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.     

Isolation and analysis of six timeless alleles that cause short- or long-period circadian rhythms in Drosophila

In genetic screens for Drosophila mutations affecting circadian locomotion rhythms, we have isolated six new alleles of the timeless (tim) gene. Two of these mutations cause short-period rhythms of 21-22 hr in constant darkness, and four result in long-period cycles of 26-28 hr. All alleles are semidominant. Studies of the genetic interactions of some of the tim alleles with period-altering period (per) mutations indicate that these interactions are close to multiplicative; a given allele changes the period length of the genetic background by a fixed percentage, rather than by a fixed number of hours. The tim(L1) allele was studied in molecular detail. The long behavioral period of tim(L1) is reflected in a lengthened molecular oscillation of per and tim RNA and protein levels. The lengthened period is partly caused by delayed nuclear translocation of TIM(L1) protein, shown directly by immunocytochemistry and indirectly by an analysis of the phase response curve of tim(L1) flies.     

A forced desynchrony study of circadian pacemaker characteristics in seasonal affective disorder

The circadian pacemaker is an endogenous clock that regulates oscillations in most physiological and psychological processes with a near 24-h period. In many species, this pacemaker triggers seasonal changes in behavior. The seasonality of symptoms and the efficacy of light therapy suggest involvement of the circadian pacemaker in seasonal affective disorder (SAD), winter type. In this study, circadian pacemaker characteristics of SAD patients were compared with those of controls. Seven SAD patients and matched controls were subjected to a 120-h forced desynchrony protocol, in which core body temperature and melatonin secretion profiles were measured for the characterization of circadian pacemaker parameters. During this protocol, which enables the study of unmasked circadian pacemaker characteristics, subjects were exposed to six 20-h days in time isolation. Patients participated twice in winter (while depressed and while remitted after light therapy) and once in summer. Controls participated once in winter and once in summer. Between the SAD patients and controls, no significant differences were observed in the melatonin-derived period or in the phase of the endogenous circadian temperature rhythm. The amplitude of this rhythm was significantly smaller in depressed and remitted SAD patients than in controls. No abnormalities of the circadian pacemaker were observed in SAD patients. A disturbance in thermoregulatory processes might explain the smaller circadian temperature amplitude in SAD patients during winter.     

ldpA encodes an iron-sulfur protein involved in light-dependent modulation of the circadian period in the cyanobacterium Synechococcus elongatus PCC 7942

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.     

The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators

ABSTRACT. The behaviour of the circadian locomotor rhythm of the New Zealand weta, Hemideina thoracica (White), supports the model that the underlying pacemaker consists of a population of weakly coupled oscillators. Certain patterns of locomotor activity, previously demonstrated almost exclusively in vertebrates, are presented here as evidence for the above hypothesis. They include after-effects of various pre-treatments, rhythm-splitting and spontaneous changes in the rhythm. After-effects, which describe the unstable behaviour of free-running circadian rhythms following particular experimental perturbations, have been observed in Hemideina following single light pulses, constant dim light, and laboratory and natural entrainment. Period changes occurred in the activity rhythm after single light pulses of 8-h and 12-h duration (25 lx). Constant dim light (0.1 lx) increased the free-running period (τ) of the activity rhythm, but the after-effect of constant dim light was either an increase or a decrease in τ. After-effects upon both τ and the active phase length of the activity rhythm were found following non-24-h light entrainment cycles with 8-h and 12-h light phases of 25 lx. Qualitative measurements of these after-effects upon τ are presented which reveal a relationship between both the direction and amount of change in τ, and the difference between entrainment cycle length (T) and pre-entrainment free-running period. The after-effect of natural entrainment was an initial short-period free-run (τ < 24h) lasting 5–10 days, generally followed by a rapid period lengthening to τ= 25–26 h. Support for the population model was provided by spontaneous dampening, recovery, and period changes of the rhythm, together with the disruption of the active phase following critical light perturbations, and rhythm-splitting. These Hemideina results are compared with the simulations of the Coupled Stochastic System of Enright (1980).     

Adult emergence rhythm of the egg-parasitoid Telenomus busseolae

The adult emergence rhythm of Telenomus busseolae, an egg parasitoid of Sesamia nonagrioides, was examined when parasitoids were exposed to different light-dark regimes. Most of the adult parasitoids emerged throughout the whole period of the photoperiodic cycle. Peak male emergence occurred 2–5 hours earlier than that of females. Adult emergence was asynchronous in continuous darkness or light. However, regimes of alternative light and dark phases such as L4:D20, L8:D16, L12:D12, L16:D8 and L20:D4 h generated a population rhythm with a period length of 24 hours. The peak of the emergence activity moves from the scotophase to the middle of the photophase with an increase of the photophase from 4 to 20 h. Rhythmical activity of adults was synchronised within 2 cycles when immature stages of parasitoid grow under continuous light conditions (LL) and then transferred to L12:D12. Moreover, emergence rhythm persisted and continued in a free-run with a period length of less than 24 hours by transferring a rhythmic culture from L12:D12 h to LL or RR (continuous red light) conditions, indicating the existence of a circadian rhythm. The ecological implications of the expression rhythm relate to better survival of the parasitoids.     

Integration of human sleep-wake regulation and circadian rhythmicity.

The human sleep-wake cycle is generated by a circadian process, originating from the suprachiasmatic nuclei, in interaction with a separate oscillatory process: the sleep homeostat. The sleep-wake cycle is normally timed to occur at a specific phase relative to the external cycle of light-dark exposure. It is also timed at a specific phase relative to internal circadian rhythms, such as the pineal melatonin rhythm, the circadian sleep-wake propensity rhythm, and the rhythm of responsiveness of the circadian pacemaker to light. Variations in these internal and external phase relationships, such as those that occur in blindness, aging, morning and evening, and advanced and delayed sleep-phase syndrome, lead to sleep disruptions and complaints. Changes in ocular circadian photoreception, interindividual variation in the near-24-h intrinsic period of the circadian pacemaker, and sleep homeostasis can contribute to variations in external and internal phase. Recent findings on the physiological and molecular-genetic correlates of circadian sleep disorders suggest that the timing of the sleep-wake cycle and circadian rhythms is closely integrated but is, in part, regulated differentially.     

Adult Emergence Rhythm of Fruit Flies Drosophila melanogaster under Seminatural Conditions

In insects, the role of circadian clocks in the temporal regulation of adult emergence rhythm under natural conditions has not previously been reported. Here we present the results of a study aimed at examining the time course and waveform of emergence rhythm in the fruit fly Drosophila melanogaster under seminatural condition (SN). We studied this rhythm in wild-type and clock mutant flies under SN in parallel with laboratory condition (LAB) to examine (1) how the rhythm differs between SN and LAB, (2) what roles the circadian clock protein PERIOD and the circadian photoreceptor CRYPTOCHROME (CRY) play in the regulation of emergence rhythm under SN, and (3) whether there is seasonality in the rhythm. Under SN, wild-type flies displayed tightly gated emergence, peaking at "dawn" and gradually tapering down toward the evening, with little or no emergence by night, while in LAB, flies emerged throughout the light phase of light-dark (LD) cycles. The period loss-of-function mutant (per ( 0 )) flies were arrhythmic in LAB but displayed weak rhythmic emergence under SN. Under SN, cry mutants displayed less robust rhythm with wider gates, greater variance in peak timing, and enhanced nighttime emergence compared to controls. Furthermore, flies showed seasonal variation in emergence rhythm, coupled either to light or to humidity/temperature depending on the severity of environmental conditions. These results suggest that adult emergence rhythm of Drosophila is more robust in nature, is coupled to environmental cycles, and shows seasonal variations.     

Effects of the duper mutation on circadian responses to light

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.     

Mutational Analysis of the Drosophila Miniature-Dusky (M-Dy) Locus: Effects on Cell Size and Circadian Rhythms

A mutational analysis has been performed to explore the function of the Drosophila melanogaster miniature-dusky (m-dy) locus. Mutations at this locus affect wing development, fertility and behavior. The genetic characterization of 13 different mutations suggests that m and dy variants are alleles of a single complex gene. All of these mutations alter wing size, apparently by reducing the volume of individual epidermal cells of the developing wing. In m mutants, epidermal cell boundaries persist in the mature wing, whereas they normally degenerate 1-2 hr after eclosion in wild-type or dy flies. This has permitted the direct visualization of cell size differences among several m mutants. Mutations at the m-dy locus also affect behavioral processes. Three out of nine dy alleles (dyn1, dyn3 and dyn4) lengthen the circadian period of the activity and eclosion rhythms by approximately 1.5 hr. In contrast, m mutants have normal circadian periods, but an abnormally large percentage of individuals express aperiodic bouts of activity. These behavior genetic studies also indicate that an existing "rhythm" mutation known as Andante is an allele of the m-dy locus. The differential effects of certain m-dy mutations on wing and behavioral phenotypes suggest that separable domains of function exist within this locus.     

Phase Resetting of the Human Circadian Pacemaker with Use of a Single Pulse of Bright Light

Since the initial studies reporting that light can alter the phase position of the human circadian system, there has been increasing interest in the use of bright light as a tool for manipulating the phase position of the circadian pacemaker. Exposure protocols typically require subjects to receive 2–5 h of exposure over several circadian cycles. As a consequence, bright light treatment can involve a considerable time investment. However, recent studies indicate that a single pulse of bright light can produce significant phase shifts in the circadian pacemaker. If a single pulse of bright light can produce significant phase-shifting effects, multiple-pulse designs may be unnecessary. This study examined the phase-shifting effects of a single 4-h pulse of bright light (12,000 lux) in 14 male and one female subject aged between 19–45 years. With use of a “constant routine” to estimate circadian phase, a single 4-h pulse of light produced significant shifts in the phase of the core temperature rhythm. The timing of the exposure, relative to the core temperature rhythm, determined the degree and direction of the phase shift. Exposure immediately prior to habitual bedtime produced a mean phase delay in the core temperature of 2.39 h (SD = 1.37 h). In contrast, exposure immediately following habitual wake-up produced a mean phase advance of 1.49 h (SD = 2.06 h). In addition, the magnitude of the shift increased the closer the light pulse was to the individual's estimated endogenous core temperature minimum. There was, however, considerable interindividual variability in this relationship. Overall, these results confirm that a single pulse of bright light can produce significant phase shifts in the phase of the circadian pacemaker controlling core temperature.     

Phase Resetting of the Human Circadian Pacemaker with Use of a Single Pulse of Bright Light

Since the initial studies reporting that light can alter the phase position of the human circadian system, there has been increasing interest in the use of bright light as a tool for manipulating the phase position of the circadian pacemaker. Exposure protocols typically require subjects to receive 2-5 h of exposure over several circadian cycles. As a consequence, bright light treatment can involve a considerable time investment. However, recent studies indicate that a single pulse of bright light can produce significant phase shifts in the circadian pacemaker. If a single pulse of bright light can produce significant phase-shifting effects, multiple-pulse designs may be unnecessary. This study examined the phase-shifting effects of a single 4-h pulse of bright light (12,000 lux) in 14 male and one female subject aged between 19-45 years. With use of a “constant routine” to estimate circadian phase, a single 4-h pulse of light produced significant shifts in the phase of the core temperature rhythm. The timing of the exposure, relative to the core temperature rhythm, determined the degree and direction of the phase shift. Exposure immediately prior to habitual bedtime produced a mean phase delay in the core temperature of 2.39 h (SD = 1.37 h). In contrast, exposure immediately following habitual wake-up produced a mean phase advance of 1.49 h (SD = 2.06 h). In addition, the magnitude of the shift increased the closer the light pulse was to the individual's estimated endogenous core temperature minimum. There was, however, considerable interindividual variability in this relationship. Overall, these results confirm that a single pulse of bright light can produce significant phase shifts in the phase of the circadian pacemaker controlling core temperature. Key Words: Bright light—Circadian rhythm—Core body temperature—Sleep-wake disorders—Chronobiology.     

Die Steuerung einer gezeitenparallelen Schlüpfrhythmik nach dem Sanduhr-Prinzip

Summary The arctic population of the intertidal midge Clunio marinus (location: Tromsö, Norway) shows a tidal rhythm of emergence (period: 12.4 hours) in midsummer. The emergence time exactly coincides with the initial exposure of the habitat during the ebb.When the animals were bred in a 24 hour light-dark cycle involving 16 hours of light, emergence occurred mainly 10–11 hours after light-on. When the animals were subsequently placed under constant light, no rhythm could be detected. When constant light was interrupted by a single period of 6 hours of darkness, only one peak of emergence was evoked, again 10–11 hours after light-on. Similar results were observed when in constant darkness a single period of 6 hours of lower temperature was offered.The mechanism of control differs from that of a southern population (location: St. Jean-de-Luz, France) which has a circadian clock mechanism and shows a free-running rhythm of emergence in constant light. It is postulated that the tidal rhythm of the Tromsö population is controlled by an hour-glass mechanism which starts its run of at least 10–11 hours during the preceding ebb.     

A phase dynamics model of human circadian rhythms

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.     

Locomotor Activity Rhythm in the Field Mouse Mus booduga Phase-shifts to Melatonin Injections in a Dose-dependent Manner

Melatonin is known to shift the phase of the locomotor activity rhythm in the field mouse Mus booduga in accordance with a type-I phase response curve (PRC), with phase delays during the subjective day and phase advances during late subjective night and the early subjective day. At CT4 (circadian time 4; i.e. 16 hr. after activity onset) and CT22 of the circadian cycle, a single dose of melatonin (1 mg/kg) is known to evoke maximum delay and maximum advance phase-shifts, respectively. We investigated the dose-dependent responses of the circadian pacemaker of these mice to a single dose of melatonin at the times for maximum delay and maximum advance. The circadian pacemaker responsible for the locomotor activity rhythm in these mice responded to various doses of melatonin in a dose-dependent manner with the magnitude of phase shifts increasing with dose.     

sn-1,2-diacylglycerol levels in the fungus Neurospora crassa display circadian rhythmicity

The fungus Neurospora crassa is a model organism for investigating the biochemical mechanism of circadian (daily) rhythmicity. When a choline-requiring strain (chol-1) is depleted of choline, the period of the conidiation rhythm lengthens. We have found that the levels of sn-1,2-diacylglycerol (DAG) increase in proportion to the increase in period. Other clock mutations that change the period do not affect the levels of DAG. Membrane-permeant DAGs and inhibitors of DAG kinase were found to further lengthen the period of choline-depleted cultures. The level of DAG oscillates with a period comparable to the rhythm of conidiation in wild-type strains, choline-depleted cultures, and frq mutants, including a null frq strain. The DAG rhythm is present at the growing margin and also persists in older areas that have completed development. The phase of the DAG rhythm can be set by the light-to-dark transition, but the level of DAG is not immediately affected by light. Our results indicate that rhythms in DAG levels in Neurospora are driven by a light-sensitive circadian oscillator that does not require the frq gene product. High levels of DAG may feed back on that oscillator to lengthen its period.     

Locomotor Activity Rhythm in the Field Mouse Mus booduga Phase-shifts to Melatonin Injections in a Dose-dependent Manner

Melatonin is known to shift the phase of the locomotor activity rhythm in the field mouse Mus booduga in accordance with a type-I phase response curve (PRC), with phase delays during the subjective day and phase advances during late subjective night and the early subjective day. At CT4 (circadian time 4; i.e. 16 hr. after activity onset) and CT22 of the circadian cycle, a single dose of melatonin (1 mg/kg) is known to evoke maximum delay and maximum advance phase-shifts, respectively. We investigated the dose-dependent responses of the circadian pacemaker of these mice to a single dose of melatonin at the times for maximum delay and maximum advance. The circadian pacemaker responsible for the locomotor activity rhythm in these mice responded to various doses of melatonin in a dose-dependent manner with the magnitude of phase shifts increasing with dose.     

Effect of light during lactation on the phasic and tonic responses of the rat pacemaker

The circadian system in mammals generates endogenous circadian rhythms and entrains them to external cycles. Here, we examine whether the lighting conditions under which rats are reared affect the properties of the circadian pacemaker. We maintained three groups of rats under constant darkness (DD-rats), constant bright light (LL-rats) or light-dark cycles of 24 hours (LD-rats) during lactation. We then studied motor activity rhythm under constant light of four intensities, and under seven light-dark cycles with periods ranging between 22 and 27 hours. Results show that neither the tau nor the phase angle to the external cycle differed between groups. Differences were found in the amplitude of the circadian rhythm and in the number of rats that became arrhythmic under LL. We conclude that the light received during lactation affects the strength of the circadian pacemaker and its sensitivity to light.     

Probing the circadian oscillator of a mammal by two-pulse perturbations

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129-136, 2000)