Genetic and phenotypically flexible components of seasonal variation in immune function.

Animals cope with seasonal variation in environmental factors by adjustments of physiology and life history. When seasonal variation is partly predictable, such adjustments can be based on a genetic component or be phenotypically flexible. Animals have to allocate limited resources over different demands, including immune function. Accordingly, immune traits could change seasonally, and such changes could have a genetic component that differs between environments. We tested this hypothesis in genotypically distinct groups of a widespread songbird, the stonechat (Saxicola torquata). We compared variation in immunity during 1 year in long-distance migrants, short-distance migrants, tropical residents and hybrids in a common garden environment. Additionally, we investigated phenotypically flexible responses to temperature by applying different temperature regimes to one group. We assessed constitutive immunity by measuring hemagglutination, hemolysis, haptoglobin and bactericidal ability against Escherichia coli and Staphylococcus aureus. Genotypic groups differed in patterns of variation of all measured immune indices except haptoglobin. Hybrids differed from, but were rarely intermediate to, parental subspecies. Temperature treatment only influenced patterns of hemolysis and bactericidal ability against E. coli. We conclude that seasonal variation in constitutive immunity has a genetic component, that heredity does not follow simple Mendelian rules, and that some immune measures are relatively rigid while others are more flexible. Furthermore, our results support the idea that seasonal variability in constitutive immunity is associated with variability in environment and annual-cycle demands. This study stresses the importance of considering seasonal variation in immune function in relation to the ecology and life history of the organism of interest.

Annual cycles of metabolic rate are genetically determined but can be shifted by phenotypic flexibility.

Birds have adjusted their life history and physiological traits to the characteristics of the seasonally changing environments they inhabit. Annual cycles in physiology can result from phenotypic flexibility or from variation in its genetic basis. A key physiological trait that shows seasonal variation is basal metabolic rate (BMR). We studied genetic and phenotypic variation in the annual cycles of body mass, BMR and mass-specific BMR in three stonechat subspecies (Saxicola torquata) originating from environments that differ in seasonality, and in two hybrid lines. Birds were kept in a common garden set-up, under annually variable day length and at constant temperature. We also studied whether stonechats use the proximate environmental factor temperature as a cue for changes in metabolic rate, by keeping birds at two different temperature regimes. We found that the different subspecies kept in a common environment had different annual cycles of body mass, BMR (variance: Kazakh 4.12, European 1.31, Kenyans 1.25) and mass-specific BMR (variance: Kazakh 0.042, European 0.003, Kenyans 0.013). Annual variation in metabolic measures of hybrids was intermediate or similar to that of parental species. Temperature treatment did not affect the shape of the annual cycles of metabolic rate, but metabolic rate was higher in birds kept under the variable temperature regime. The distinct annual cycles in body mass and metabolic rate in stonechat subspecies kept in a common environment indicate different genetic backgrounds rather than merely a phenotypically flexible response to proximate environmental cues. Phenotypic effects of temperature are superimposed on this genetically orchestrated annual cycle.

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula).

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.

Complex bird clocks.

The circadian pacemaking system of birds comprises three major components: (i) the pineal gland, which rhythmically synthesizes and secretes melatonin; (ii) a hypothalamic region, possibly equivalent to the mammalian suprachiasmatic nuclei; and (iii) the retinae of the eyes. These components jointly interact, stabilize and amplify each other to produce a highly self-sustained circadian output. Their relative contribution to overt rhythmicity appears to differ between species and the system may change its properties even within an individual depending, for example, on its state in the annual cycle or its photic environment. Changes in pacemaker properties are partly mediated by changes in certain features of the pineal melatonin rhythm. It is proposed that this variability is functionally important, for instance, for enabling high-Arctic birds to retain synchronized circadian rhythms during the low-amplitude zeitgeber conditions in midsummer or for allowing birds to adjust quickly their circadian system to changing environmental conditions during migratory seasons. The pineal melatonin rhythm, apart from being involved in generating the avian pacemaking oscillation, is also capable of retaining day length information after isolation from the animal. Hence, it appears to participate in photoperiodic after-effects. Our results suggest that complex circadian clocks have evolved to help birds cope with complex environments.

Seasonal variations of in vivo and in vitro melatonin production in a passeriform bird, the house sparrow (Passer domesticus).

Melatonin, released from the pineal gland, is an important signal within the circadian pacemaking system of passeriform birds. Until now, seasonal variations in melatonin production have only been examined in a few avian species and the role of melatonin in the regulation of annual rhythms in birds is unclear. We investigated plasma melatonin in a group of house sparrows kept in an outside aviary in spring (March/April), summer (May/June), autumn (September/October), and winter (December/January). The durations of elevated melatonin values mirrored the seasonal changes in night length to a certain degree, the melatonin signal being longest in winter and shortest in summer. Additionally, plasma melatonin peak amplitudes differed significantly among seasons, with highest values in spring and summer and lowest values in winter. Cultured explanted pineal glands obtained from animals in winter and summer showed patterns of in vitro melatonin release comparable to in vivo circulating melatonin with different durations of elevated melatonin and peak amplitude values. These data indicate that the circadian pacemaking system of the house sparrow changes properties seasonally, either as a result of endogenous mechanisms or in response to environmental conditions. These properties are maintained in the pineal gland even after isolation from the animal.

Predators as stressors? Physiological and reproductive consequences of predation risk in tropical stonechats (Saxicola torquata axillaris).

Tropical birds usually lay smaller clutches and are less likely to initiate a second brood than their temperate-zone relatives. This reduction in annual fecundity is generally explained as an adaptation either to higher rates of nest predation or to a more limited food supply concurrent with higher adult survival in the tropics. However, the physiological parameters associated with lower annual fecundity in tropical birds have not been well investigated. We compared the annual fecundity, behaviour and a number of physiological parameters of stonechat parents feeding fledged juveniles in territories with and without fiscal shrikes, a predator on adult and fledged birds. Stonechat pairs in territories with shrikes were less likely to initiate a second brood and delayed successive broods compared to pairs in territories without shrikes. After fledging of their young, males showed a greater propensity than females to initiate distraction calls after a human intrusion into their territory and, therefore, invested more in the defence of their young. In territories with shrikes stonechat males had higher initial plasma corticosterone levels and lower body conditions than males in territories without shrikes, suggesting that they were chronically stressed. In contrast, the females from both types of territory had low initial plasma corticosterone levels. We conclude that shrike presence might account for the delay in initiation of a second brood and the reduction in the tendency to initiate a second brood. Whether these effects are mediated by the elevated levels of corticosterone remains to be demonstrated.

Photoperiodic information acquired and stored in vivo is retained in vitro by a circadian oscillator, the avian pineal gland.

Endogenous circadian rhythms have been described in a wide range of organisms from prokaryotes to man. Although basic circadian mechanisms at the molecular level are genetically fixed, certain properties of circadian rhythms at the organismic level can be modified by environmental conditions and subsequently retained for some time, even in organisms shielded from 24-hr environmental variations. To investigate the capacity of animals to acquire and store photoperiodic information, we examined activity and melatonin rhythms in house sparrows during synchronization to two different photoperiods and during subsequent prolonged darkness. Under constant environmental conditions, intact animals continued to have long feeding activity times when previously exposed to long days and short feeding activity times when previously exposed to short days. Correspondingly, significantly different durations of elevated melatonin in the plasma directly reflected the differences in night length during synchronization as well as during prolonged darkness. Additionally, we found a significant difference in the amplitude of the nocturnal melatonin signal, which also was conserved in prolonged darkness. To investigate whether the photoperiodic experience of an intact animal can be "memorized" by an isolated component of its circadian pacemaking system, we have investigated in vitro melatonin release during continuous darkness from explanted pineal glands of house sparrows after in vivo synchronization to two distinct photoperiods. Differences in the durations of elevated melatonin occurred during the first two cycles in culture and a difference in melatonin amplitude was detectable during the first night in culture. Our data indicate that photoperiodic patterns imposed on sparrows during in vivo synchronization can be maintained as an internal representation of time within the isolated pineal gland. Hence, the pineal gland, as one of the most significant components of the songbird circadian pacemaker, not only has the capacity to autonomously produce circadian rhythms of melatonin release but also is capable of storing biologically meaningful information experienced during previous cycles.

Circadian rhythms of melatonin in European starlings exposed to different lighting conditions: relationship with locomotor and feeding rhythms.

In passerine birds, the periodic secretion of melatonin by the pineal organ represents an important component of the pacemaker that controls overt circadian functions. The daily phase of low melatonin secretion generally coincides with the phase of intense activity, but the precise relationship between the melatonin and the behavioral rhythms has not been studied. Therefore, we investigated in European starlings (Sturnus vulgaris) (1) the temporal relationship between the circadian plasma melatonin rhythm and the rhythms in locomotor activity and feeding; (2) the persistence of the melatonin rhythm in constant conditions; and (3) the effects of light intensity on synchronized and free-running melatonin and behavioral rhythms. There was a marked rhythm in plasma melatonin with high levels at night and/or the inactive phase of the behavioral cycles in almost all birds. Like the behavioral rhythms, the melatonin rhythm persisted for at least 50 days in constant dim light. In the synchronized state, higher day-time light intensity resulted in more tightly synchronized rhythms and a delayed melatonin peak. While all three rhythms usually assumed a rather constant phase relationship to each other, in one bird the two behavioral rhythms dissociated from each other. In this case, the melatonin rhythm retained the appropriate phase relationship with the feeding rhythm.

Exogenous melatonin reduces the resynchronization time after phase shifts of a nonphotic zeitgeber in the house sparrow (Passer domesticus).

Continuous melatonin administration via silastic implants accelerates the resynchronization of the circadian locomotor activity rhythm in house sparrows (Passer domesticus) after exposure to phase shifts of a weak light-dark cycle. Constant melatonin might induce this effect either by increasing the sensitivity of the visual system to a light zeitgeber or by reducing the degree of self-sustainment of the circadian pacemaker. To distinguish between these two possible mechanisms, two groups of house sparrows, one carrying melatonin implants and the other empty implants, were kept in constant dim light and subjected to advance and delay shifts of a 12-h feeding phase. The resynchronization times of their circadian feeding rhythm following the phase shifts were significantly shorter when the birds carried melatonin implants than when they carried empty implants. In a second experiment, melatonin-implanted and control birds were released into food ad libitum conditions 2 days after either a delay or an advance phase shift. The number of hours by which the activity rhythms had been shifted on the second day in food ad libitum conditions was assessed. Melatonin-implanted house sparrows had significantly larger phase shifts in their circadian feeding rhythm than control birds. This is in accordance with the first experiment since a larger phase shift at a given time reflects accelerated resynchronization. Additionally, the second experiment also excludes any possible masking effects of the nonphotic zeitgeber. In conclusion, constant melatonin accelerates resynchronization even after phase shifts of a nonphotic zeitgeber, indicating that constant high levels of melatonin can reduce the degree of self-sustainment of the circadian pacemaker independent of any effects on the photoreceptive system.

Influence of pinealectomy and pineal stalk deflection on circadian gastrointestinal tract melatonin rhythms in zebra finches (Taeniopygia guttata).

The authors examined levels of melatonin in the plasma and various tissues in intact, pinealectomized, and pineal stalk-deflected zebra finches kept under 12:12 LD to determine if the melatonin found in the gastrointestinal tract is secreted in a circadian manner. In intact and pineal stalk-deflected birds, there is a clear day-night rhythm in melatonin content of the plasma, pineal gland, eyes, proventriculus, crop, duodenum, jejunum/ileum, colon, heart, and liver. In contrast, pinealectomy abolished the day-night rhythm. These results indicate that most of the melatonin present in the gastrointestinal tract of zebra finches is of pineal origin. However, some melatonin remained. This suggests that this melatonin may be locally synthesized and has paracrine and/or autocrine functions. Nonetheless, the results do not lend support to the contention that this putative melatonin secretion by the gastrointestinal tract is circadian.

Perinatal development of circadian melatonin production in domestic chicks.

In contrast to the situation in mammals, in which circadian melatonin production by the pineal gland does not begin until some time after birth, the development of pineal gland rhythmicity is an embryonic event in the precocial domestic fowl. A distinct melatonin rhythm was found in 19-d-old chick embryos maintained under light:dark (LD) 16:8. No significant variation in melatonin levels was detected in embryos exposed to LD 8:16. The melatonin rhythm in the pineal gland and plasma of chick embryos incubated for 18 d in LD 12:12 persisted for 2 d in constant darkness indicating that melatonin production is under circadian control at least from the end of embryonic life. A 1-d exposure to a LD cycle during the first postembryonic day was sufficient to entrain the melatonin rhythm, and previous embryonic exposure to either LD or constant darkness (DD) neither modified this rapid synchronization nor did it affect the melatonin pattern during the two subsequent days in DD. It is suggested that, in contrast to the situation in mammals, the avian embryo has evolved its own early circadian melatonin-producing system because, as a consequence of its extrauterine development, it cannot use the system of its mother.

Adjustment of house sparrow circadian rhythms to a simultaneously applied light and food zeitgeber.

Periodic food availability has been shown to be an effective circadian zeitgeber in many vertebrates. It is still unclear, however, i) whether light-active species like most birds can synchronize with food cycles in the presence of a strong light-dark (LD) cycle and ii) whether it is common among non-mammalian vertebrates to use a separate circadian oscillator to synchronize with food cycles as most mammals do. We investigated these questions experimentally by exposing house sparrows simultaneously to two zeitgebers: light and food. The LD cycle was set at 1410 h; food was always available for 12 hour per day, but at different phases of the LD cycle. The effects of the two zeitgebers were analyzed by observing two behavioral outputs of the birds' circadian system, the rhythms of locomotion and feeding. The data revealed that light acted as the dominant zeitgeber in most conditions. Food cycles affected the phase of the behavioral rhythms of the birds only when the food was presented no later than 3 h after the onset of light. Apart from their synchronizing actions both light and food cycles also exerted direct (masking) effects on the behavioral rhythms of the birds. The results suggest that the circadian system of house sparrows can indeed adjust to two simultaneous environmental periodicities, i.e. light and food. We propose that light is the stronger zeitgeber and plays a 'permissive' role in determining the phases at which synchronization with food cycles comes into effect. We did not find evidence that the house sparrows' behavioral rhythms are controlled by a food-entrainable circadian oscillator that is distinct from the light-entrainable system as is the case in most mammals. The differences in the patterns of food synchronization and organization of the circadian system that appear to exist between different species can be interpreted in two ways: i) species of different phylogenetic origin (e.g., mammals versus birds) evolved different circadian system or ii) regardless of phylogeny, species with different ecological requirements show a specialization in their circadian organization which is adjusted to the importance of zeitgebers in nature.

Melatonin: generation and modulation of avian circadian rhythms.

The pineal organ and its hormone melatonin are significant components of avian circadian pacemaking systems. In songbirds, pinealectomy results in the abolition or destabilization of overt circadian rhythms such as the rhythm of locomotor activity, feeding, or body temperature. A stable rhythmicity can be restored either by reimplanting a pineal organ, by periodic injections or infusions of melatonin, or by applying melatonin rhythmically through the drinking water. Several results suggest that the pineal melatonin rhythm acts on at least one other oscillator within the circadian pacemaking system, presumably the SCN, which in turn, feeds back to the pineal. As described by the "Neuroendocrine Loop" and "Internal Resonance" models, overall pacemaker output thus depends on the relative strengths of the oscillations in the pineal and the SCN. Investigations on migratory birds have shown that the amplitude of the 24-h plasma melatonin rhythm is reduced during the migratory seasons compared with the nonmigratory seasons. According to the models mentioned above, such a reduced melatonin amplitude should result in a reduction in the degree of self-sustainment of the pacemaker as a whole. This, in turn, should facilitate adjustment to the altered Zeitgeber conditions encountered by these birds as a result of their own migratory flights. A seasonal reduction in melatonin amplitude also occurs in some high-latitude birds during midsummer and midwinter. Under such conditions a less self-sustained circadian pacemaker may enhance entrainability to weak zeitgeber conditions. These examples suggest that the properties of the circadian system may be adjusted to match the changing requirements for synchronization, and that this is achieved by altering the melatonin amplitude.

Synchronization by low-amplitude light-dark cycles of 24-hour pineal and plasma melatonin rhythms of hatchling European starlings (Sturnus vulgaris).

In young European starlings, as in other avian species, high-amplitude 24-hr rhythms in plasma and pineal melatonin are already present around the time of hatching. In chickens this rhythmicity results at least partly from the light sensitivity of the melatonin-producing and -secreting system. In contrast to the chicken, the starling is a hole-nesting bird, and it seemed questionable whether the low light intensities in the nest are sufficient to synchronize perinatal melatonin rhythms. We therefore exposed starling eggs to light cycles roughly simulating those measured in nest-boxes, i.e., an 11-hr phase of complete darkness and a 13-hr phase consisting of 15 min of dim light (10 lux) alternating with 30 min of darkness. For one group the photophase lasted from 0600 to 1900 hr; for the other group the photophase lasted from 1800 to 0700 hr. In approximately 10-hr-old hatchlings of both groups, plasma and pineal melatonin concentrations were high during the dark phase and low during the light phase. We conclude that perinatal low-amplitude light intensity changes of the kind experienced by hatching starlings in the field are sufficient for synchronizing the melatonin-producing and -secreting system in the pineal and possibly other organs.

Hippocampal volume in migratory and non-migratory warblers: effects of age and experience.

We tested the hypothesis that experience of migration from Europe to tropical Africa by Garden Warblers is associated with changes in the relative volume of the hippocampus, a brain region thought to be involved in processing spatial information, including that used in navigation. Relative hippocampal volume was larger in birds at least one year old that had migrated to and from Africa, than in naive birds approx. 3 months old. Further comparisons between groups of differing age and experience of migration suggested that both experience and age during the first year have an effect of relative hippocampal volume. The increase in relative hippocampal volume was mainly due to a decrease in the size of the telencephalon; however, the comparison between young, naive birds and older, experienced birds also suggests a possible increase in absolute hippocampal volume. The latter is associated with an increase in number and density of neurons, whilst the former is associated with an increase in density but no change in total number of neurons. In a non-migratory close relative of the garden warbler, the Sardinian warbler, older birds had a smaller telencephalon but there was no change in hippocampal volume, which supports the view that changes in the hippocampus may be associated with migratory experience, whilst changes in the telencephalon are not.

Food as a circadian Zeitgeber for house sparrows: the effect of different food access durations.

House sparrows (Passer domesticus) can synchronize their circadian rhythms of locomotion and feeding with times of periodic food availability. In contrast to most mammals, which synchronize only a specific part of their circadian system with feeding cycles and thus express two distinct activity components, house sparrows synchronize their circadian activity rhythms as a whole with the food zeitgeber. Previous results had indicated that feeding cycles act as comparatively weak zeitgebers for house sparrows. In the present study, therefore, we investigate whether feeding schedules are weak zeitgebers in general or whether their impact on the circadian system of the birds depends on the degree of food restriction. A detailed analysis of the synchronization pattern under the different experimental conditions should help to clarify whether house sparrows use a different mechanism for food-synchronization than mammals. House sparrows were kept in continuous dim light and exposed to different feeding schedules with daily food access durations ranging from 8 to 20 h. Many birds lost synchronization and exhibited free-running rhythms in locomotor and feeding activity when the daily food access duration was lengthened but became synchronized when the feeding duration was shortened. The interpretation that short food access durations represent stronger zeitgebers than long food access durations was supported by the occurrence of large negative phase-angle differences during long daily feeding schedules, contrasting with small and sometimes positive phase-angle differences under short food access durations. There were no indications that house sparrows possess a specific food-entrainable circadian oscillator as mammals do. Rather, periodic food availability seems to be a zeitgeber for the whole circadian system, which, hence, can be synchronized both by light and food. An explanation for such different mechanisms of food-synchronization is offered in the feeding ecology of these animals. Birds may evaluate the importance of a specific feeding schedule as a zeitgeber either from temporal information on the duration of the daily food access time or from energetic considerations. The phase-angle differences associated with the different feeding schedules and the maintenance of daily activity times may ensure an appropriate temporal integration of behavior with specific conditions. Non-synchronized birds exhibited masking-induced feeding activity, which might represent an alternative means of adjusting to feeding cycles when synchronization cannot occur.

Development of post-hatching melatonin rhythm in zebra finches (Poephila guttata).

We examined levels of melatonin in the pineal, eyes and plasma over a 24 h period during development in the altricial zebra finch. Beginning as early as 2 days after hatching there was a distinct 24 h rhythm in melatonin in the pineal and plasma. Beginning at day seven after hatching there was also a 24 h rhythm present in the eyes. In the pineal and eyes the amplitude of the 24 h rhythm increased with age. In contrast, the amplitude of the plasma melatonin rhythm at 2 days was already within the range of adults and did not increase with age. These results confirm and expand earlier findings in the European starling and parallel those from precocial birds indicating that the circadian system is already competent at or shortly after hatching even in atricial birds.

Circadian and circannual programmes in avian migration

In migratory birds, endogenous daily (circadian) and annual (circannual) rhythms serve as biological clocks that provide the major basis for their temporal orientation. Circannual rhythms are responsible for the initiation of migration both in autumn and spring. This function of timing migrations is particularly important for birds that spend the winter close to the equator where the environment is too constant or irregular to provide accurate timing cues. In addition, circannual rhythms produce programmes that determine both the temporal and the spatial course of migration. In Sylvia warblers, the time programmes controlling autumn migration are organized in a species- or population-specific manner. It has been proposed that, in first-year migrants, the time programme for autumn migration plays a major role in determining migratory distance, thus providing the vector component in a mechanism of vector navigation. It is not yet clear, however, whether this programme does indeed determine migratory distance or whether it only provides the temporal framework within which other factors determine how far a bird flies. Evidence against the first alternative comes from findings indicating that migratory activity can be drastically modified by a constellation of rather specific, but highly relevant, factors and that the resulting changes in migratory activity are not compensated by subsequent increases or decreases of migratory activity. In normally day-active but nocturnally migrating birds, circannual signals cause alterations in the circadian system leading to the development of nocturnal activity. Although the nature of these signals is unknown, there is evidence that changes in the diurnal pattern of melatonin secretion by the pineal gland are associated with, and possibly causally involved in, the waxing and waning of nocturnal activity. These changes in the melatonin pattern presumably also affect general synchronization properties of the circadian system to Zeitgebers in such a way that circadian rhythms adjust faster to new conditions after long transmeridian flights.

Synchronization of circadian rhythms of house sparrows by oral melatonin: effects of changing period.

House sparrows (Passer domesticus) whose circadian rhythms of locomotor activity and feeding had been abolished by pinealectomy were held in constant dim light and periodically exposed to melatonin in the drinking water. By alternating 8 h of melatonin water with variable phases of tap water, rhythms with periods (T) ranging from 21 to 27 h were produced. When melatonin was administered in rhythms with periods of 23, 24, and 25 h, feeding and locomotion behavior of most birds were rhythmic and synchronized with the exogenous melatonin rhythm. The rest phase coincided approximately with the phase of melatonin availability. Under melatonin cycles < 23 h and > 25 h, fewer birds had synchronized rhythms. Nonsynchronized birds were either arrhythmic or they expressed free-running rhythms. Under melatonin rhythms with periods between 23 and 26 h, the phase-angle difference between defined phases of the behavioral rhythms and the melatonin rhythm became more positive with increasing T. These data are consistent with the hypothesis (a) that periodic exogenous melatonin can substitute, at least to a certain degree, for the endogenous plasma melatonin rhythm normally resulting from the periodic melatonin secretion by the pineal gland, and (b) that this melatonin rhythm acts on another oscillator, possibly the SCN, as part of the overall circadian pacemaking system.