Multiple Effects of Melatonin on Rhythmic Clock Gene Expression in the Mammalian Pars Tuberalis
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《内分泌学杂志》
School of Biological Sciences (J.D.J, D.G.H.), University of Aberdeen, Zoology Building, Aberdeen AB24 2TZ, Scotland, United Kingdom
Neurobiologie des Rhythmes laboratory, Unite Mixte de Recherche 7518 Centre National de la Recherche Scientifique/Universite Louis Pasteur (B.B.T., M.M.-P.),67084 Strasbourg, France
Medical Research Council Human Reproductive Sciences Unit (H.A., G.A.L.), Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom
Abstract
In mammals, changing day length modulates endocrine rhythms via nocturnal melatonin secretion. Studies of the pituitary pars tuberalis (PT) suggest that melatonin-regulated clock gene expression is critical to this process. Here, we considered whether clock gene rhythms continue in the PT in the absence of melatonin and whether the effects of melatonin on the expression of these genes are temporally gated. Soay sheep acclimated to long photoperiod (LP) were transferred to constant light for 24 h, suppressing endogenous melatonin secretion. Animals were infused with melatonin at 4-h intervals across the final 24 h, and killed 3 h after infusion. The expression of five clock genes (Per1, Per2, Cry1, Rev-erb, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erb showed pronounced temporal variation despite the absence of melatonin, with peak times occurring earlier than predicted under LP. The time of peak Bmal1 expression remained LP-like, whereas Cry1 expression was continually low. Melatonin infusion induced Cry1 expression at all times and suppressed other genes, but only when they showed high expression in sham-treated animals. Hence, 3 h after melatonin treatment, clock gene profiles were driven to a similar state, irrespective of infusion time. In contrast to the PT, melatonin infusions had no clear effect on clock gene expression in the suprachiasmatic nuclei. Our results provide the first example of acute sensitivity of multiple clock genes to one endocrine stimulus and suggest that rising melatonin levels may reset circadian rhythms in the PT, independently of previous phase.
Introduction
MELATONIN, THE PRINCIPAL product of the vertebrate pineal gland, undergoes marked temporal variation in secretion and drives a wide range of rhythmic physiology. In mammals, the synthesis and secretion of pineal melatonin occurs at night, under the control of the circadian clock present in the suprachiasmatic nuclei (SCN) of the hypothalamus. The duration of this nocturnal signal varies in proportion to the length of the night, and thence melatonin secretion transduces both daily and seasonal time throughout the body (1).
The pituitary pars tuberalis (PT) is strongly implicated in the photoperiodic regulation of prolactin secretion and, because of its high density of melatonin receptors, has become a key model tissue for the understanding of melatonin signal readout (2, 3, 4). It is now clear that melatonin is a key regulator of clock gene expression in the mammalian PT, because modulation of the endogenous profile of melatonin secretion, by manipulation of ambient photoperiod or by administration of exogenous melatonin, changes PT clock gene profiles (5, 6, 7, 8, 9).
To date, the documented effects of melatonin in the PT can be divided into those occurring in the morning (Per1) and those occurring in the evening (Cry1, Rev-rb). The former depend on melatonin withdrawal at the end of the night, probably mediated by the resultant increase in cAMP production (2, 5). In rodents unable to synthesize melatonin, either because of genetic deficiency (10) or pinealectomy (11), rhythmic expression of Per1 expression is absent, suggesting that melatonin is necessary for maintained rhythmic gene expression in this tissue.
Collectively, these observations suggest that the PT contains a circadian oscillator that is either driven or modulated by melatonin and raise questions about 1) whether a rhythmic melatonin signal is necessary for the 24-h variation of clock gene expression in the PT and 2) whether melatonin actions on gene expression in the PT is restricted to time windows around the morning and evening changes in illumination. To test these ideas, we returned to the sheep as a model because this species has been extensively used for investigations into the molecular basis of melatonin-dependent photoperiodic responses (2, 6, 12). We placed animals in constant light for one 24-h cycle (thereby suppressing endogenous melatonin secretion) and treated them at 4-h intervals with either melatonin-containing sc implants or sham procedure.
Materials and Methods
Animals and housing
All animals were treated in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Forty-eight pubertal female Soay sheep, obtained from specialist breeders in Scotland (12), were brought indoors in winter (November). They were housed in light-sealed rooms and fed a standardized diet of grass pellets (500 g per animal; Vitagrass, Cumbria, UK) given daily 1 h into the light phase. Hay and water were available ad libitum. White fluorescent strip lights provided approximately 160 lux at the animals’ eye level during the light phase, whereas dim red light (<5 lux) was provided during the dark phase.
Light and melatonin treatment
The animals were initially acclimatized under a winter-like short photoperiod (SP; 8 h light, 16 h darkness) for 4 wk and then exposed to long photoperiod (LP; 16 h light, 8 h dark) for 6 wk. Blood samples were collected twice weekly from the jugular vein from representative animals (n = 12), and the blood plasma separated by centrifugation within 30 min and stored at –20 C, to measure prolactin concentration as evidence of photoresponsiveness.
Over the final 24 h of the experiment, lights were left on to suppress endogenous pineal melatonin secretion. Animals were treated with a single sc implant of melatonin (Regulin; CEVA Animal Health, Chesham, Bucks, UK) placed on the inside of the hind leg using a standard injector, or a sham treatment with no implant as control at 4-h intervals across the final 24 h (n = 4 per time point), and killed 3 h after treatment by pentobarbital injection. Immediately before death, blood samples were collected from the jugular vein and processed as above to permit analysis of the effect of treatments on melatonin concentration. The hypothalamus and upper pituitary gland were removed within 10 min of death and snap frozen in isopentane kept at –30 C by dry ice. The expression of five clock genes (Per1, Per2, Cry1, Rev-erb, and Bmal1) was measured in the PT and SCN by in situ hybridization.
RIA
Prolactin concentrations were measured by a validated RIA (13). The lower limit of sensitivity was 0.5 ng/ml prolactin standard (NIH-PRL-S13), and the intraassay coefficient of variation was 8.3%. Melatonin concentrations were also measured by a standard RIA (14), using a commercial antibody (PF-1288; SPI-BIO, Paris, France). The sensitivity of the assay was 5 pg/ml, and the intraassay coefficient of variation was 12.0%.
In situ hybridization
Coronal cryosections (20 μm) cut through the SCN and PT were thaw-mounted onto poly-L-lysine-coated glass slides and stored at –80 C. Expression of the clock genes Per1, Per2, Cry1, Bmal1, and Rev-erb was then detected using radioactive riboprobes and procedures described previously (15).
Statistics
Weekly changes in plasma prolactin concentration during acclimation to LP were analyzed by one-way ANOVA with repeated measures. Clock gene expression profiles in sham-treated animals were analyzed by one-way ANOVA. The comparison between melatonin and sham treatment was analyzed by two-way ANOVA and Bonferroni’s post hoc testing when appropriate.
Results
Photosensitivity of experimental animals
The pretreatment exposure to LP induced a significant (P < 0.01, one-way ANOVA with repeated measures) increase in plasma prolactin concentrations from 19.2 ± 4.6, at the change from SP to LP, to 52.4 ± 11.1 ng/ml at wk 6, illustrating the physiological and photosensitive state of the experimental animals before the final day on constant light (LL) (Fig. 1).
Effect of treatment on endocrine profiles
LL treatment resulted in suppressed plasma melatonin concentration across the 24-h cycle (Fig. 2A; melatonin concentration in sham group was 10.4 ± 0.9 pg/ml, mean ± SEM, n = 24). Melatonin implants significantly increased plasma melatonin concentration within 3 h (Fig. 2A; P < 0.001 for effect of treatment, two-way ANOVA). This effect was independent of infusion time (P > 0.05 for treatment x time interaction, two-way ANOVA). The resulting plasma melatonin concentrations (216.9 ± 28.0 pg/ml, mean ± SEM, n = 24) were within the nocturnal physiological range commonly observed in this species (12).
Although prolactin concentration appeared to be low in sham-treated animals killed at zeitgeber time 16 (ZT16) (Fig. 2B), there was no statistically significant 24-h variation in either the sham- or melatonin-treated animals (P > 0.05 for effect of time in both experimental groups, one-way ANOVA). Administration of melatonin during LL decreased mean plasma prolactin concentration within 3 h of treatment (Fig. 2B; plasma prolactin concentration was 29.8 ± 4.0 vs. 18.6 ± 2.6 ng/ml, sham vs. melatonin-treated group, mean ± SEM, n = 24; P < 0.01 for effect of treatment, two-way ANOVA). This acute response to melatonin was not time dependent (P > 0.05 for treatment x time interaction, two-way ANOVA).
Clock gene expression in the PT
Despite the suppressed plasma melatonin concentrations in sham-treated animals, the expression of four of the five clock genes analyzed in the PT exhibited significant (P < 0.05, one-way ANOVA) variation over the 24-h cycle (Fig. 3). For Rev-erb and Per1, which show peak PT expression in the early light phase in LP acclimated animals (6, 8), a melatonin-independent expression peak was seen after approximately 20 h of constant light treatment. For Per2, which peaks slightly later than Per1 under LP (6), a melatonin-independent increase in expression was seen after 24 h of exposure to LL. Similar to the case in LP acclimated animals (6), Bmal1 expression peaked between ZT12 and ZT16 in LL.
In contrast to the other clock genes, Cry1 expression in sham-treated animals did not vary significantly over the LL sampling period (P > 0.05, one-way ANOVA) and was low at all sampling points (Fig. 4). Melatonin infusion for a period of 3 h caused a large increase in the expression of Cry1 mRNA in the PT (Fig. 4) (P < 0.001 for effect of treatment, two-way ANOVA). This increase occurred at all sampling points throughout the 24-h cycle (P < 0.05 melatonin vs. sham, two-way ANOVA with Bonferroni’s post hoc test). The expression of Per1, Per2, Rev-erb, and Bmal1 was also sensitive to melatonin treatment, but in these instances the effects of melatonin were inhibitory and time dependent (P < 0.05 melatonin vs. sham, two-way ANOVA with Bonferroni’s post hoc test) (Fig. 5). As a result of these effects, the expression of clock genes in the PT 3 h after melatonin treatment was qualitatively similar, irrespective of the time of infusion; Cry1 mRNA was elevated, whereas all other mRNAs were suppressed (Figs. 4 and 5).
Clock gene expression in the SCN
The expression of all five clock genes was rhythmic in the SCN over 24 h of LL exposure (P < 0.001 for effect of time, two-way ANOVA). Melatonin had a significant effect on Rev-erb expression (P = 0.05 for effect of treatment, two-way ANOVA), although this effect was not dependent upon time of melatonin treatment (P > 0.05 for treatment x time interaction, two-way ANOVA). For all other genes, melatonin had no significant effect on mRNA expression (P > 0.05 effect of treatment, two-way ANOVA) (Fig. 6).
Discussion
Recent studies have identified rhythmic expression of multiple clock genes in the mammalian PT and proposed models by which these genes may mediate the photoperiodic effects of melatonin (16). Here, we have used photosensitive Soay sheep to demonstrate that clock gene rhythms persist in the absence of endogenous melatonin secretion. Furthermore, in these animals, melatonin infusions acutely alter the expression of multiple clock genes at all phases of the 24-h cycle in the PT, without any corresponding effects in the SCN. These data are consistent with the possibility that the PT functions as a circadian oscillator that can be synchronized by an increase in melatonin concentration in the bloodstream, irrespective of when this occurs relative to lights on. More generally, this study provides the first example of acute responsiveness of multiple clock genes to a single defined endocrine stimulus.
Since the classical lesioning experiments performed over 30 yr ago (17, 18), the SCN has been viewed as a master circadian pacemaker that drives and synchronizes overt rhythms of physiology and behavior. The recent identification of circadian oscillators in peripheral tissues and in immortalized cell lines suggests that the role of the SCN is primarily to coordinate circadian oscillations throughout the organism (19) and focuses attention on the mechanisms through which such circadian synchronization takes place. The precise manner by which a given SCN-dependent circadian signal influences peripheral oscillator function may be expected to reflect the physiological functions of the tissues upon which it acts. In mammals, melatonin is one such SCN-dependent circadian output, whose primary function is to modulate neuroendocrine physiology, by relaying information about changes in day length (20). The pineal melatonin signal communicates the phase of night onset and of the subsequent dawn; melatonin is secreted continuously between these transitions, giving a signal whose duration is proportional to the length of the night. This ability of the melatonin signal to represent night duration is crucial for photoperiodic responses in seasonal mammals (21).
The PT is a largely homogeneous population of type 1 melatonin (MT1) receptor-expressing thyrotrophic cells (22, 23), making it an ideal tissue for the study of melatonin action. Long-term loss of melatonin synthesis or loss of functional MT1 receptors abolishes rhythmic clock gene expression in the mammalian PT (10, 11, 24, 25), indicating that the molecular clockwork of this tissue is ultimately melatonin dependent. The data presented here, however, demonstrate clear temporal variation in PT expression of four core clock genes over a 24-h period in the absence of a rhythmic melatonin signal and thus strongly support the existence of an endogenous PT oscillator. We therefore suspect that arrhythmic Per1 gene expression in the PT of hamsters that have been pinealectomized for 7 d (11) derives from progressive damping of the PT circadian oscillator. Based on recent studies of damping in cell culture systems (26, 27, 28), we speculate that this is a result of desynchronization of circadian oscillators in individual PT cells. Accordingly, in genetically melatonin-synthesis- or melatonin-receptor-deficient mice (10, 24, 25), PT gene expression is likely arrhythmic because PT cells never become synchronized during fetal or postnatal development.
The present study has examined only one aspect of the information inherent in the endogenous melatonin signal: rising melatonin levels associated with night onset. Our data suggest that a dusk-like response (high Cry1, low Per, Rev-erb, and Bmal1) can be elicited by increasing melatonin levels occurring at any phase throughout the 24-h cycle. This suggests that PT cells exhibit a type 0 resetting response (29) to rising melatonin levels, that is to say that the PT molecular clockwork resets to a dusk state in response to rising melatonin, independent of its state immediately beforehand. This dusk state is transient, because our previous data show that a state of low Cry1 and high Bmal1 expression develops during continued melatonin presence through the middle to the end of the night (6). Because the effects of melatonin on cAMP-dependent signal transduction and gene expression persist during prolonged melatonin exposure (2), it is likely that cAMP-independent mechanisms account for disappearance of the dusk state under physiological conditions.
Interestingly, the PT of the Japanese quail also rhythmically expresses clock genes, of which Cry1 may be acutely induced by light. However, photic induction of Cry1 is subject to circadian gating in this tissue (30), suggesting that the avian PT may contain a more robust oscillator than is the case in mammals.
For sheep in a natural environment, the times of dawn and dusk move in opposite directions during the annual cycle, raising the possibility of a similar flexibility in the resetting response of the PT oscillator to declining melatonin levels associated with dawn. Although we have yet to perform an experiment explicitly to address this, our published data agree with this prediction; the timing of the dawn state, characterized by high Per1, can be delayed by an acute light manipulation in the preceding evening that delays the dawn-associated decline in melatonin secretion (8). Hence the circadian clockwork of the sheep PT shows a remarkable plasticity in its response to changing patterns of melatonin secretion.
Early work in the mammalian PT focused on the ability of melatonin to modulate adenylate cyclase activity (2). Melatonin-regulated expression of Per1, which is considered a cAMP-regulated immediate-early response gene in the PT (31), is likely to occur through this pathway (24). However, other clock genes are not directly sensitive to cAMP signaling, suggesting that melatonin-dependent signaling may occur by additional and as yet undefined signaling pathways. Consistent with this possibility, previous studies of many tissues, including the ovine PT, have revealed a wide range of intracellular pathways that are sensitive to melatonin (32). At present, we cannot entirely rule out the possibility that induction of Cry1 drives changes in the expression of other clock genes. However, we feel that this putative mechanism is unlikely, because it would require not only rapid transcription and translation of Cry1 but also a high level of mRNA instability for other clock genes to be affected within the short interval (3 h) between melatonin infusion and tissue collection used in this study.
Additional to its role in seasonal neuroendocrine control, melatonin may influence the function of the SCN itself. Exogenous melatonin administration has been shown to synchronize circadian behavioral rhythmicity in rodents (33, 34) and in humans (35), and melatonin can reportedly phase-shift circadian rhythms of electrical activity in mouse SCN slice preparations (36). Knockout of the MT1 and MT2 melatonin receptor subtypes abolishes phase-shifting responses to melatonin in mice and acute effects of melatonin on SCN electrical activity (37, 38). Hence it is possible that circadian effects of melatonin are mediated through melatonin receptors located in the SCN. Nevertheless, a recent study reported no effect of melatonin injection on clock gene expression in the rat SCN (39). In sheep, melatonin receptors are at best weakly expressed in the SCN (40), and in the present study a mild effect of melatonin injection was seen for only one gene, Rev-erb, at one time point only. Possibly our data and that of Poirel et al. (39) reflect different coupling between melatonin receptors and circadian molecular clockwork in SCN compared with PT cells. Alternatively the heterogeneous cellular composition of the SCN and its sensitivity to multiple inputs in addition to melatonin (41) may account for these results.
A final point of interest is the regulation of plasma prolactin concentration in this study. We have previously described a LP-induced increase in prolactin concentration within 24 h (8). In this study, the animals required approximately 4–6 wk of LP exposure before showing such a rise. This difference can be explained by two reasons. In the current study, animals were likely to exhibit early refractoriness to shortening winter photoperiod when they were transferred to controlled lighting regimes; furthermore, animals were not habituated to blood sampling and it is therefore likely that initial prolactin concentrations were affected by stress of experimental manipulation at this stage. During the final 24 h of this experiment, melatonin caused an acute inhibition of prolactin release in the sheep under constant light. This is unlikely to reflect a direct effect on lactotroph cells, which do not appear to express melatonin receptors (22, 42) and are unresponsive to melatonin in primary cell culture (43). It is unclear whether melatonin acts at the level of the PT or hypothalamus to acutely suppress prolactin concentration. Although hypothalamic monoamines are not believed to drive photoperiodic changes of prolactin secretion (44, 45), there is some evidence that melatonin may stimulate secretion of the prolactin-inhibiting factor dopamine from hypothalamic neurons (46, 47). Alternatively, melatonin infusions may have acutely inhibited the secretion of prolactin-releasing factor(s), termed tuberalin, from the PT (43, 48, 49, 50, 51). However, the acute effects of melatonin on tuberalin secretion are as yet unknown.
In summary, we propose that the PT may become an important model tissue to address the function of peripheral circadian oscillators as well as the mechanisms of photoperiodic time measurement. A strength of the PT as a circadian model is that it has a well defined circadian input, melatonin. As well as testing the generality of the results described here in other species, we are currently developing in vitro techniques to further evaluate the effect of melatonin on the PT circadian clockwork. These studies will extend beyond the scope of the current in vivo work and will hopefully provide novel insights into the chronotherapeutic effects of melatonin.
Acknowledgments
We are grateful to Marshall Building staff (Edinburgh) and Nigel Graham and Jean-Michel Fustin (Aberdeen) for technical assistance and to Prof. Ueli Schibler (University of Geneva, Switzerland) for the generous gift of the Rev-erb probe template. Purified preparations of ovine prolactin were provided by the National Hormone and Peptide Program.
Footnotes
Present address for J.D.J.: School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom.
This work was supported by the Medical Research Council (to H.A. and G.A.L.) and a grant from the Biotechnology and Biological Sciences Research Council (to D.G.H.).
First Published Online November 3, 2005
1 J.D.J. and B.B.T. are joint first authors.
Abbreviations: LL, Constant light; LP, long photoperiod; MT1, melatonin type 1; PT, pars tuberalis; SCN, suprachiasmatic nuclei; SP, short photoperiod; ZT, zeitgeber time.
Accepted for publication October 26, 2005.
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Neurobiologie des Rhythmes laboratory, Unite Mixte de Recherche 7518 Centre National de la Recherche Scientifique/Universite Louis Pasteur (B.B.T., M.M.-P.),67084 Strasbourg, France
Medical Research Council Human Reproductive Sciences Unit (H.A., G.A.L.), Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom
Abstract
In mammals, changing day length modulates endocrine rhythms via nocturnal melatonin secretion. Studies of the pituitary pars tuberalis (PT) suggest that melatonin-regulated clock gene expression is critical to this process. Here, we considered whether clock gene rhythms continue in the PT in the absence of melatonin and whether the effects of melatonin on the expression of these genes are temporally gated. Soay sheep acclimated to long photoperiod (LP) were transferred to constant light for 24 h, suppressing endogenous melatonin secretion. Animals were infused with melatonin at 4-h intervals across the final 24 h, and killed 3 h after infusion. The expression of five clock genes (Per1, Per2, Cry1, Rev-erb, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erb showed pronounced temporal variation despite the absence of melatonin, with peak times occurring earlier than predicted under LP. The time of peak Bmal1 expression remained LP-like, whereas Cry1 expression was continually low. Melatonin infusion induced Cry1 expression at all times and suppressed other genes, but only when they showed high expression in sham-treated animals. Hence, 3 h after melatonin treatment, clock gene profiles were driven to a similar state, irrespective of infusion time. In contrast to the PT, melatonin infusions had no clear effect on clock gene expression in the suprachiasmatic nuclei. Our results provide the first example of acute sensitivity of multiple clock genes to one endocrine stimulus and suggest that rising melatonin levels may reset circadian rhythms in the PT, independently of previous phase.
Introduction
MELATONIN, THE PRINCIPAL product of the vertebrate pineal gland, undergoes marked temporal variation in secretion and drives a wide range of rhythmic physiology. In mammals, the synthesis and secretion of pineal melatonin occurs at night, under the control of the circadian clock present in the suprachiasmatic nuclei (SCN) of the hypothalamus. The duration of this nocturnal signal varies in proportion to the length of the night, and thence melatonin secretion transduces both daily and seasonal time throughout the body (1).
The pituitary pars tuberalis (PT) is strongly implicated in the photoperiodic regulation of prolactin secretion and, because of its high density of melatonin receptors, has become a key model tissue for the understanding of melatonin signal readout (2, 3, 4). It is now clear that melatonin is a key regulator of clock gene expression in the mammalian PT, because modulation of the endogenous profile of melatonin secretion, by manipulation of ambient photoperiod or by administration of exogenous melatonin, changes PT clock gene profiles (5, 6, 7, 8, 9).
To date, the documented effects of melatonin in the PT can be divided into those occurring in the morning (Per1) and those occurring in the evening (Cry1, Rev-rb). The former depend on melatonin withdrawal at the end of the night, probably mediated by the resultant increase in cAMP production (2, 5). In rodents unable to synthesize melatonin, either because of genetic deficiency (10) or pinealectomy (11), rhythmic expression of Per1 expression is absent, suggesting that melatonin is necessary for maintained rhythmic gene expression in this tissue.
Collectively, these observations suggest that the PT contains a circadian oscillator that is either driven or modulated by melatonin and raise questions about 1) whether a rhythmic melatonin signal is necessary for the 24-h variation of clock gene expression in the PT and 2) whether melatonin actions on gene expression in the PT is restricted to time windows around the morning and evening changes in illumination. To test these ideas, we returned to the sheep as a model because this species has been extensively used for investigations into the molecular basis of melatonin-dependent photoperiodic responses (2, 6, 12). We placed animals in constant light for one 24-h cycle (thereby suppressing endogenous melatonin secretion) and treated them at 4-h intervals with either melatonin-containing sc implants or sham procedure.
Materials and Methods
Animals and housing
All animals were treated in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Forty-eight pubertal female Soay sheep, obtained from specialist breeders in Scotland (12), were brought indoors in winter (November). They were housed in light-sealed rooms and fed a standardized diet of grass pellets (500 g per animal; Vitagrass, Cumbria, UK) given daily 1 h into the light phase. Hay and water were available ad libitum. White fluorescent strip lights provided approximately 160 lux at the animals’ eye level during the light phase, whereas dim red light (<5 lux) was provided during the dark phase.
Light and melatonin treatment
The animals were initially acclimatized under a winter-like short photoperiod (SP; 8 h light, 16 h darkness) for 4 wk and then exposed to long photoperiod (LP; 16 h light, 8 h dark) for 6 wk. Blood samples were collected twice weekly from the jugular vein from representative animals (n = 12), and the blood plasma separated by centrifugation within 30 min and stored at –20 C, to measure prolactin concentration as evidence of photoresponsiveness.
Over the final 24 h of the experiment, lights were left on to suppress endogenous pineal melatonin secretion. Animals were treated with a single sc implant of melatonin (Regulin; CEVA Animal Health, Chesham, Bucks, UK) placed on the inside of the hind leg using a standard injector, or a sham treatment with no implant as control at 4-h intervals across the final 24 h (n = 4 per time point), and killed 3 h after treatment by pentobarbital injection. Immediately before death, blood samples were collected from the jugular vein and processed as above to permit analysis of the effect of treatments on melatonin concentration. The hypothalamus and upper pituitary gland were removed within 10 min of death and snap frozen in isopentane kept at –30 C by dry ice. The expression of five clock genes (Per1, Per2, Cry1, Rev-erb, and Bmal1) was measured in the PT and SCN by in situ hybridization.
RIA
Prolactin concentrations were measured by a validated RIA (13). The lower limit of sensitivity was 0.5 ng/ml prolactin standard (NIH-PRL-S13), and the intraassay coefficient of variation was 8.3%. Melatonin concentrations were also measured by a standard RIA (14), using a commercial antibody (PF-1288; SPI-BIO, Paris, France). The sensitivity of the assay was 5 pg/ml, and the intraassay coefficient of variation was 12.0%.
In situ hybridization
Coronal cryosections (20 μm) cut through the SCN and PT were thaw-mounted onto poly-L-lysine-coated glass slides and stored at –80 C. Expression of the clock genes Per1, Per2, Cry1, Bmal1, and Rev-erb was then detected using radioactive riboprobes and procedures described previously (15).
Statistics
Weekly changes in plasma prolactin concentration during acclimation to LP were analyzed by one-way ANOVA with repeated measures. Clock gene expression profiles in sham-treated animals were analyzed by one-way ANOVA. The comparison between melatonin and sham treatment was analyzed by two-way ANOVA and Bonferroni’s post hoc testing when appropriate.
Results
Photosensitivity of experimental animals
The pretreatment exposure to LP induced a significant (P < 0.01, one-way ANOVA with repeated measures) increase in plasma prolactin concentrations from 19.2 ± 4.6, at the change from SP to LP, to 52.4 ± 11.1 ng/ml at wk 6, illustrating the physiological and photosensitive state of the experimental animals before the final day on constant light (LL) (Fig. 1).
Effect of treatment on endocrine profiles
LL treatment resulted in suppressed plasma melatonin concentration across the 24-h cycle (Fig. 2A; melatonin concentration in sham group was 10.4 ± 0.9 pg/ml, mean ± SEM, n = 24). Melatonin implants significantly increased plasma melatonin concentration within 3 h (Fig. 2A; P < 0.001 for effect of treatment, two-way ANOVA). This effect was independent of infusion time (P > 0.05 for treatment x time interaction, two-way ANOVA). The resulting plasma melatonin concentrations (216.9 ± 28.0 pg/ml, mean ± SEM, n = 24) were within the nocturnal physiological range commonly observed in this species (12).
Although prolactin concentration appeared to be low in sham-treated animals killed at zeitgeber time 16 (ZT16) (Fig. 2B), there was no statistically significant 24-h variation in either the sham- or melatonin-treated animals (P > 0.05 for effect of time in both experimental groups, one-way ANOVA). Administration of melatonin during LL decreased mean plasma prolactin concentration within 3 h of treatment (Fig. 2B; plasma prolactin concentration was 29.8 ± 4.0 vs. 18.6 ± 2.6 ng/ml, sham vs. melatonin-treated group, mean ± SEM, n = 24; P < 0.01 for effect of treatment, two-way ANOVA). This acute response to melatonin was not time dependent (P > 0.05 for treatment x time interaction, two-way ANOVA).
Clock gene expression in the PT
Despite the suppressed plasma melatonin concentrations in sham-treated animals, the expression of four of the five clock genes analyzed in the PT exhibited significant (P < 0.05, one-way ANOVA) variation over the 24-h cycle (Fig. 3). For Rev-erb and Per1, which show peak PT expression in the early light phase in LP acclimated animals (6, 8), a melatonin-independent expression peak was seen after approximately 20 h of constant light treatment. For Per2, which peaks slightly later than Per1 under LP (6), a melatonin-independent increase in expression was seen after 24 h of exposure to LL. Similar to the case in LP acclimated animals (6), Bmal1 expression peaked between ZT12 and ZT16 in LL.
In contrast to the other clock genes, Cry1 expression in sham-treated animals did not vary significantly over the LL sampling period (P > 0.05, one-way ANOVA) and was low at all sampling points (Fig. 4). Melatonin infusion for a period of 3 h caused a large increase in the expression of Cry1 mRNA in the PT (Fig. 4) (P < 0.001 for effect of treatment, two-way ANOVA). This increase occurred at all sampling points throughout the 24-h cycle (P < 0.05 melatonin vs. sham, two-way ANOVA with Bonferroni’s post hoc test). The expression of Per1, Per2, Rev-erb, and Bmal1 was also sensitive to melatonin treatment, but in these instances the effects of melatonin were inhibitory and time dependent (P < 0.05 melatonin vs. sham, two-way ANOVA with Bonferroni’s post hoc test) (Fig. 5). As a result of these effects, the expression of clock genes in the PT 3 h after melatonin treatment was qualitatively similar, irrespective of the time of infusion; Cry1 mRNA was elevated, whereas all other mRNAs were suppressed (Figs. 4 and 5).
Clock gene expression in the SCN
The expression of all five clock genes was rhythmic in the SCN over 24 h of LL exposure (P < 0.001 for effect of time, two-way ANOVA). Melatonin had a significant effect on Rev-erb expression (P = 0.05 for effect of treatment, two-way ANOVA), although this effect was not dependent upon time of melatonin treatment (P > 0.05 for treatment x time interaction, two-way ANOVA). For all other genes, melatonin had no significant effect on mRNA expression (P > 0.05 effect of treatment, two-way ANOVA) (Fig. 6).
Discussion
Recent studies have identified rhythmic expression of multiple clock genes in the mammalian PT and proposed models by which these genes may mediate the photoperiodic effects of melatonin (16). Here, we have used photosensitive Soay sheep to demonstrate that clock gene rhythms persist in the absence of endogenous melatonin secretion. Furthermore, in these animals, melatonin infusions acutely alter the expression of multiple clock genes at all phases of the 24-h cycle in the PT, without any corresponding effects in the SCN. These data are consistent with the possibility that the PT functions as a circadian oscillator that can be synchronized by an increase in melatonin concentration in the bloodstream, irrespective of when this occurs relative to lights on. More generally, this study provides the first example of acute responsiveness of multiple clock genes to a single defined endocrine stimulus.
Since the classical lesioning experiments performed over 30 yr ago (17, 18), the SCN has been viewed as a master circadian pacemaker that drives and synchronizes overt rhythms of physiology and behavior. The recent identification of circadian oscillators in peripheral tissues and in immortalized cell lines suggests that the role of the SCN is primarily to coordinate circadian oscillations throughout the organism (19) and focuses attention on the mechanisms through which such circadian synchronization takes place. The precise manner by which a given SCN-dependent circadian signal influences peripheral oscillator function may be expected to reflect the physiological functions of the tissues upon which it acts. In mammals, melatonin is one such SCN-dependent circadian output, whose primary function is to modulate neuroendocrine physiology, by relaying information about changes in day length (20). The pineal melatonin signal communicates the phase of night onset and of the subsequent dawn; melatonin is secreted continuously between these transitions, giving a signal whose duration is proportional to the length of the night. This ability of the melatonin signal to represent night duration is crucial for photoperiodic responses in seasonal mammals (21).
The PT is a largely homogeneous population of type 1 melatonin (MT1) receptor-expressing thyrotrophic cells (22, 23), making it an ideal tissue for the study of melatonin action. Long-term loss of melatonin synthesis or loss of functional MT1 receptors abolishes rhythmic clock gene expression in the mammalian PT (10, 11, 24, 25), indicating that the molecular clockwork of this tissue is ultimately melatonin dependent. The data presented here, however, demonstrate clear temporal variation in PT expression of four core clock genes over a 24-h period in the absence of a rhythmic melatonin signal and thus strongly support the existence of an endogenous PT oscillator. We therefore suspect that arrhythmic Per1 gene expression in the PT of hamsters that have been pinealectomized for 7 d (11) derives from progressive damping of the PT circadian oscillator. Based on recent studies of damping in cell culture systems (26, 27, 28), we speculate that this is a result of desynchronization of circadian oscillators in individual PT cells. Accordingly, in genetically melatonin-synthesis- or melatonin-receptor-deficient mice (10, 24, 25), PT gene expression is likely arrhythmic because PT cells never become synchronized during fetal or postnatal development.
The present study has examined only one aspect of the information inherent in the endogenous melatonin signal: rising melatonin levels associated with night onset. Our data suggest that a dusk-like response (high Cry1, low Per, Rev-erb, and Bmal1) can be elicited by increasing melatonin levels occurring at any phase throughout the 24-h cycle. This suggests that PT cells exhibit a type 0 resetting response (29) to rising melatonin levels, that is to say that the PT molecular clockwork resets to a dusk state in response to rising melatonin, independent of its state immediately beforehand. This dusk state is transient, because our previous data show that a state of low Cry1 and high Bmal1 expression develops during continued melatonin presence through the middle to the end of the night (6). Because the effects of melatonin on cAMP-dependent signal transduction and gene expression persist during prolonged melatonin exposure (2), it is likely that cAMP-independent mechanisms account for disappearance of the dusk state under physiological conditions.
Interestingly, the PT of the Japanese quail also rhythmically expresses clock genes, of which Cry1 may be acutely induced by light. However, photic induction of Cry1 is subject to circadian gating in this tissue (30), suggesting that the avian PT may contain a more robust oscillator than is the case in mammals.
For sheep in a natural environment, the times of dawn and dusk move in opposite directions during the annual cycle, raising the possibility of a similar flexibility in the resetting response of the PT oscillator to declining melatonin levels associated with dawn. Although we have yet to perform an experiment explicitly to address this, our published data agree with this prediction; the timing of the dawn state, characterized by high Per1, can be delayed by an acute light manipulation in the preceding evening that delays the dawn-associated decline in melatonin secretion (8). Hence the circadian clockwork of the sheep PT shows a remarkable plasticity in its response to changing patterns of melatonin secretion.
Early work in the mammalian PT focused on the ability of melatonin to modulate adenylate cyclase activity (2). Melatonin-regulated expression of Per1, which is considered a cAMP-regulated immediate-early response gene in the PT (31), is likely to occur through this pathway (24). However, other clock genes are not directly sensitive to cAMP signaling, suggesting that melatonin-dependent signaling may occur by additional and as yet undefined signaling pathways. Consistent with this possibility, previous studies of many tissues, including the ovine PT, have revealed a wide range of intracellular pathways that are sensitive to melatonin (32). At present, we cannot entirely rule out the possibility that induction of Cry1 drives changes in the expression of other clock genes. However, we feel that this putative mechanism is unlikely, because it would require not only rapid transcription and translation of Cry1 but also a high level of mRNA instability for other clock genes to be affected within the short interval (3 h) between melatonin infusion and tissue collection used in this study.
Additional to its role in seasonal neuroendocrine control, melatonin may influence the function of the SCN itself. Exogenous melatonin administration has been shown to synchronize circadian behavioral rhythmicity in rodents (33, 34) and in humans (35), and melatonin can reportedly phase-shift circadian rhythms of electrical activity in mouse SCN slice preparations (36). Knockout of the MT1 and MT2 melatonin receptor subtypes abolishes phase-shifting responses to melatonin in mice and acute effects of melatonin on SCN electrical activity (37, 38). Hence it is possible that circadian effects of melatonin are mediated through melatonin receptors located in the SCN. Nevertheless, a recent study reported no effect of melatonin injection on clock gene expression in the rat SCN (39). In sheep, melatonin receptors are at best weakly expressed in the SCN (40), and in the present study a mild effect of melatonin injection was seen for only one gene, Rev-erb, at one time point only. Possibly our data and that of Poirel et al. (39) reflect different coupling between melatonin receptors and circadian molecular clockwork in SCN compared with PT cells. Alternatively the heterogeneous cellular composition of the SCN and its sensitivity to multiple inputs in addition to melatonin (41) may account for these results.
A final point of interest is the regulation of plasma prolactin concentration in this study. We have previously described a LP-induced increase in prolactin concentration within 24 h (8). In this study, the animals required approximately 4–6 wk of LP exposure before showing such a rise. This difference can be explained by two reasons. In the current study, animals were likely to exhibit early refractoriness to shortening winter photoperiod when they were transferred to controlled lighting regimes; furthermore, animals were not habituated to blood sampling and it is therefore likely that initial prolactin concentrations were affected by stress of experimental manipulation at this stage. During the final 24 h of this experiment, melatonin caused an acute inhibition of prolactin release in the sheep under constant light. This is unlikely to reflect a direct effect on lactotroph cells, which do not appear to express melatonin receptors (22, 42) and are unresponsive to melatonin in primary cell culture (43). It is unclear whether melatonin acts at the level of the PT or hypothalamus to acutely suppress prolactin concentration. Although hypothalamic monoamines are not believed to drive photoperiodic changes of prolactin secretion (44, 45), there is some evidence that melatonin may stimulate secretion of the prolactin-inhibiting factor dopamine from hypothalamic neurons (46, 47). Alternatively, melatonin infusions may have acutely inhibited the secretion of prolactin-releasing factor(s), termed tuberalin, from the PT (43, 48, 49, 50, 51). However, the acute effects of melatonin on tuberalin secretion are as yet unknown.
In summary, we propose that the PT may become an important model tissue to address the function of peripheral circadian oscillators as well as the mechanisms of photoperiodic time measurement. A strength of the PT as a circadian model is that it has a well defined circadian input, melatonin. As well as testing the generality of the results described here in other species, we are currently developing in vitro techniques to further evaluate the effect of melatonin on the PT circadian clockwork. These studies will extend beyond the scope of the current in vivo work and will hopefully provide novel insights into the chronotherapeutic effects of melatonin.
Acknowledgments
We are grateful to Marshall Building staff (Edinburgh) and Nigel Graham and Jean-Michel Fustin (Aberdeen) for technical assistance and to Prof. Ueli Schibler (University of Geneva, Switzerland) for the generous gift of the Rev-erb probe template. Purified preparations of ovine prolactin were provided by the National Hormone and Peptide Program.
Footnotes
Present address for J.D.J.: School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom.
This work was supported by the Medical Research Council (to H.A. and G.A.L.) and a grant from the Biotechnology and Biological Sciences Research Council (to D.G.H.).
First Published Online November 3, 2005
1 J.D.J. and B.B.T. are joint first authors.
Abbreviations: LL, Constant light; LP, long photoperiod; MT1, melatonin type 1; PT, pars tuberalis; SCN, suprachiasmatic nuclei; SP, short photoperiod; ZT, zeitgeber time.
Accepted for publication October 26, 2005.
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