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.     

Neurochemical basis for the photic control of circadian rhythms and seasonal reproductive cycles: role for acetylcholine.

A pharmacological approach was used to examine the role of acetylcholine in the photic control of circadian rhythms and seasonal reproductive cycles. The experimental protocol was designed to determine whether the administration of carbachol, a cholinergic agonist, could mimic the effects of brief light pulses on gonadal function and/or the circadian rhythm of wheel-running activity in golden hamsters. Intraventricular injections of carbachol, administered singularly at discrete phase points throughout the circadian cycle, induced phase-dependent shifts in the free-running rhythm of activity similar to those caused by a brief light exposure. Injections of carbachol once every 23.33 hr for 9 weeks entrained the activity rhythm and stimulated the neuroendocrine-gonadal axis in a manner similar to that observed after the presentation of 1-hr light pulses at this frequency. In contrast, the administration of carbachol once every 24 hr did not consistently provide an entraining signal for the activity rhythm and did not stimulate reproductive function. Importantly, the effects of carbachol on the seasonal reproductive response were correlated with the timing of the injections relative to the activity rhythm. These findings suggest that acetylcholine may play an important role in the mechanism by which light regulates circadian rhythms and seasonal reproductive cycles.     

Plasma melatonin in the horse: Measurements in natural photoperiod and in acutely extended darkness throughout the year

Abstract: Plasma melatonin was measured at the winter and summer solstices and the autumn and spring equinoxes in four mares held under natural conditions at 35°S. At all seasons the onset of the nightly elevated melatonin was coincident with or after the time of sunset and the melatonin offset after the time of sunrise. The duration of elevated melatonin was not different from the duration of natural scotophase for each season, with the duration of elevated melatonin longer in winter than the other seasons. Immediately following each 24 hr sampling two mares were resampled in acutely extended darkness to determine the melatonin profile of the endogenous rhythm of the circadian pacemaker, originating from the suprachiasmatic nuclei (SCN). At each season melatonin secretion commenced earlier and decreased later than that measured under the natural photoperiodic condition, suggesting that the expression of the melatonin rhythm is normally gated by natural environmental light both at dusk and dawn. The interval from the onset of melatonin measured under acutely extended darkness to the time of sunset was greater in the spring/ summer than the autumn/winter suggesting a possible alternating signal throughout the year. Thus the mare appears to exhibit a similar interaction between endogenous circadian rhythmic activity and the natural photoperiod as the ewe which may underlie the mechanism for timing reproductive activity through the year.     

Melatonin phase-shifts human circadian rhythms with no evidence of changes in the duration of endogenous melatonin secretion or the 24-hour production of reproductive hormones

The pineal hormone melatonin is a popular treatment for sleep and circadian rhythm disruption. Melatonin administered at optimal times of the day for treatment often results in a prolonged melatonin profile. In photoperiodic (day length-dependent) species, changes in melatonin profile duration influence the timing of seasonal rhythms. We investigated the effects of an artificially prolonged melatonin profile on endogenous melatonin and cortisol rhythms, wrist actigraphy, and reproductive hormones in humans. Eight healthy men took part in this double-blind, crossover study. Surge/sustained release melatonin (1.5 mg) or placebo was administered for 8 d at the beginning of a 16-h sleep opportunity (1600 h to 0800 h) in dim light. Compared with placebo, melatonin administration advanced the timing of endogenous melatonin and cortisol rhythms. Activity was reduced in the first half and increased in the second half of the sleep opportunity with melatonin; however, total activity during the sleep opportunities and wake episodes was not affected. Melatonin treatment did not affect the endogenous melatonin profile duration, pituitary/gonadal hormone levels (24-h), or sleepiness and mood levels on the subsequent day. In the short term, suitably timed sustained-release melatonin phase-shifts circadian rhythms and redistributes activity during a 16-h sleep opportunity, with no evidence of changes in the duration of endogenous melatonin secretion or pituitary/gonadal hormones.     

A test of the coincidence and duration models of melatonin action in Siberian hamsters: the effects of 1-hr melatonin infusions on testicular development in intact and pinealectomized prepubertal Phodopus sungorus

The pineal hormone melatonin is known to play an important role in mediating photoperiodic messages to the reproductive system in seasonal breeding animals. Our goal was to test, in a single experimental paradigm, two hypotheses that have been forwarded to describe how the circadian rhythm of pineal melatonin transmits photoperiodic information to the reproductive system: 1) induction, i.e., a short-day effect, occurs when secreted melatonin and a circadian rhythm of sensitivity to melatonin coincide in time; 2) induction occurs following exposure to elevated circulating melatonin levels for a prescribed duration. In order to determine the relative validity of these hypotheses, we investigated the testicular maturation response to 1-hr daily infusions of 10, 25, and 50 ng of melatonin in pinealectomized intact and prepubertal Siberian hamsters (Phodopus sungorus). Animals received, beginning on day 15 of life, programmed subcutaneous infusions of melatonin or vehicle at one of five time points (19:00-20:00, 20:00-21:00, 21:00-22:00, 24:00-01:00, and 03:00-04:00 hr) for 15 days. In animals gestated and raised in a long photoperiod (LD16:8 = 16L, where L is the duration of light in hours, and D that of dark), melatonin infusion right after lights off (20:00-21:00 hr) significantly retarded gonadal maturation; this dose was ineffective at other times tested. Doses of 10 and 25 ng melatonin were ineffective at all time points. Identical results were obtained in prepubertal hamsters gestated in a short photoperiod (LD10:14 = 10L) and raised in 16L; these results were independent of the presence or absence of the pineal gland. In animals gestated and raised in 10L, melatonin infusions failed to suppress testicular development beyond that induced by the photoperiod; testicular development was maximally suppressed in all groups. The results of these investigations are best explained under the experimental conditions employed here: 1) the photoperiodic gonadal response in juvenile Siberian hamsters is regulated by the coincidence in time of exogenously administered melatonin with an intrinsic rhythm of sensitivity to melatonin, which, under the constraints imposed by our experimental design, occurred at 20:00-21:00 hr; and 2) the duration of the melatonin signal alone, equal in all groups, cannot explain the results.     

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.     

Continuous light and melatonin: Daily and seasonal variations of brain binding sites and plasma concentration during the first reproductive cycle of sea bass

The present study reports on the daily and seasonal variations in plasma melatonin concentration, and also in optic tectum and hypothalamus melatonin binding sites, in male European sea bass maintained under natural photoperiod (NP) or continuous light (LL) from early stages of development. Samples were collected on a 24-h cycle, at four physiological phases of their first annual reproductive cycle, i.e., pre-spermatogenesis, spermatogenesis, spermiation and post-spermiation. Under NP, (1) plasma melatonin levels were higher at night than during the day regardless of the year period, and the duration of the signal matched the duration of the dark phase; (2) daily variations in Kd and Bmax were found in the optic tectum, but only during spermiation, with the acrophase being 180° out of phase with the plasma melatonin variations; and (3) significant seasonal Kd and Bmax changes were seen in the hypothalamus. Under LL, (1) plasma melatonin showed no elevation during the subjective night; and (2) Kd and Bmax exhibited seasonal variations in the hypothalamus. These results led to the conclusion that long-term exposure to LL affected both plasma melatonin and receptor oscillations; particularly, LL disrupted the receptor density circadian oscillation found in the optic tectum during spermiation under NP. This oscillation appears to be important for sea bass to pursue gametogenesis until full spermiation. The persistence of both daily and seasonal variation of receptor affinity and density in the hypothalamus under LL indicates that these variations are controlled by internal circadian and circannual clocks that do not involve melatonin.     

Kisspeptin: A key link to seasonal breeding

In seasonal species, photoperiod (i.e. daylength) tightly regulates reproduction to ensure that birth occurs at the most favorable time of year. In mammals, a distinct photoneuroendocrine circuit controls this process via the pineal hormone melatonin. This hormone is responsible for the seasonal regulation of reproduction, but the anatomical substrate and the cellular mechanism through which melatonin modulates sexual activity is far from understood. The Syrian hamster is widely used to explore the photoneuroendocrine system, because it is a seasonal model in which sexual activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Recent evidences indicate that the products of the KiSS-1 gene, kisspeptins, and their specific receptor GPR54, represent potent stimulators of the sexual axis. We have shown that melatonin impacts on KiSS-1 expression to control reproduction in the Syrian hamster. In this species, KiSS-1 is expressed in the antero-ventral-periventricular and arcuate nuclei of the hypothalamus at significantly higher levels in hamsters kept in LD as compared to SD. In the arcuate nucleus, the downregulation of KiSS-1 expression in SD appears to be mediated by melatonin and not by secondary changes in gonadal hormones. Remarkably, a chronic administration of kisspeptin restores testicular activity in SD hamsters, despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that the photoperiod, via melatonin, modulates KiSS-1 neurons to drive the reproductive axis.     

Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro.

The pineal gland of birds contains one or more circadian oscillators that play a major role in overall temporal organization. We have developed a flow-through culture system for the isolated pineal by which we can measure the release of melatonin continuously from superfused glands over long periods of time. Chicken pineals release melatonin rhythmically, and these rhythms persist in vitro with a circadian oscillation. In light cycles the release of melatonin is strongly rhythmic; however, in constant conditions the amplitude of the rhythm is lower and appears to be damping. Light has at least two effects upon the isolated pineal: cyclic light input synchronizes the rhythm, and acute light exposure at night rapidly inhibits melatonin release. The cultured avian pineal clearly offers great potential as a model system for the study of vertebrate circadian oscillators and may open the way for an analysis of mechanism.     

Long‐term effects of maternal separation on the responsiveness of the circadian system to melatonin in the diurnal nonhuman primate (Macaca mulatta)

Depression is often linked to early‐life adversity and circadian disturbances. Here, we assessed the long‐term impact of early‐life adversity, particularly preweaning mother–infant separation, on the circadian system's responsiveness to a time giver or synchronizer (Zeitgeber). Mother‐reared (MR) and peer‐reared (PR) rhesus monkeys were subjected to chronic jet‐lag, a forced desynchrony protocol of 22 hr T‐cycles [11:11 hr light:dark (LD) cycles] to destabilize the central circadian organization. MR and PR monkeys subjected to the T‐cycles showed split locomotor activity rhythms with periods of ~22 hr (entrained) and ~24 hr (free‐running), simultaneously. Continuous melatonin treatment in the drinking water (20 μg/mL) gradually increased the amplitude of the entrained rhythm at the expense of the free‐running rhythm, reaching complete entrainment by 1 wk. Upon release into constant dim light, a rearing effect on anticipation for both the predicted light onset and food presentation was observed. In MR monkeys, melatonin did not affect the amplitude of anticipatory behavior. Interestingly, however, PR macaques showed light onset and food anticipatory activities in response to melatonin treatment. These results demonstrate for the first time a rearing‐dependent effect of maternal separation in macaques, imprinting long‐term plastic changes on the circadian system well into late adulthood. These effects could be counteracted by the synchronizer molecule melatonin. We conclude that the melatonergic system is targeted by early‐life adversity of maternal separation and that melatonin supplementation ameliorates the negative impact of stress on the circadian system.     

Trans-pineal microdialysis in the Djungarian hamster (Phodopus sungorus): a tool to study seasonal changes of circadian clock activities

The Djungarian hamster is a highly seasonal small mammal. The rhythmic secretion of melatonin by the pineal gland is under control of the circadian clock, conveying the photoperiodic message to the organism. Trans-pineal microdialysis permits the in vivo study of this well-defined and precise clock output by measuring melatonin release directly in the pineal gland. The aim of this study was to adapt this method to the Djungarian hamster in order to monitor clock properties during photoperiodic changes. Male adult Djungarian hamsters were kept in a long photoperiod (LD 16:8) and melatonin release was measured hourly during the dark period for several weeks. Melatonin showed a regular secretion between ZT 17 and ZT 23.5 whereas the amplitude became stable only after the third day of perfusion. To test how quickly changes in melatonin profile can be measured, 15-min light pulses were given at different time points throughout the scotophase. Light-pulses immediately interrupted melatonin secretion at any time point during the scotophase and the temporal resolution for measurement could be reduced to 30 min. In accordance with studies in the rat, long-term effects of light on the clock could only be observed when a light pulse was administered in the second half of the night. For the first time we established a method to measure precisely a direct and reliable clock-output in a highly seasonal species which allows us now to study the circadian and seasonal properties of the clock in detail.     

Photoperiodic regulation of the hamster testis: dependence on circadian rhythms.

The testes of hamsters exposed to short days (10 hr of light per day) regress within 13 weeks. Administration of 7.5% deuterium oxide to hamsters lengthens the period of free running circadian activity rhythms by 2.2% and prevents testicular regression during short-day exposure. This consistent with predictions derived from an external coincidence model for photoperiodic time measurement: Deuterium oxide changes phase relationships between the light-dark cycle and the circadian system, the hamster's daily photosensitive phase is stimulated with light during short days, and the testes remain large. Conservation of the period of circadian rhythms within narrow limits has adaptive significance for hamster photoperiodism and for the occurrence and phasing of the annual reproductive cycle.     

Melatonin receptor density in Area X of European starlings is correlated with reproductive state and is unaffected by plasma melatonin concentration

Several of the song control nuclei of songbirds, including HVc (higher vocal center) and Area X, contain melatonin receptor (MelR). In laboratory-housed male starlings, the densities of MelR in Area X change markedly according to reproductive state. MelR are down-regulated when starlings are photostimulated (in full breeding condition) and are subsequently up-regulated when starlings become photorefractory (reproductively quiescent). However, seasonal regulation of MelR densities in Area X has only been investigated during the light phase of the light:dark cycle. Variation in MelR densities are physiologically relevant only if they also occur during the dark phase, when melatonin is present in the circulation. Brains from male starlings that were in different reproductive states but exposed to the same 18L:6D photoperiod were collected during either the mid-point of the light phase or the dark phase. Melatonin receptor distribution was assessed in vitro by 125Iodomelatonin (IMEL) receptor autoradiography. All photostimulated birds exhibited down-regulation of MelR in Area X, and all photorefractory birds exhibited high MelR density in Area X, regardless of time of sampling or plasma melatonin concentration. Thus, within each reproductive state, MelR density in Area X did not differ over the course of a circadian cycle. The functional significance of seasonal regulation of MelR in this song control nucleus remains unclear, but it is likely to involve a release of cellular inhibition by melatonin during photostimulation, with possible consequences for song learning, memory consolidation or regulation of the context of song production.     

Pineal gland interface between the photoperiodic environment and the endocrine system.

The photoperiodic message that the pineal gland conveys to the organism is encoded in the circadian melatonin rhythm. Melatonin is a ubiquitously acting hormone that mediates seasonal changes in reproduction in nonhuman mammals and may have reproductive consequences in humans as well. Additionally, melatonin may relate to the function o f the immune system, hormone-responsive tumor growth, circadian rhythm disturbances, and a number of other processes.     

Temporal and histological evaluation of melatonin patterns in a 'basal' metazoan

Abstract: While recent advances suggest functional pleiotropy of melatonin in higher organisms, an understanding of the biological significance of this ancient molecule in early evolutionary groups is lacking. Here, endogenous melatonin production was identified for the first time in the sea anemone Actinia equina, a nonsymbiotic hexacorallian cnidarian. Day/night activity profiles of melatonin in this anemone indicated that melatonin levels oscillate with significant nocturnal peaks. However, dynamic changes in melatonin concentration did not persist under constant dark conditions and therefore were not circadian in nature. Thus, the oscillating pattern of melatonin in A. equina is presumed to be the result of alternative, simpler melatonin control mechanism that likely involves direct regulation by the daily photocycle. As nocturnal melatonin signals still potentially provide ‘time‐of‐day’ information and can illustrate the seasonally changing length of the biological night, we hypothesize that melatonin may be relevant to temporal coordination of timed processes also in anthozoans. Spatial patterns of melatonin distribution found in this study indicate abundant melatonin distribution in the endodermal filaments wrapped around gametes. This finding supports the possibility that one of the melatonin‐responsive processes in this basal metazoan species may involve reproductive functions.     

Does melatonin alter pituitary responsiveness to gonadotropin-releasing hormone in the ewe?

The diurnal secretion of melatonin from the pineal gland transduces information about day length to the reproductive axis of many seasonal breeders including the ewe. In the sheep the target for melatonin is thought to be neural, such that the hormone acts through the GnRH pulse generator to produce seasonal alterations in the frequency of pulsatile LH secretion. These effects on the pulse generation mechanism take approximately 50 days to become evident. It is possible that melatonin also exerts direct effects at the level of the pituitary gland to alter responsiveness to GnRH. Such effects have been noted in other species. The site of action of melatonin to regulate pulsatile LH secretion was assessed in the ewe by determining whether the animal's endogenous melatonin acutely modifies pituitary responsiveness to sustained pulsatile administration of GnRH. Using an animal model in which endogenous GnRH was blocked, pituitary responsiveness to hourly pulses of exogenous GnRH was assessed under conditions of both high (dark period) and low (light period) melatonin. No evidence for acute effects of melatonin on pituitary response to GnRH was found. In another experiment, the amplitude and frequency of endogenously generated LH pulses in ovariectomized ewes was found not to change during the 24-hour light/dark cycle. These data lead to the conclusion that melatonin does not act at the pituitary gland to produce acute effects on LH secretion. Rather, our findings are consistent with the hypothesis that the action of melatonin, in this short-day breeder is long term, and is directed towards the neural elements of the hypothalamic pulse-generating mechanism.     

The circadian basis of winter depression

The following test of the circadian phase-shift hypothesis for patients with winter depression (seasonal affective disorder, or SAD) uses low-dose melatonin administration in the morning or afternoon/evening to induce phase delays or phase advances, respectively, without causing sleepiness. Correlations between depression ratings and circadian phase revealed a therapeutic window for optimal alignment of circadian rhythms that also appears to be useful for phase-typing SAD patients for the purpose of administering treatment at the correct time. These analyses also provide estimates of the circadian component of SAD that may apply to the antidepressant mechanism of action of appropriately timed bright light exposure, the treatment of choice. SAD may be the first psychiatric disorder in which a physiological marker correlates with symptom severity before, and in the course of, treatment in the same patients. The findings support the phase-shift hypothesis for SAD, as well as suggest a way to assess the circadian component of other psychiatric, sleep, and chronobiologic disorders.     

Hormonal regulation of the annual reproductive cycle of golden hamsters

The seasonally breeding golden hamster, Mesocricetus auratus, is a photoperiodic species. Reproduction is confined to the spring and summer months and is curtailed by the short days of fall and winter. The photoperiodic response is dependent on an endogenous circadian sensitivity to light. Reacquisition of reproductive activity occurs spontaneously, even in the complete absence of light. These animals are refractory to the inhibitory effects of short days. Refractoriness is terminated by exposure to long days for a period of 11 or more weeks, after which the animals are once again sensitive to short days. The short-day response in the male is characterized by testicular regression induced by a decline in pituitary and circulating titers of LH, FSH, and PRL. These animals demonstrate an increased sensitivity to negative-feedback effects of exogenous (and, presumably, endogenous) testosterone that is associated with a decreased castration response. The reproductively inactive female fails to ovulate. Estrous cycles are absent, supplanted by a daily surge of LH and FSH. Spontaneous recovery of testicular function is associated with an increase in circulating titers of LH, FSH, PRL, and testosterone, and a resumption of spermatogenic activity. In the female the daily surge of LH and FSH is replaced by the 4-day estrous cycle. The pineal gland is essential for the occurrence of photoperiodically induced changes in the hamster's reproductive cycle. The pineal produces the indole, melatonin, with peak synthesis and release occurring during the subjective night. Administration of melatonin, by injection, at the appropriate time of day, will induce gonadal regression in both intact and pinealectomized hamsters maintained on normally stimulatory photoperiods. Subcutaneous melatonin implants, however, prevent gonadal regression induced by short photoperiods or melatonin injections. Melatonin implants also prevent photoinduced gonadal recrudescence. Constant release melatonin implants may function by interfering with target tissue sensitivity to endogenous melatonin. The target tissue for melatonin and the mechanism by which the pineal is involved in the photoperiodic response remain unknown.