Melatonin rhythm observed throughout a three-cycle bright-light stimulus designed to reset the human circadian pacemaker.

Exposure to light and darkness can rapidly induce phase shifts of the human circadian pacemaker. A type 0 phase response curve (PRC) to light that has been reported for humans was based on circadian phase data collected from constant routines performed before and after a three-cycle light stimulus, but resetting data observed throughout the entire resetting protocol have not been previously reported. Pineal melatonin secretion is governed by the hypothalamic circadian pacemaker via a well-defined neural pathway and is reportedly less subject to the masking effects of sleep and activity than body temperature. The authors reasoned that observation of the melatonin rhythm throughout the three-cycle light resetting trials could provide daily phase-resetting information, allowing a dynamic view of the resetting response of the circadian pacemaker to light. Subjects (n = 12) living in otherwise dim light (approximately 10-15 lux) were exposed to a noncritical stimulus of three cycles of bright light (approximately 9500 lux for 5 h per day) timed to phase advance or phase delay the human circadian pacemaker; control subjects (n = 11) were scheduled to the same protocols but exposed to three 5-h darkness cycles instead of light. Subjects underwent initial and final constant routine phase assessments; hourly melatonin samples and body temperature data were collected throughout the protocol. Average daily phase shifts of 1 to 3 h were observed in 11 of 12 subjects receiving the bright light, supporting predictions obtained using Kronauer's phase-amplitude model of the resetting response of the human circadian pacemaker. The melatonin rhythm in the 12th subject progressively attenuated in amplitude throughout the resetting trial, becoming undetectable for >32 hours preceding an abrupt reappearance of the rhythm at a shifted phase with a recovered amplitude. The data from control subjects who remained in dim lighting and darkness delayed on average -0.2 h per day, consistent with the daily delay expected due to the longer than 24-h intrinsic period of the human circadian pacemaker. Both temperature and melatonin rhythms shifted by equivalent amounts in both bright light-treated and control subjects (R = 0.968; p<0.0001; n = 23). Observation of the melatonin rhythm throughout a three-cycle resetting trial has provided a dynamic view of the daily phase-resetting response of the human circadian pacemaker. Taken together with the observation of strong type 0 resetting in humans in response to the same three-cycle stimulus applied at a critical phase, these data confirm the importance of considering both phase and amplitude when describing the resetting of the human circadian pacemaker by light.     

Melatonin as a time-meaningful signal in circadian organization of immune response

Melatonin is synthesized and secreted during the dark period of the light/dark cycle. The rhythmic nocturnal melatonin secretion is directly generated by the circadian clock, located within the suprachiasmatic nuclei in mammals and is entrained to a 24-hour period by the light-dark cycle. The periodic secretion of melatonin may be used as a circadian mediator to any system that can 'read' the message. Melatonin seems to act as an arm of the circadian clock, giving a time-related signal to a number of body functions; one of these, the circadian organization of the defense of the organism, is discussed in some detail as an example.     

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.     

Contribution of circadian physiology and sleep homeostasis to age-related changes in human sleep

The circadian pacemaker and sleep homeostasis play pivotal roles in vigilance state control. It has been hypothesized that age-related changes in the human circadian pacemaker, as well as sleep homeostatic mechanisms, contribute to the hallmarks of age-related changes in sleep, that is, earlier wake time and reduced sleep consolidation. Assessments of circadian parameters in healthy young (∼20-30 years old) and older people (∼65-75 years old)—in the absence of the confounding effects of sleep, changes in posture, and light exposure—have demonstrated that an earlier wake time in older people is accompanied by about a 1h advance of the rhythms of core body temperature and melatonin. In addition, older people wake up at an earlier circadian phase of the body temperature and plasma melatonin rhythm. The amplitude of the endogenous circadian component of the core body temperature rhythm assessed during constant routine and forced desynchrony protocols is reduced by 20-30% in older people. Recent assessments of the intrinsic period of the human circadian pacemaker in the absence of the confounding effects of light revealed no age-related reduction of this parameter in both sighted and blind individuals. Wake maintenance and sleep initiation are not markedly affected by age except that sleep latencies are longer in older people when sleep initiation is attempted in the early morning. In contrast, major age-related reductions in the consolidation and duration of sleep occur at all circadian phases. Sleep of older people is particularly disrupted when scheduled on the rising limb of the temperature rhythm, indicating that the sleep of older people is more susceptible to arousal signals genernpated by the circadian pacemaker. Sleep-homeostatic mechanisms, as assayed by the sleep-deprivation-induced increase of EEG slow-wave activity (SWA), are operative in older people, although during both baseline sleep and recovery sleep SWA in older people remains at lower levels. The internal circadian phase advance of awakening, as well as the age-related reduction in sleep consolidation, appears related to an age-related reduction in the promotion of sleep by the circadian pacemaker during the biological night in combination with a reduced homeostatic pressure for sleep. Early morning light exposure associated with this advance of awakening in older people could reinforce the advanced circadian phase. Quantification of the interaction between sleep homeostasis and circadian rhythmicity contributes to understanding age-related changes in sleep timing and quality. (Chronobiology International, 17(3), 285-311, 2000)     

A cluster of Arabidopsis genes with a coordinate response to an environmental stimulus

Vernalization, the promotion of flowering after prolonged exposure to low temperatures, is an adaptive response of plants ensuring that flowering occurs at a propitious time in the annual seasonal cycle. In Arabidopsis, FLOWERING LOCUS C (FLC), which encodes a repressor of flowering, is a key gene in the vernalization response; plants with high-FLC expression respond to vernalization by downregulating FLC and thereby flowering at an earlier time. Vernalization has the hallmarks of an epigenetically regulated process. The downregulation of FLC by low temperatures is maintained throughout vegetative development but is reset at each generation. During our study of vernalization, we have found that a small gene cluster, including FLC and its two flanking genes, is coordinately regulated in response to genetic modifiers, to the environmental stimulus of vernalization, and in plants with low levels of DNA methylation. Genes encoded on foreign DNA inserted into the cluster also acquire the low-temperature response. At other chromosomal locations, FLC maintains its response to vernalization and imposes a parallel response on a flanking gene. This suggests that FLC contains sequences that confer changes in gene expression extending beyond FLC itself, perhaps through chromatin modification.     

Prenatal melatonin influences developmental changes of tachykinins in response to estradiol benzoate

The developmental changes of hypothalamic, pituitary, striatum and pineal gland tachykinin concentrations, as well as the response to estradiol-benzoate (EB) administration, were studied in offspring of control and melatonin (MEL) treated mother rats. Female rats were studied throughout different phases of the sexual development: infantile, prepubertal and pubertal periods, in the four following groups; control-offspring+vehicle; control-offspring+EB; MEL-offspring+vehicle; MEL-offspring+EB. Hypothalamic NKA in control-offspring+ vehicle was significantly increased only at 27 days of age and in control-offspring+EB at 27 days of age and during the infantile period. Hypothalamic SP levels increased similarly in control-offspring+EB during the infantile period but the EB influence was more pronounced with significantly increased concentrations at 32 days of age. Prenatal melatonin treatment produced major alterations in these patterns of postnatal development. In MEL-offspring+EB tachykinins concentrations in the hypothalamus during infantile and prepubertal periods did not increase, however at 37 days of age, they showed significantly higher values than in control-offspring+EB groups. The developmental pattern of pituitary NKA and SP concentrations in both; control-offspring+vehicle and control-offspring+EB groups, showed similar values from the infantile period to puberty, indicating that NKA and SP concentrations remained at similar levels independently of the sexual stage, only at 27 days of age in control-offspring+EB significantly increased values were found as compared to MEL-offspring+EB. Prenatal melatonin did not produce marked modifications, only significantly lower NKA and SP concentrations in MEL-offspring+EB group were observed at 25 days of age in comparison to control-offspring+EB group. Striatal NKA and SP concentrations showed a similar developmental pattern. In control-offspring, EB treatment produced NKA and SP decreased concentrations at the infantile period than in control-offspring+vehicle and significantly increased concentrations during the prepubertal period, then during the pubertal period NKA and SP concentrations decreased in control-group+EB. However, prenatal melatonin treatment reduced the levels of striatal NKA and SP during the prepubertal period after EB treatment and delayed until pubertal period the increase previously observed in control group during the prepubertal period. In MEL-offspring+vehicle group striatal concentrations of both tachykinins remained at low levels from infantile period until pubertal period. Prenatal melatonin and EB did not produce major alterations in SP pineal concentrations throughout sexual development. Plasma estradiol concentrations were significantly higher in the groups that received EB treatment than in those that received vehicle during prepubertal and juvenile periods in control-offspring+EB group and during the pubertal period in MEL-offspring+EB group. These data indicate that prenatal MEL treatment may influence NKA and SP developmental pattern from the infantile period until adulthood in the female rat.     

Testicular response to melatonin or suprachiasmatic nuclei ablation in the spotted skunk

Testes of the Western spotted skunk enlarge and regress seasonally. The pineal hormone, melatonin, may be important in timing this seasonal reproductive activity. Likewise, the suprachiasmatic nuclei (SCN) have been implicated as possible neural regulators of pineal and reproductive events. These experiments were conducted to determine whether ablation of the SCN or constant administration of melatonin alters timing of the seasonal pattern of testicular regression and recrudescence. Male skunks (n = 24) were treated as follows: six received two empty Silastic capsules, six received two melatonin-filled Silastic capsules, six received sham lesions to the SCN, and six received lesions to the SCN (SCNx). All skunks were exposed to a natural photoperiod and had regressed testes at the onset of the experiment. Four of six males from the SCNx group had an average of 94 +/- 11.3% of these nuclei destroyed. Sham SCNx, animals with less than 40% of the SCN ablated, and males with empty capsules did not have fully enlarged testes until October. SCNx and melatonin-treated skunks exhibited a hastening of testicular recrudescence with maximal testis size being reached in June. Skunks with lesions to the SCN maintained enlarged testes for 5 months while all other groups exhibited rapid regression after attaining maximal testis size. Testicular regression occurred from July through October in animals receiving continuous melatonin, while controls exhibited recrudescence during this same period. Our data suggest that the SCN, melatonin, and thus the pineal gland, play a role in regulating the seasonal testicular cycle of the spotted skunk.     

Melatonin and photorefractoriness: loss of response to the melatonin signal leads to seasonal reproductive transitions in the ewe

Experiments were conducted to examine whether the refractoriness of the Suffolk ewe to the reproductive effects of day length is associated with a deficit in the generation of the circadian rhythm of melatonin secretion or in the postpineal processing of this photoperiodic message. Using serum luteinizing hormone (LH) concentrations in ovariectomized ewes bearing constant-release estradiol implants as a marker of reproductive induction, ewes with intact pineal glands were found to become unresponsive to fixed artificial photoperiods that initially had been either inductive (short days) or inhibitory (long days). The loss of the photoperiodic response was not associated with notable changes in the 24-h secretory pattern of melatonin, which remained characteristically low throughout the day and rose at night. In pinealectomized ewes, nightly infusion of a stimulatory pattern of melatonin (simulating that seen on short days) initially provoked reproductive induction; this response then lessened over much the same time course that pineal intact ewes became refractory to short days. These results support the hypothesis that photorefractoriness reflects a deficit in the postpineal processing of the photoperiodic message. Further, in view of recent evidence that photorefractoriness normally leads to both onset and cessation of the breeding season in Suffolk ewes maintained outdoors, these findings suggest that the loss of response to the melatonin signal contributes to at least one of these reproductive transitions, the cessation of the breeding season, under natural environmental conditions.     

Altered circadian rhythms of corticosterone, melatonin, and phagocytic activity in response to stress in rats

Corticosterone is thought to be the main glucocorticoid secreted in response to stressful exercise, while melatonin buffers the adverse immunological effects of stress. The present work was aimed to evaluate whether swimming-exercise-induced stress leads to changes in the chronobiology parameters of the circadian rhythms of melatonin and corticosterone, and in the number and phagocytosis of peritoneal macrophages in 3-month-old male Wistar rats. The animals were subjected to a physical activity trial consisting of 2 h of free swimming. Radioimmunoassay was used to determine the plasma levels of melatonin and corticosterone. Phagocytosis was measured by the latex-bead phagocytosis index (PI), i.e., the number of latex beads ingested by 100 macrophages, the phagocytosis percentage (PP), i.e., the percentage of cells that had phagocytosed at least one latex bead, and the phagocytosis efficiency (PE), i.e., the ratio PI: PP which indicates how effectively the phagocytes ingested the particles. Stress significantly decreased the MESOR and amplitude of the melatonin rhythm, and significantly increased the MESOR of the corticosterone rhythm. The control animals' peritoneal macrophage number and PI showed a circadian rhythm with maxima at 02:00 and 03:00, respectively. The stressed group displayed higher values of PI than the controls at most hours of the night, but the number of cells in the peritoneal cavity was practically the same at all hours studied. These data confirm that melatonin and corticosterone act as modulators of the innate immune response, and that the circadian rhythm of the two hormones are altered in situations of stress.     

Effects of a synchronizer phase-shift on circadian rhythms in response of mice to ethanol or ouabain

The percentage of mice dying from a toxic dose of ethanol or ouabain was determined at 4-hour intervals during the first 3 days after inversion of an LD(12:12) lighting regimen. In comparison to unshifted control animals at similar stages of the synchronizer schedule, spans of relative advantage alternated with spans of relative disadvantage due to a gradual phase-shift of the circadian susceptibility rhythm. Overall mortality, determined for an integral number of day, was not obviously affected by the regimen-shift.Supported by grants from the United States Public Health Service (5-K6-GM-13,981), NASA (NGR-24-005-006) and the U.S. Air Force (F29600-69-C-0011).     

The epigenetic calnexin-independent state is induced in response to environmental changes

Yeasts have evolved numerous responsive pathways to survive in fluctuating and stressful environments. The endoplasmic reticulum (ER) is sensitive to adverse conditions, which are detected by response pathways to ensure correct protein folding. Calnexin is an ER transmembrane chaperone acting in both quality control of folding and response to persistent stress. Calnexin is a key protein required for viability in certain organisms such as mammals and the fission yeast Schizosaccharomyces pombe . Nevertheless, S. pombe calnexin-independent (Cin) cells were obtained after transient expression of a particular calnexin mutant. The Cin state is dominant, is stably propagated by an epigenetic mechanism and segregates in a non-Mendelian fashion to the meiotic progeny. The nucleolar protein Cif1p was identified as an inducer of the Cin state in a previous genetic screen. Here, we report the identification of novel inducers isolated in an overexpression genetic screen: pyruvate kinase (Pyk1p) and phosphoglycerate kinase (Pgk1p). Addition of pyruvate, the end product of pyruvate kinase and glycolysis, also induced calnexin independence in a dose-dependent manner. Remarkably, growth in respiration media or cold temperatures induced the appearance of Cin cells at high frequencies. Taken together, our results indicate that the Cin state can be triggered by extracellular changes, suggesting that this state represents an epigenetic adaptative response to environmental modifications.     

Melatonin and porphyrin in the harderian glands of the Syrian hamster: circadian patterns and response to autumnal conditions

1. Adult male Syrian hamsters were killed at nine intervals during a 24 hr period in the autumn, after 2 months either indoors in controlled conditions or in natural outdoor conditions. 2. Harderian glands were taken for determination of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities and melatonin and porphyrin concentrations. 3. Mean 24 hr Harderian NAT and melatonin values were lower outside than inside. 4. Twenty-four hour melatonin rhythms were detected with similar daytime (afternoon) acrophases in both environmental conditions. 5. An NAT rhythm was seen only in animals kept inside, with a circadian maximum in the late dark phase. 6. Mean 24 hr HIOMT activity was slightly higher outdoors than indoors, and 24 hr rhythms were not detected in either condition. 7. Mean porphyrin concentrations were higher outdoors, with 24 hr rhythms detected in both conditions and a significantly earlier nocturnal circadian maximum outdoors.     

Foraminiferal response to Holocene environmental changes of a tidal estuary in Victoria, southeastern Australia

The Holocene strata in the Anderson Inlet area in Victoria can be stratigraphically divided into four units, Unit I, Unit II, Unit III, and Unit IV. Unit I and Unit IV lack fossils and were deposited in non-marine, probably fluvial environments. Unit II and Unit III contain abundant foraminifera with molluscs, ostracods and bryozoans. Foraminiferal analysis suggests that Unit III was formed in a partially sheltered marine environment, while the high plankton content and relatively high diversity of benthic species in Unit II indicate that this unit was deposited in an open bay at water depths possibly less than 5 m. The foraminiferal data are integrated with radiocarbon dates to arrive at the following Holocene palaeoenvironmental history in this area: (1) low alluvial plain stage (10,000–7000 yr B.P.); (2) open bay environment stage (7000–5500 yr B.P.); (3) partially sheltered marine environment stage (5500–4500 yr B.P.); (4) alluvial plain and coastal lagoon environment stage (since about 4500 yr B.P.). The foraminiferal fauna show a clear response to these palaeoenvironmental changes. Globigerina bulloides can be used as an indicator for cold water marine environments. The high concentration of this species in these middle Holocene sediments shows a strong cold water influence on the coastal environments which reduced the effect of regional warm currents during this period. The Holocene palaeoenvironmental changes in the area were controlled by the Holocene sea-level fluctuations associated with the deglaciation history during this period. Similar integrated studies of shallow to marginal marine strata in southern Africa, America and New Zealand will lead to a better understanding of Holocene relative sea-level change and the interplay between Holocene cold and warm water regimes in the Southern Hemisphere.     

Evidence for age-related changes in the circadian activity rhythm of the diurnal primate Callithrix jacchus: a case report

Many cross-sectional studies have shown that circadian rhythms change with age, but such age-related modifications are gradual and may be insufficiently described by cross-sectional studies. In this case study, circadian activity rhythm (CAR) was evaluated longitudinally, in both LD (12:12) and LL conditions, on two occasions in a single male marmoset: when ‘adult’ (3 y.o.) and when ‘old’ (9 y.o.). When adult, the CAR synchronized with positive phase angles for the onset and offset of activity. In LL, the rhythm free-ran with τ < 24 h. When old, the animal showed a significant phase delay of its activity rhythm with respect to the LD cycle (t-test, p < 0.01) and a reduction on total daily activity (t-test, p < 0.01), with signs of lesser stability, greater fragmentation and some loss of photic synchronization. In LL, the period free-ran with τ > 24 h. We conclude that aging is associated with attenuation of photic synchronization and expression of CAR in LL in the marmoset. Further studies with a larger number of individuals are needed to confirm these findings.