Influence of photoperiod duration and light–dark transitions on entrainment of Per1 and Per2 gene and protein expression in subdivisions of the mouse suprachiasmatic nucleus

The circadian clock located within the suprachiasmatic nuclei (SCN) of the hypothalamus responds to changes in the duration of day length, i.e. photoperiod. Recently, changes in phase relationships among the SCN cell subpopulations, especially between the rostral and caudal region, were implicated in the SCN photoperiodic modulation. To date, the effect of abrupt, rectangular, light-to-dark transitions have been studied while in nature organisms experience gradual dawn and twilight transitions. The aim of this study was to compare the effect of a long (18 h of light) and a short (6 h of light) photoperiod with twilight relative to that with rectangular light-to-dark transition on the daily profiles of Per1 and Per2 mRNA ( in situ hybridization) and PER1 and PER2 protein (immunohistochemistry) levels within the rostral, middle and caudal regions of the mouse SCN. Under the short but not under the long photoperiod, Per1 , Per2 and PER1, PER2 profiles were significantly phase-advanced under the twilight relative to rectangular light-to-dark transition in all SCN regions examined. Under the photoperiods with rectangular light-to-dark transition, Per1 and Per2 mRNA profiles in the caudal SCN were phase-advanced as compared with those in the rostral SCN. The phase differences between the SCN regions were reduced under the long, or completely abolished under the short, photoperiods with twilight. The data indicate that the twilight photoperiod provides stronger synchronization among the individual SCN cell subpopulations than the rectangular one, and the effect is more pronounced under the short than under the long photoperiod.     

Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis of the Siberian hamster (Phodopus sungorus)

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.     

Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.     

Using Per gene expression to search for photoperiodic oscillators in the hamster suprachiasmatic nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) is also believed to underlie photoperiodic (seasonal) timekeeping in mammals. This clock has been modeled as a complex pacemaker composed of two coupled circadian oscillators; variability in their mutual phase relationship could account for the ability to measure daylength, with putative morning and evening oscillators synchronized to dawn and dusk, respectively. Recently, several genes have been identified that are believed to be part of the clock's core oscillatory mechanism. Here, we investigate how such molecular oscillations are altered as a function of photoperiod by analyzing Period (Per1, Per2, and Per3) gene expression at the mRNA level using SCN tissue sections and in situ hybridization. Golden hamsters were entrained to complete 24-h light-dark (LD) cycles with either a long (16 h) or a short (8 h) photophase, or they were entrained to the long complete photoperiod and then allowed to free-run in constant darkness. The results show large photoperiod-dependent changes in the duration of high daytime SCN Per1 and Per2 mRNA levels and small changes in the phase difference between their rhythms.     

Effect of photic stimuli disturbing overt circadian rhythms on the dorsomedial and ventrolateral SCN rhythmicity

To ascertain how photic stimuli disturbing overt circadian rhythms affect the endogenous rhythmicity of the suprachiasmatic nucleus (SCN), rats were subjected to constant light (LL) or to a 9-h light pulse encompassing midnight, and rhythms of abundance of the c-Fos-immunoreactive (c-Fos-ir) and the PER1-immunoreactive (PER1-ir) cells were studied during the first 1-2 cycles following release into LL or darkness (DD) within the whole SCN as well as in its ventrolateral (vl) and the dorsomedial (dm) part. LL seemingly abolished the c-Fos rhythm in the whole SCN, while the rhythm persisted in the vl- and dm-SCN. In the dm-SCN, the rhythm of c-Fos-ir was phase-delayed by about 4 h in LL, whereas the rhythm of PER1-ir was affected just slightly. In the vl-SCN, the rhythm of c-Fos photo-induction might be delayed by 5-6 h as compared with the reported rhythm [A. Sumova and H. Illnerova, Am. J. Physiol. 274 (1998) R857-R863], whereas the PER1 profile appeared to be out of phase with that in DD. After a 9-h light pulse encompassing midnight, the rhythm of PER1-ir in the dm-SCN changed just slightly, whereas the PER1 rhythm in the vl-SCN was abolished and there was just an indication of extension of elevated PER1-ir. Altogether, the data indicate that photic stimuli disturbing circadian rhythms affect more dramatically the vl- than the dm-SCN rhythmicity within the first cycles and that in the dm-SCN shifting of the c-Fos rhythm proceeds more rapidly than that of the Per1 rhythm.     

Entrainment of the rat circadian clock controlling the pineal N-acetyltransferase rhythm depends on photoperiod.

Entrainment of the circadian clock as a function of time when a light stimulus is presented has been studied in detail while little attention has been paid to a role photoperiod may play in the resetting. To find out whether and how photoperiod affects the entrainment, resetting of the rat circadian pacemaker by delays in the evening light offset and by advances in the morning light onset, respectively, was studied in rats maintained either under a short photoperiod, with 8 h of light and 16 h of darkness per day (LD 8:16) or under a long, LD 18:6 photoperiod. To assess phase shifts of the clock, the suprachiasmatic nucleus controlled rhythm in the pineal N-acetyltransferase (NAT), namely the time of the evening NAT rise and the time of the morning decline, were followed. One day after a delay in lights off, on LD 8:16 the NAT rhythm with a normal amplitude was retained following longer delays of the light offset and the maximum phase delay of the NAT rise was 3 times larger than on LD 18:6. One day after an advance in lights on, the NAT decline was phase advanced under both photoperiods; on LD 8:16 the maximum shift was 3 times as large as on LD 18:6. On LD 8:16, the NAT rise was not shifted after shorter advances in lights on and became phase delayed only when the light onset was brought forward to before midnight while on LD 18:6 the NAT rise was phase delayed after any, even a mere 1 h, advance in lights on. The data show that magnitude and direction of phase shifts of the NAT rhythm depend not only on the time of light presentation but on photoperiod as well. Difference in resetting of the rhythm under various photoperiods may reflect photoperiod-dependent changes of an underlying pacemaker.     

Tissue-specific abolition of Per1 expression in the pars tuberalis by pinealectomy in the Syrian hamster

Melatonin secretion by the pineal gland transduces photoperiod into a neuroendocrine signal. In the pars tuberalis (PT), we have shown that photoperiod modifies the amplitude of the clock gene Per1. The aim of this study was to test whether the endogenous melatonin signal is required for rhythmic expression of Per1 in the PT. Male Syrian hamsters housed in long days (LD, 16:8h light:dark) were pinealectomized and Per1 mRNA expression studied by in situ hybridization. Pinealectomy abolished the rhythm of Per1 expression in the PT, but had no effect on Per1 expression in the suprachiasmatic nucleus (SCN), or the ventromedial nucleus (VMH) of the hypothalamus. Interestingly, a single night-time injection of melatonin (25 microg), given to pinealectomized animals, failed to restore Per1 expression in the PT. These data demonstrate that Per1 expression in the PT is driven by melatonin, and that the features of the endogenous signal through which the Per1 expression is achieved cannot be reproduced by a single melatonin injection.     

The photoperiod entrains the molecular clock of the rat pineal

The suprachiasmatic nucleus-pineal system acts as a neuroendocrine transducer of seasonal changes in the photoperiod by regulating melatonin formation. In the present study, we have investigated the extent to which the photoperiod entrains the nonself-cycling oscillator in the Sprague-Dawley rat pineal. For this purpose, the 24-h expression of nine clock genes (bmal1, clock, per1, per2, per3, cry1, cry2, dec1 and dec2) and the aa-nat gene was monitored under light-dark 8 : 16 and light-dark 16 : 8 in the rat pineal by using real-time RT-PCR. The 24-h pattern of the expression of only per1, dec2 and aa-nat genes was affected by photoperiod. In comparison with the short photoperiod, the duration of elevated expression under the long photoperiod was elongated for per1 and shortened for dec2 and aa-nat. For each of the genes, photoperiod-dependent variations partly persisted under constant darkness. Therefore, the pineal clockwork appears to memorize the photoperiod of prior entrained cycles. The findings of the present study indicate that the nonself-cycling oscillator of the rat pineal is entrained by photoperiodic information and therefore that it participates in seasonal timekeeping.     

Day‐length encoding through tonic photic effects in the retinorecipient SCN region

The circadian clock in the suprachiasmatic nucleus (SCN) plays a critical role in seasonal processes by sensing ambient photoperiod. To explore how it measures day‐length, we assessed the state of SCN oscillators using markers for neuronal activity (c‐FOS) and the clock protein (PER1) in Syrian hamsters housed in long (LD, 16 : 8 h light : dark) vs. short days (SD, 8 : 16 h light : dark). During SD, there was no detectable phase dispersion across the rostrocaudal extent of the nucleus. In contrast, during LD, rhythms in the caudal SCN phase led those in the mid‐ and rostral SCN by 4–8 h and 8–12 h, respectively. Importantly, some neurons in the retinorecipient core SCN were unique in that they were FOS‐positive during the dark phase in LD, but not SD. Transfer of LD animals to constant darkness or skeleton photoperiod revealed that dark‐phase FOS expression depends on tonic light exposure rather than on intrinsic clock properties. By transferring animals from SD to LD, we next discovered that there are two separate populations of SCN cells, one responding to acute and the other to tonic light exposure. The results suggest that the seasonal encoding of day‐length by the SCN entails reorganization of its constituent oscillators by a subgroup of neurons in the SCN core that respond to tonic photic cues.     

Development of circadian rhythmicity and photoperiodic response in subdivisions of the rat suprachiasmatic nucleus

To ascertain whether the circadian rhythmicity of the ventrolateral (vl) suprachiasmatic nucleus (SCN) develops concurrently with that of the dorsomedial (dm) SCN and when the rhythmicity starts to respond to day length, i.e., to the photoperiod, rats with their offspring were maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The rhythms of spontaneous c-Fos immunoreactivity in the dm-SCN and of the light-induced c-Fos immunoreactivity in the vl-SCN were studied in the pups. In 3- and 10-day-old rats, the dm-SCN rhythm in spontaneous c-Fos immunoreactivty was already well expressed but a response to a photoperiod similar to that in adult rats has not yet been developed. The vl-SCN gate for insensitivity of c-Fos production to light at certain times was detected in 10-day but not yet in 3-day-old rats: in the latter, light exposure at any daytime induced high c-Fos immunoreactivity. In the 10-day-old pups, similarly as with adult rats, the gate was shorter under LD 8:16 than under LD 16:8, but the difference in the gate duration between the short and the long photoperiod did not yet attain that of adult animals. The data indicate that the circadian rhythmicity may develop sooner in the dm-SCN, than in the vl-SCN, whereas the photoperiodic response may develop sooner in the vl-SCN.     

Effect of acute and chronic photoperiod modulation on pentylenetetrazole-induced clonic seizure threshold in mice

Changes in circadian rhythms have been shown to alter seizure susceptibility and anticonvulsant properties of drugs. The present study attempts to elucidate the effect of acute and chronic light/dark (LD) cycle alterations on pentylenetetrazol-induced clonic seizure threshold (CST) in male NMRI mice. The acute effect was tested by comparing the effects of abrupt 6-h phase shifts that resulted in 6-h and 18-h photoperiods, during the 24-h period before CST determination, with the controls that were maintained on 12h/12h LD cycle. In order to test the effect of chronic LD cycle alteration on CST, three groups of mice were maintained on 12h/12h, 6h/18h and 18h/6h LD cycles for 2 weeks prior to CST testing. The effect of administration of exogenous melatonin (5, 10 and 20mg/kg, i.p.) was also assessed on LD cycle related changes of CST. The results indicate that acute photoperiod change from 12h/12h to 18h/6h LD cycle lowers CST, while keeping animals under shorter photoperiod does not produce a significant effect. The pro-convulsant effect of acute increase in light period is reversed by a single injection of melatonin (10 and 20mg/kg). Animals chronically maintained on both shorter and longer photoperiods show a significant decrease in CST compared to animals under 12h/12h LD cycle. However, in both groups chronic administration of melatonin (20mg/kg) reversed the effect of LD cycle alteration on CST. In conclusion, our data demonstrate that acute increase and chronic modulation of the photoperiod increase seizure susceptibility in mice. Moreover, the pro-convulsant effect of LD cycle alteration could be reversed by exogenous melatonin administration.     

The effect of different photoperiods on the methylating capacity of the pineal gland of adult,male golden hamsters,with special reference to 5-methoxyindoles

Summary Testes weight, plasma FSH and LH concentration and pineal methylating capacity were compared in hamsters housed under either long (LD 1410) or short (LD 816) photoperiods.Hamsters housed for 14 weeks under short photoperiod showed gonadal atrophy, which was complete after 6 weeks. Also plasma FSH and LH concentration showed a marked decline after transfer to short photoperiod. However, after 14 weeks the concentration of FSH and LH as well as testes weight increased again.Under both photoperiods day/night rhythms in plasma FSH and LH concentration were measured. Under both light regimes the concentrations did not show significant differences.Under long as well as short photoperiods in the pineal gland of animals no significant differences were found in the daily synthesis of various MI tested. Only the synthesis of ML was significantly higher in the pineal of hamsters housed under short photoperiod. The function of this higher synthesis of ML remains unknown.Although the maxima of the rhythm for the various MI found under different LD regimes did not differ in magnitude or duration, their location in respect to the onset of darkness was different. It is suggested that this specific location is of more physiological importance than the quantity or duration of synthesis, concentration or release of MI.At the moment the day/night rhythms were determined there were indications that recrudescence of the testes had already started. It is suggested that this recrudescence is responsible for the fact that no differences in the synthesis of MI were found comparing the influence of both photoperiods.After 14 weeks of exposure to short photoperiod, aML synthesis was, in contrast to the synthesis of the other MI, (not significantly) higher under LD 816. Moreover, opposite results for aMT and aML synthesis during darkness were found. It is suggested that the ratio of synthesis of these compounds is of physiological significance.Abbreviations have been used (Smith, 1982) MI 5-methoxyindoles - MW 5-methoxytryptophah - MT 5-methoxytryptamine - aMT melatonin - ML 5-methoxytryptophol - aML O-acetyl-5-methoxytryptophol - MA 5-methoxyindole-3-acetic acid - HIOMT hydroxyindole-O-methyltransferase - FSH follicle-stimulating hormone - LH luteinizing hormone - LD light/dark     

Altered Rhythm of Adrenal Clock Genes, StAR and Serum Corticosterone in VIP Receptor 2-Deficient Mice

The circadian time-keeping system consists of clocks in the suprachiasmatic nucleus (SCN) and in peripheral organs including an adrenal clock linked to the rhythmic corticosteroid production by regulating steroidogenic acute regulatory protein (StAR). Clock cells contain an autonomous molecular oscillator based on a group of clock genes and their protein products. Mice lacking the VPAC2 receptor display disrupted circadian rhythm of physiology and behaviour, and therefore, we using real-time RT-PCR quantified (1) the mRNAs for the clock genes Per1 and Bmal1 in the adrenal gland and SCN, (2) the adrenal Star mRNA and (3) the serum corticosterone concentration both during a light/dark (L/D) cycle and at constant darkness in wild type (WT) and VPAC2 receptor-deficient mice (VPAC2-KO). We also examined if PER1 and StAR were co-localised in the adrenal steroidogenic cells. Per1 and Bmal1 mRNA showed a 24-h rhythmic expression in the adrenal of WT mice under L/D and dark conditions. During a L/D cycle, the adrenal clock gene rhythm in VPAC2-KO mice was phase-advanced by approximately 6?h compared to WT mice and became arrhythmic in constant darkness. A significant 24-h rhythmic variation in the adrenal Star mRNA expression and circulating corticosterone concentration was similarly phase-advanced during the L/D cycle. The loss of adrenal clock gene rhythm in the VPAC2 receptor knockout mice after transfer into constant darkness was accompanied by disappearance of rhythmicity in Star mRNA expression and serum corticosterone concentration. Double immunohistochemistry showed that the PER1 protein and StAR were co-localised in the same steroidogenic cells. Circulating corticosterone plays a role in the circadian timing system and the misaligned corticosterone rhythm in the VPAC2 receptor knockout mice could be involved in their abnormal rhythms of physiology.     

Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)

In mammals, day length (photoperiod) is read and encoded in the main circadian clock, the suprachiasmatic nuclei (SCN). In turn, the SCN control the seasonal rhythmicity of various physiological processes, in particular the secretion pattern of the pineal hormone melatonin. This hormone then operates as an essential mediator for the control of seasonal physiological functions on some tissues, especially the pars tuberalis (PT). In the European hamster, both hormonal (melatonin) and behavioral (locomotor activity) rhythms are strongly affected by season, making this species an interesting model to investigate the impact of the seasonal variations of the environment. The direct (on SCN) and indirect (via melatonin on PT) effect of natural short and long photoperiod was investigated on the daily expression of clock genes, these being expressed in both tissues. In the SCN, photoperiod altered the expression of all clock genes studied. In short photoperiod, whereas Clock mRNA levels were reduced, Bmal1 expression became arrhythmic, probably resulting in the observed dramatic reduction in the rhythm of Avp expression. In the PT, Per1 and Rev-erbalpha expressions were anchored to dawn in both photoperiods. The daily profiles of Cry1 mRNA were not concordant with the daily variations in plasma melatonin although we confirmed that Cry1 expression is regulated by an acute melatonin injection in the hamster PT. The putative role of such seasonal-dependent changes in clock gene expression on the control of seasonal functions is discussed.     

Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus

The circadian clock cells of the mammalian suprachiasmatic nucleus (SCN) generate oscillations in physiology and behavior that are synchronized (entrained) by the external light/dark (LD) cycle. The mechanisms that mediate the effect of light on the core molecular mechanism of the clock are not well understood, but evidence suggests that the Period2 gene, which encodes a key clock regulator (PER2), might be involved. We assessed the expression of PER2 immunoreactivity in the retinorecipient core and shell compartments of the SCN of rats entrained to cycles of discrete light pulses presented at the early subjective day (dawn) or night (dusk), or housed in constant light. We found that in animals entrained to a 0.5 h:23.5-h LD cycle (light falls near dawn), PER2 expression is rhythmic both in the shell and in the core regions of the SCN and indistinguishable from that seen in the SCN of control rats housed in complete darkness. Similarly, the pattern of PER2 expression in the SCN of rats entrained to a 0.5-h:25.5-h LD cycle (light falls near dusk) resembled that in dark-housed controls. We also found that presentation of a discrete light pulse in the early subjective night did not induce PER2 protein expression in the SCN, even 6 h after photic stimulation. Finally, in constant light-housed, behaviorally arrhythmic rats, PER2 expression in the SCN was low and nonrhythmic. These results show that rhythmic PER2 expression occurs both in the shell and core regions of the rat SCN. Furthermore, they show that the expression of PER2 in the SCN is not regulated by entraining light. Finally, constant light-induced behavioral arrhythmicity is associated with a disruption of rhythmic PER2 expression in the whole SCN. Together, the results are consistent with a proposed role for PER2 in the core mechanism of the circadian clock but argue against an important role for PER2 in the mechanism mediating photic entrainment.     

Photoperiodic Modulation of Clock Gene Expression in the Avian Premammillary Nucleus

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual‐oscillation in dopamine (DA)/melatonin (MEL) as well as c‐fos mRNA expression that is associated with the daily photo‐inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase‐related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase‐delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light‐induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase‐shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.