Photoperiod Differentially Regulates Circadian Oscillators in Central and Peripheral Tissues of the Syrian Hamster

Current Biology, Vol. 13, 1543–1548, September 2, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S 09 60 - 98 22 ( 03 )0 0 61 9- 5

Photoperiod Differentially Regulates Circadian Oscillators in Central and Peripheral Tissues of the Syrian Hamster Amanda-Jayne F. Carr,1 Jonathan D. Johnston,1 Andrei G. Semikhodskii, Tania Nolan, Felino R.A. Cagampang, J. Anne Stirland, and Andrew S.I. Loudon* School of Biological Sciences University of Manchester Oxford Road Manchester M13 9P United Kingdom

Summary In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD) [1]. After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology [2, 3]. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals [4]. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod [1]. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks. Results and Discussion In order to test the hypothesis that central and peripheral circadian clocks measure seasonal time, male Syrian hamsters were housed on either 16 hr light: 8 hr dark (LD group) or 8 hr light: 16 hr dark for 12 or 28 weeks (SD and SD-R groups, respectively). Following photoperiodic treatment, hamsters were sacrificed every 2 hr across the 24 hr light-dark cycle under the appropriate lighting conditions. Measurement of three factors confirmed the expected response to photoperiodic exposure: the paired testes weight, plasma prolactin concentration, and pineal melatonin content. After 12 weeks of photoperiod treatment, SD hamsters *Correspondence: [email protected] 1 These authors contributed equally to this work.

exhibited significantly reduced paired testes weight (Figure 1A) and 24 hr plasma prolactin concentration (Figure 1B) compared to the LD group. However, following exposure to SD for 28 weeks (SD-R group), the paired testes weight and plasma prolactin concentration were significantly elevated back to LD-like values. There was no significant daily rhythm of prolactin over the 24 hr sampling period for any of the groups (Figure 1B). Pineal melatonin concentration was elevated between zeitgeber time (ZT) 20 and 24 in LD and between ZT14 and ZT22 in both SD groups (Figure 1C). This finding is consistent with previous studies in this species [5, 6] and indicates refractoriness of the reproductive and neuroendocrine axis to the SD photoperiod and prevailing SD melatonin profile. Having established neuroendocrine responsiveness of all hamsters to photoperiodic treatment, we next determined the temporal pattern of expression of three canonical circadian clock genes (Per1, Per2, and Cry2) in the SCN of hamsters in the LD, SD, and SD-R groups. Strong diurnal expression of Per1, Per2, and Cry2 was observed in the SCN of both LD- and SD-housed animals, and expression peaked during the light phase for all genes (Figure 2). Photoperiod had a significant effect on the timing of expression, such that all three genes were expressed for a longer duration in LD hamsters compared to both SD groups. Reduced duration of expression was retained in SD-R animals. On the basis of theoretical modeling of the rodent SCN pacemaker, some authors have proposed a dual oscillator model in which two mutually coupled oscillators track ambient light, with a morning oscillator (M) synchronized to dawn and accelerated by light, and an evening (E) oscillator synchronized to dusk and decelerated by light [7–13]. Recently, different molecular models have sought to relate the E-M oscillator model to circadian clock gene rhythms [14, 15]. These models suggest that either the relative phasing or the duration of clock gene rhythms will depend upon photoperiod. In the current study, peaks of Per gene expression are too broad to permit accurate resolution of phase relationships. However, our data do support the hypothesis that photoperiod regulates the duration of Per and Cry gene expression; this finding is consistent with findings of previous studies of seasonal sheep [16] and hamsters [17–19]. Critically, the duration of SCN gene expression does not differ between SD and SD-R animals, and this similarity demonstrates a clear dissociation between photoperiodic time measurement in the SCN and “downstream” seasonal physiology. Recent studies have revealed strong circadian gene expression in multiple peripheral tissues, including the heart and lung [4, 20]. We therefore used TAQMAN RTPCR to define changes in the level of clock gene transcripts in the same animals. In the lung, there was diurnal variation of Per1 expression in all groups, with a shortduration, low-amplitude rhythm in SD, and a prolonged, high-amplitude rhythm of 14 hr in LD (Figure 3, top row). In both photoperiods, transcripts rose at approximately

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Figure 1. Photoperiodic and Refractory Responses in the Syrian Hamster Hamsters were exposed to LD photoperiods for 12 weeks (LD group) and SD photoperiods for 12 (SD group) and 28 weeks (SD-R group). All values are mean ⫾ SEM. The white and black bars indicate the light and dark phases, respectively. (A) Mean paired testes weight (PTW) after exposure to LD for 12 weeks and SD for 12 and 28 weeks (n ⫽ 48 [LD], 47 [SD], 45 [SD-R]). There was no significant difference between the LD and SD-R PTW; however, both were significantly different from the SD PTW (P ⬍ 0.001). (B) 24 hr profiles of plasma prolactin in LD (solid line, diamond), SD (dotted line, square), and SD-R (dashed line, triangle) hamsters (n ⫽ 3–4 per data point). Data at ZT0 and ZT24 are double plotted. There was no significant effect of time on prolactin expression in any photoperiod group. 24 hr profiles of the LD and SD-R group were significantly different from the SD profile (P ⬍ 0.001), but not from each other. (C) 24 hr profiles of pineal melatonin in LD (solid line, diamond), SD (dotted line, square), and SD-R (dashed line, triangle) hamsters (n ⫽ 3–4 per data point). Data at ZT0 and ZT24 are double plotted. There was a significant effect of time on pineal melatonin in all photoperiod groups (P ⬍ 0.001). SD and SD-R profiles were not significantly different from each other, but they were both significantly different from the LD profile (P ⬍ 0.05).

the same ZT but were truncated in the SD hamster. Thus, photoperiod clearly modulates the amplitude and wave form of gene expression in this peripheral tissue. In SD-R hamsters, there was a clear long-duration rhythm,

which was significantly elevated compared to the SD group but, importantly, was not significantly different from the LD group profile. In order to confirm these observations in another tissue, we also studied the Figure 2. Effects of Photoperiod on Clock Gene Expression in the SCN Following in situ hybridization for Per1, Per2, and Cry2, SCN optical density was expressed relative to the cingulate cortex of the same section. The relative optical density was plotted for LD (solid line, diamond), SD (dotted line, square), and SD-R (dashed line, triangle) hamsters (n ⫽ 3–4 per data point). The white and black bars indicate the light and dark phases, respectively. There was a significant effect of time on all clock gene expression profiles in the SCN (P ⬍ 0.001) and a significant effect of photoperiod (P ⬍ 0.05). There were not significant difference between SD and SD-R gene profiles, but both were significantly different from the LD profiles for all genes examined (P ⬍ 0.005).

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Figure 3. Effect of Photoperiod on Per1 and Dbp Expression in the Lung and Heart Gene expression was analyzed by quantitative PCR and was plotted relative to Gapdh expression. Mean ⫾ SEM of n ⫽ 3–4 hamsters per data point are plotted for LD (first column), SD (second column), SD-R (third column). SD (dotted line, diamond) and SD-R (solid line, square) data are replotted in the fourth column. The white and black bars indicate the light and dark phases, respectively. In the lung, there was a significant effect of time on Per1 expression of all groups (P ⬍ 0.05) and a significant effect of photoperiod between LD and SD animals (P ⬍ 0.05). Per1 expression in SD-R lung was significantly different from the SD animals (P ⬍ 0.001), but it was not significantly different from LD animals. In the heart, there was a significant effect of time on Per1 and Dbp expression for all groups (P ⬍ 0.01) and significant effects of photoperiod for LD, SD, and SD-R animals (P ⬍ 0.01). Both Per1 and Dbp expression in SD-R animals was significantly different from that of SD animals (P ⬍ 0.01).

heart. In LD, there was also a significant diurnal rhythm of Per1 expression, which peaked in the early dark phase. There was a marked attenuation of the heart Per1 rhythm in SD, with a small but statistically significant elevation confined to the late-night portion of the cycle (Figure 3, middle row). In marked contrast, SD-R animals exhibited a high-amplitude nocturnal peak of Per1 with a 6-fold higher level compared to the SD group. Thus, in both the lung and heart, the Per1 rhythm exhibits a significant increase in duration and/or amplitude in animals that become refractory to SD. In many tissues, Per1 can function as an immediateearly gene, induced by different stimuli [21–24], and thus may not reflect an altered core circadian oscillator per se. In order to test further the hypothesis that altered Per1 expression is due to a change in peripheral clock function, we extended our study to include Dbp, a clockcontrolled gene whose circadian expression is regulated by the action of core clock proteins on E box promoter elements and therefore reflects clock output [25, 26]. In the LD heart, there was a pronounced Dbp rhythm, with

a peak at ZT14 (Figure 3, bottom row). Similar to Per1, there was a loss of overt rhythmicity of Dbp in SD, but a high-amplitude rhythm in SD-R animals (Figure 3, bottom RH panel). The phasing of this SD-R rhythm was similar to that of Per1 for LD/SD-R animals, with a peak in expression at ZT12. The loss of overt rhythmicity of Dbp and the generation of a low-amplitude Per1 rhythm in the heart on SD may reflect a significant loss or dampening of circadian regulation in this tissue on short day lengths. Our data now show that photoperiod regulates both clock and clock-controlled gene expression in peripheral tissues. Furthermore, in SD-R hamsters, the amplitude and/or duration of expression of both Per1 and Dbp are markedly increased compared to SD animals, despite both SCN and pineal rhythms remaining “locked” to the ambient photoperiod. These observations therefore strongly implicate an intermediary component(s) driving these peripheral oscillators. A number of physiological systems are now implicated in the regulation of peripheral oscillator function, includ-

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Figure 4. Model of Seasonal Time Measurement in the SCN and Peripheral Circadian Clocks Photoperiod regulates the duration of expression of multiple SCN clock genes and downstream pineal melatonin secretion. These rhythms remain locked to photoperiod, irrespective of physiological state. A well-defined melatonin target tissue, the PT, responds to melatonin signal duration with altered amplitude of Per1 gene expression, which remains locked to melatonin signal duration, irrespective of PT endocrine activity. The lung and heart, peripheral oscillators that do not express melatonin receptors, respond to photoperiod with altered duration and amplitude of Per1 and Dbp expression. In contrast to the SCN and PT region of the pituitary gland, gene expression in these peripheral tissues tracks seasonal changes in physiological state, rather than ambient photoperiod.

ing body temperature rhythms and feeding. In rodents, food restriction can phase shift rhythms in peripheral tissues, including the lung and heart, although these organs are less sensitive to food restriction than the liver [27, 28]. In seasonally breeding animals, photoperiod regulates body weight, although much of the seasonal weight change in Syrian hamsters is accomplished without altered food intake [29], and our hamsters were provided with food ad libitum throughout the experiment. It is possible that changes in the relative timing of feeding in SD-R hamsters, albeit without an overall change in food intake, is responsible for the changes in peripheral Per1 and Dbp expression. However, altered neuroendocrine output may offer a more attractive pathway responsible for photoperiodic modulation of peripheral oscillators, and hormones such as glucocorticoids both regulate the phasing of peripheral oscillators [30, 31] and are strongly modulated by photoperiod in seasonal mammals, including hamsters [32]. We have re-

cently shown that within the melatonin target site of the pituitary gland (the pars tuberalis, PT), exposure to SD results in an SD-like clock gene rhythm within this tissue, irrespective of refractory state [6]. In contrast, the prolactin-releasing activity of the PT cells reverts to an LD phenotype in the SD-R hamster [6]; this phenotype makes PT cells prime candidates for seasonal calendar cells, which drive annual cycles of this hormone system [33]. An intriguing possibility is that changes in the neuroendocrine axis of refractory animals, but not necessarily prolactin, may drive seasonal changes in peripheral circadian oscillators. The regulation of peripheral clocks may then contribute toward the adaptive changes observed in major body organs of seasonally breeding mammals [34–37]. Conclusions The outcome of our experiments is shown as a summary model (Figure 4). A major conclusion from our study is

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that both central (SCN) and peripheral oscillators exhibit clear photoperiodic modulation of clock gene rhythms. However, peripheral oscillators may also measure seasonal time, while the role of the central SCN oscillator and pineal melatonin rhythm is to track ambient photoperiod, irrespective of physiology. Decoding the pathways and molecular mechanisms that link the SCN and periphery may reveal important information relating to photoperiodic time measurement in mammals and perhaps other vertebrates. Experimental Procedures Animals Animals were housed as previously described [6] and in accord with the provisions of the 1986 Animal Procedures Act (UK). Assays and In Situ Hybridizations Prolactin and melatonin radioimmunoassays were performed as previously described [38, 39]. Prolactin assay sensitivity was 0.78 ng/ml, and intra- and interassay coefficients of variation were 6.7% and 5.1%, respectively. Melatonin assay sensitivity was 11.03 pg/ ml, and the mean intra- and interassay coefficients of variance were 17.9% and 8.1%, respectively. In situ hybridization was conducted by using antisense riboprobes for Per1, Per2, and Cry2 labeled with 33 P-UTP and hybridized to 20 ␮m coronal brain sections and was quantified as previously described [6]. TAQMAN Q-PCR cDNAs were prepared from 3 ␮g DNase-treated total lung and heart RNA by using Superscript II Reverse Transcriptase (GIBCO-BRL) according to the manufacturer’s instructions. Primers and probes were synthesized as follows, with probes labeled with 6-FAM and TAMRA at the 5⬘ and 3⬘ ends, respectively (MWG, AE): Per1 forward, 5⬘-GCCTTCCTCAACCGCTTCA-3⬘; Per1 reverse, 5⬘-GAGGGAGCC ACAGAAGAGTTGT-3⬘; Per1 probe, 5⬘-TCCACGGAGCCTGCCAAG GTCT-3⬘; Gapdh forward, 5⬘-CACCCGAGGACCAGGTTGTCT-3⬘; Gapdh reverse, 5⬘-CATACCAGGAGATGAGCTTTACGA-3⬘; Gapdh probe, 5⬘-CAAGAGTGACTCCCACTCTTCCACCTTTGA-3⬘; Dbp forward, 5⬘-CGAGACGCACGAAGACTCAA-3⬘; Dbp reverse, 5⬘-CACCAC CTCCTGTCGCAATA-3⬘; Dbp probe, 5⬘-TCGGTGCGGGCCGCCTT-3⬘. Amplification was performed on an ABI 7700 Sequence Detection System (Applied Biosciences) by using the TAQMAN 1000 RxN Gold (Applied Biosicences) with buffer A kit according to the manufacturer’s instructions. The parameters of the amplification were 50⬚C for 2 min, 95⬚C for 10 min, followed by 40 cycles of 95⬚C for 15 s and 60⬚C for 1 min. PCR amplification of lung samples was performed alongside gene-specific standards of known concentration. These were prepared by amplifying hamster-specific gene sequences by using the following primers: Per1 forward, 5⬘-GCAAAAAGAAGAG CAAGC-3⬘; Per1 reverse, 5⬘-TGGTGAAGATAGCATCAGC-3⬘; Gapdh forward, 5⬘-CTGCCCAGAACATCATCC-3⬘; Gapdh reverse, 5⬘-GCC GAATTCATTGTCATACC-3⬘; Dbp forward, 5⬘-CAAGAACAATGAGG CAGC-3⬘; Dbp reverse, 5⬘-AAGAGCACACACAGGACC-3⬘. The PCR products were purified and diluted to produce a set of standards ranging from 3 ⫻ 101 to 3 ⫻ 107 double-stranded molecules. The number of molecules of Per1 and Dbp was expressed as a percentage of Gapdh. Statistical Analysis Differences between experimental groups were compared by analysis of variance with SigmaStat 2.033 (SPSS). Acknowledgments We thank Dr. Urs Albrecht for the gifts of hamster Per1, Per2, and Cry2 probes, Dr. Richard Preziosi for statistical advice, and Dr. Michael Hastings for helpful comments on an earlier draft. This work was supported by Biotechnology and Biological Sciences Research Council, UK (BBSRC) studentships (to A-J.F.C. and J.D.J.) and a BBSRC grant (to A.S.I.L.).

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