Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.     

Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.     

Photorefractoriness in mammals: dissociating a seasonal timer from the circadian-based photoperiod response

In seasonal animals, prolonged exposure to constant photoperiod induces photorefractoriness, causing spontaneous reversion in physiology to that of the previous photoperiodic state. This study tested the hypothesis that the onset of photorefractoriness is correlated with a change in circadian expression of clock genes in the suprachiasmatic nucleus (circadian pacemaker) and the pars tuberalis (PT, a melatonin target tissue). Soay sheep were exposed to summer photoperiod (16-h light) for either 6 or 30 wk to produce a photostimulated and photorefractory physiology, and seasonal changes were tracked by measuring the long-term prolactin cycles. Animals were killed at 4-h intervals throughout 24 h. Contrary to the hypothesis, the 24-h rhythmic expression of clock genes (Rev-erbalpha, Per1, Per2, Bmal1, Cry1) in the suprachiasmatic nucleus and PT reflected the ambient photoperiod/melatonin signal and not the changing physiology. Contrastingly, the PT expression of alpha-glycoprotein hormone subunit (alphaGSU) and betaTSH declined in photorefractory animals toward a short day-like endocrinology. We conclude that the generation of long-term endocrine cycles depends on the interaction between a circadian-based, melatonin-dependent timer that drives the initial photoperiodic response and a non-circadian-based timer that drives circannual rhythmicity in long-lived species. Under constant photoperiod the two timers can dissociate, leading to the apparent refractory state.     

Photoperiod regulates clock gene rhythms in the ovine liver

To investigate the photoperiodic entrainment of peripheral rhythms in ruminants, we studied the expression of clock genes in the liver in the highly seasonal Soay sheep. Animals were kept under long (LD 16:8) or short photoperiod (LD 8:16). Daily rhythms in locomotor activity were recorded, and blood concentrations of melatonin and cortisol were measured by RIA. Per2, Bmal1, and Cry1 gene expression was determined by Northern blot analyses using ovine RNA probes in liver collected every 4h for 24h. Liver Per2 and Bmal1, but not Cry1, expression was rhythmic in all treatments. Under long days, peak Per2 expression occurred at end of the night with a similar timing to Bmal1, whereas, under short days the Per2 maximum was in the early night with an inverse pattern to Bmal1. There was a photoperiodxtime interaction for only Per2 (P < 0.001). The 24-h pattern in plasma cortisol matched the observed phasing of Per2 expression, suggesting that it may act as an endocrine entraining factor. The clock gene rhythms in the peripheral tissues were different in timing compared with the ovine suprachiasmatic nucleus (SCN, central pacemaker) and pars tuberalis (melatonin target tissue), and the hepatic rhythms were of lower amplitude compared with photoperiodic rodents. Thus, there are likely to be important species differences in the way the central and peripheral clockwork encodes external photoperiod.     

Multiple effects of melatonin on rhythmic clock gene expression in the mammalian pars tuberalis

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-erbalpha, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erbalpha 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.     

Photoinducible phase-specific light induction of Cry1 gene in the pars tuberalis of Japanese quail

Prolactin (PRL) secretion is regulated by photoperiod in mammals and birds. In mammals, the pars tuberalis (PT) in the pituitary is involved in the regulation of photoperiodic regulation of PRL secretion. In birds, however, hypothalamic vasoactive intestinal peptide is implicated in PRL secretion, and physiological roles of the avian PT remain unknown. In the present study, we show that PRL secretion increases under long days and short days with a night interruptive schedule, both of which also cause gonadal growth in Japanese quail. We have also found Cry1 gene expression in the PT of Japanese quail. Cry1 expression was rhythmic under long and short photoperiods in the PT, and the peak was phase delayed under a lengthened photoperiod. Moreover, expression of Cry1 gene was induced by a light pulse but only when given during the photoinducible phase. In our previous study, we have shown rhythmic Per2 gene expression with a peak in the PT during the early day under various photoperiods. When taken together with the results from the present study, different phase relationships between Per2 and Cry1 in the Japanese quail PT under different photoperiods may decode photoperiodic information and regulate photoperiodic PRL secretion in a manner similar to that of mammals.     

Redefining the limits of day length responsiveness in a seasonal mammal

At temperate latitudes, increases in day length in the spring promote the summer phenotype. In mammals, this long-day response is mediated by decreasing nightly duration of melatonin secretion by the pineal gland. This affects adenylate cyclase signal transduction and clock gene expression in melatonin-responsive cells in the pars tuberalis of the pituitary, which control seasonal prolactin secretion. To define the photoperiodic limits of the mammalian long day response, we transferred short day (8 h light per 24 h) acclimated Soay sheep to various longer photoperiods, simulating those occurring from spring to summer in their northerly habitat (57 degrees N). Locomotor activity and plasma melatonin rhythms remained synchronized to the light-dark cycle in all photoperiods. Surprisingly, transfer to 16-h light/day had a greater effect on prolactin secretion and oestrus activity than shorter (12 h) or longer (20 and 22 h) photoperiods. The 16-h photoperiod also had the largest effect on expression of circadian (per1) and neuroendocrine output (betaTSH) genes in the pars tuberalis and on kisspeptin gene expression in the arcuate nucleus of the hypothalamus, which modulates reproductive activity. This critical photoperiodic window of responsiveness to long days in mammals is predicted by a model wherein adenylate cyclase sensitization and clock gene phasing effects of melatonin combine to control neuroendocrine output. This adaptive mechanism may be related to the latitude of origin and the timing of the seasonal transitions.     

The rat suprachiasmatic nucleus is a clock for all seasons.

Seasonal changes of daylength (photoperiod) affect the expression of hormonal and behavioral circadian rhythms in a variety of organisms. In mammals, such effects might reflect photoperiodic changes in the circadian pace-making system [located in the suprachiasmatic nucleus (SCN) of the hypothalamus] that governs these rhythms, but to date no functionally relevant, intrinsic property of the SCN has been shown to be photoperiod dependent. We have analyzed the temporal regulation of light-induced c-fos gene expression in the SCN of rats maintained in long or short photoperiods. Both in situ hybridization and immunohistochemical assays show that the endogenous circadian rhythm of light responsiveness in the SCN is altered by photoperiod, with the duration of the photosensitive subjective night under the short photoperiod 5-6 h longer than under the long photoperiod. Our results provide evidence that a functional property of the SCN is altered by photoperiod and suggest that the nucleus is involved in photoperiodic time measurement.     

The circadian clock stops ticking during deep hibernation in the European hamster

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.     

Serum melatonin profiles and endocrine responses of ewes exposed to a pulse of light late in the dark phase

To determine whether effects of light pulses on the photoperiodic time measuring system involve changes in pineal gland function, melatonin profiles were determined in groups of ewes maintained under 10-h light, 14-h dark (10L:14D) or 10L:10D:1L:3D. Ewes exposed to 10L:14D had a significantly (P less than 0.01) longer duration of melatonin secretion (15.0 +/- 0.4 h, mean +/- SE) than ewes under 10L:10D:1L:3D (9.0 +/- 0.4 h). The 1-h pulse of light therefore acted as a dawn signal in the latter group. During a period of extended darkness imposed to study endogenous control of melatonin release, there was no change in the duration of elevated melatonin in control ewes (16.1 +/- 0.5 h), but a significant (P less than 0.05) lengthening occurred in pulsed ewes (13.2 +/- 1.4 h). PRL responses to a bolus iv injection of TRH (50 ng/kg BW) were significantly (P less than 0.01) smaller in control ewes (478 +/- 134 ng/ml) compared with pulsed ewes (1578 +/- 175 ng/ml), with responses in the latter group resembling those observed in ewes on long days. A 1-h pulse of light late in the dark phase, therefore, resulted in a melatonin pattern normally observed under long days in ewes, and this was associated with other endocrine functions also characteristic of sheep on long days. It is concluded that pulses of light modify activity of the pineal gland which in turn interacts with the photoperiodic time-measuring system via melatonin. The increase in duration of melatonin secretion observed in pulsed ewes under extended darkness suggests that the melatonin rhythm is under the control of two oscillators coupled to dusk and dawn, and that these oscillators interact more strongly when compressed by an interrupted dark phase.     

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light–dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN′s subregional organization to both photic input processing and rhythmic output control.     

Physical and inflammatory stressors elevate circadian clock gene mPer1 mRNA levels in the paraventricular nucleus of the mouse

Stress induces secretion of corticosterone through activation of the hypothalamic-pituitary-adrenal axis. This corticosterone secretion is thought to be controlled by a circadian clock in the suprachiasmatic nucleus (SCN). The hypothalamic paraventricular nucleus (PVN) receives convergent information from both stress and the circadian clock. Recent reports demonstrate that mammalian orthologs (Per1, Per2, and Per3) of the Drosophila clock gene Period are expressed in the SCN, PVN, and peripheral tissues. In this experiment, we examined the effect of physical and inflammatory stressors on mPer gene expression in the SCN, PVN, and liver. Forced swimming, immobilization, and lipopolysaccharide injection elevated mPer1 gene expression in the PVN but not in the SCN or liver. A stress-induced increase in mPer1 expression was observed in the corticotropin-releasing factor-positive cells of the PVN; however, the stressors used in this study did not affect mPer2 expression in the PVN, SCN, or liver. The present study suggests that a stress-induced disturbance of circadian corticosterone secretion may be associated with the stress-induced expression of mPer1 mRNA in the PVN.