2 mRNA expression in whole peripheral tissues. brain, eye, and heart both in LD 12:12 (Fig. 2). In In this study, to investigate the functional involvement pinealectomized rats as well as in sham control rats, the of rhythmically secreted pineal melatonin in the expres- robust circadian expression of rPer2 mRNA with a peak sions of mammalian clock genes in peripheral tissues, we between ZT 14 and 17, was observed in each tissue. We examined the temporal expression patterns of rPer2 also examined the mRNA expression patterns of rPer1 and mRNA in tissues from pinealectomized rats under a daily BMAL1 genes, and observed that the pinealectomy had no light–dark cycle and under constant darkness by Northern significant effect on the mRNA expression patterns in each blot analysis. tissue (data not shown).
Male Wistar rats (8-weeks-old) obtained from Clea To evaluate whether the diurnal fluctuation of rPer2 Japan, Inc. (Tokyo) were pinealectomized as described mRNA is a response to a light–dark periodicity or reflects [14]. The rats were housed under a 12 h light–12 h dark a circadian clock, we examined the mRNA levels under cycle [LD 12:12; lights on at Zeitgeber time (ZT) 0]. A DD (Fig. 3). However, the rhythmic expression of rPer2 white fluorescent lamp was used as a source of light during
the day (150–200 lux at the level of the cages).
We measured blood melatonin concentrations of rats under LD 12:12, to confirm that the operation for pinealec-tomy had been completely successful. Two weeks after the operation, we collected blood samples using a heparinized microcapillary from the tail vein. The plasma fraction was gained by centrifugation, then the circulating melatonin levels were measured with commercially available ELISA
¨
kit (Buhlmann Laboratories Ag, Allschwil, Switzerland) that has a sensitivity of 3.6 pg / ml. When we drew the blood at night, a dim red light was used in order to avoid a possible light influence on the melatonin profile.
Four weeks after the operation, rats were decapitated, and tissues were dissected, quickly frozen, and kept in liquid nitrogen until used. In darkness, dissections were carried out under a dim red light. To investigate whether the expression of rPer2 mRNA was influenced by the masking effect of light, we examined the effect of pinealectomy under a constant darkness (DD) as well as in an LD condition. The experiment in DD was performed after free-running for 2 days in DD. Total RNA was isolated from tissues and separated on a 1% agarose / 0.7 M formaldehyde gel as described [22]. RNA was transferred to a nylon membrane (GeneScreen Plus; DuPont, USA) by
Fig. 1. Effects of pinealectomy on daily changes in plasma melatonin passive capillary transfer. Each lane contained 20 mg of
concentrations. Values represent the mean6S.D. from four animals. Rats total RNA from each tissue or 5mg of total SCN RNA. A
were housed in LD 12:12 (lights on at ZT 0) for 2 weeks after
32
P-labeled random primed probe was generated from a pinealectomy. Filled circles indicate values from the pinealectomized
Fig. 2. Daily expression of rPer2 mRNA in the SCN (n53), whole brain (n51), and peripheral tissues (eye: n53, heart: n51) from pinealectomized or sham rats. (a) Representative Northern blot analyses of rPer2 mRNA. Rats were housed in LD 12:12 (lights on at ZT 0) for 4 weeks after the ectomy. The animals were sacrificed at each time-point indicated above the horizontal bar. The open bar indicates lights-on, and the dark bar indicates lights-off. (b) Quantification of rPer2 mRNA expression from the Northern blots. Filled circles indicate values from the pinealectomized group and open circles from the sham group. The maximum value of the sham group was expressed as 100% in each tissue.
mRNA was observed in pinealectomized rats as well as in cAMP accumulation via the G protein-coupled receptors sham control rats. The ectomy did not cause any significant [11,13,15]. Recently, in vitro experiments using rat-1 differences between the groups. fibroblasts have implied the involvement of cAMP/ protein In this study, we showed that the pineal gland is not kinase A signaling pathway in the regulation of rPer1 indispensable for the rhythmic expression of mammalian expression [25]. Although the entraining mechanism of clock genes in rat tissues. This is the first report that exogenous melatonin is still unknown, the exogenous described the relationship between the pineal grand and the melatonin might change the expression levels of clock circadian expression of clock genes in rats. genes, and consequently entrain the circadian rhythms of A number of studies have indicated that the administra- mammals. However, our results suggest that the physiolog-tion of exogenous melatonin can entrain the circadian ical pineal melatonin is not essential for the circadian locomotor patterns of mammals [1,4]. Since cyclic AMP expression of clock genes either in the SCN or in peripher-(cAMP)-mediated mechanisms are important for the phase- al tissues.
Fig. 3. Circadian expression of rPer2 mRNA in the SCN (n52), whole brain (n51), and peripheral tissues (eye: n52, heart: n51) from pinealectomized or sham rats in constant darkness. (a) Representative Northern blot analyses of rPer2 mRNA. Rats were housed in LD 12:12 (lights on at ZT 0) for 4 weeks after the ectomy, then transferred into constant darkness (DD). The animals were sacrificed at each time-point indicated above the horizontal bar. The shaded bar indicates subjective day, and the dark bar indicates subjective night. (b) Quantification of rPer2 mRNA expression from the Northern blots. Filled circles indicate values from the pinealectomized group and open circles from the sham group. The maximum value of the sham group was expressed as 100% in each tissue.
homologs in peripheral tissues through humoral factors tissues may provide an understanding of mammalian [18,23]. It has been shown that humoral factors, released circadian rhythms.
by the SCN, mediate circadian locomotor activity, since the circadian locomotor activity can be rescued in
SCN-lesioned mammals by implants of fetal SCN tissue [6,24] Acknowledgements or immortalized SCN cell lines [8]. Therefore, some
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