CIRCADIAN CONTROL OF MIGRATORY RESTLESSNESS AND THE EFFECTS OF EXOGENOUS MELATONIN IN THE BRAMBLING,FRINGILLA MONTIFRINGILLA

Circadian pacemakers control both “daytime” activity and nocturnal restlessness of migratory birds, and the daily rhythm of melatonin release from the pineal has been suggested to be involved in the control of migratory activity. To study the phase relations between the two activity components during entrainment and when free running, locomotor activity of bramblings (Fringilla montifringilla) was recorded continuously under a 12:12 “cool light” to “warm light” cycle (CL:WL, ca. 5000 K and ca. 2500 K, respectively) or blue light to red light cycle (BL:RL, maxima at 440 and 650 nm, respectively) at different irradiance ratios. Migratory activity was expressed primarily during the WL or RL phase of the light cycles. Under free-running conditions, the circadian periods τ correlated with the phase relations between day and night (migratory) activity components during preceding entrainment. Bramblings with migratory activity had significantly longer τ at constant light intensity than the same individuals without migratory activity. Birds with migratory activity reentrained faster after a 6h phase shift of the CL:WL cycle than birds without migratory activity. When exogenous melatonin was given in the drinking water (200 μg/mL 1% ethanol or 0.86 mM) to bramblings exposed to 12:12 CL:WL cycles with constant irradiance, the amounts of activity, which were initially higher during the WL phase of the light cycle, were suppressed to similar low levels during both light phases. The systematic changes in the amounts of activity during melatonin treatment were not correlated with consistent changes in entrainment status. The data support the hypothesis that changes in the amplitude and level of the daily melatonin cycle are involved in regulating migratory restlessness, by either allowing or inhibiting nocturnal activity. (Chronobiology International, 17(4), 471–488, 2000)     

Timely administration of melatonin accelerates reentrainment to phase-shifted light-dark cycles in the field mouse Mus booduga

The effect of melatonin on the rate of reentrainment after a 6h phase delay and a 6h phase advance in the light-dark (LD) cycle was assayed in the nocturnal field mouse Mus booduga. After a phase delay of 6h in the LD cycle, a single dose of melatonin (1 mg/kg) was administered for three consecutive days at about CT4 (circadian time 4). After a phase advance of 6h in the LD cycle, melatonin was administered for three consecutive days at about CT22. Melatonin was found to accelerate reentrainment in both cases. Melatonin-treated animals took significantly fewer cycles to reentrain compared to vehicle-treated (50% dimethylsulfoxide [DMSO]) and nontreated control animals.     

Circadian rhythms of corneal mitotic rate,retinal melatonin and immunoreactive visual pigments,and the effects of melatonin on the rhythms in the Japanese quail

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 1212), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 1212. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 1212, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 1212. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 1212, but did not induce a rhythm under LL. The ratio of photoreceptor cells with positive staining to anti-visual pigment antibodies at the Golgi region was not affected by melatonin injection.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - DD constant darkness - Io-mAb monoclonal antibodies against chicken iodospin - LD light-dark - LL constant light - mRNA messenger ribonucleic acid - PBS phosphate buffer solution - Rh-As antiserum against bovine rhodopsin - SCN suprachiasmatic nucleus - T transducin - T transducin      

Zeitgeber Hierarchy in Humans: Resetting the Circadian Phase Positions of Blind People Using Melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free‐running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.     

Zeitgeber hierarchy in humans: resetting the circadian phase positions of blind people using melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free-running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.     

Chronobiotic effects of the melatonin agonist LY 156735 following a simulated 9h time shift: results of a placebo-controlled trial

Introduction: The melatonin agonist LY 156735 (LY) is a new investigational drug under development to treat circadian rhythm disorders. The present study assessed the efficacy of LY to alleviate the symptoms of shift lag and to enhance readaptation of desynchronized circadian rhythms to a new time zone.

Subjects and methods: Eight healthy male volunteers of age 25-35 yr participated in three identical trials of 13d duration in a temporal isolation unit separated by washout intervals. A high dose (HD) of 5 mg and a low dose (LD) of 0.5 mg of LY and placebo (PL) were administered double-blinded in a three-period cross-over design. Each trial consisted of an adaptation period, a pre-shift period for baseline measurements, a simulated 9h phase-advance shift, and a post-shift period for follow-up. The time shift was performed at 23:00h of day 6 by advancing the laboratory time to 08:00h of day 7. Double-blind study medication was administered at 14:30h on day 6, and at 22:30h on days 7-10. Subjective ratings of jet lag, alertness, tenseness, and daytime fatigue were assessed using visual analog scales (VAS) and standardized questionnaires. The objective markers of readaptation included core body temperature, wrist actigraphy, cortisol and electrolyte excretion, and a battery of computerized performance tests.

Results: HD but not LD enhanced the readaptation speed of all physiological rhythms investigated, as demonstrated by a significantly faster movement of acrophases towards the post-shift target time. HD (p=0.05) significantly blunted the post-shift deterioration of performance in those tests that were sensitive to shift lag. Parameters of subjective well-being were not significantly affected by either dose.

Conclusion: This pilot study demonstrates the chronobiotic efficacy of LY when taken at a dose of 5 mg/d.     

Circadian rhythms of oxidative phosphorylation: effects of rotenone and melatonin on isolated rat brain mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Circadian rhythms of oviposition and feeding activity in Japanese quail: effects of cyclic administration of melatonin

The aim of these experiments was to test the effect of a cyclic administration of melatonin, by mimicking the daily rhythm of hormone levels, on the circadian organization of two distinct functions in quail: oviposition and feeding activity. Laying and feeding rhythms under photoperiodic conditions and constant darkness (DD) were investigated. Under DD, where the two rhythms were free running, a daily rhythm of melatonin was administered. In LD 14h:10h, two different individual profiles of laying were established, with stable females laying at the same time each day and delayed females laying progressively later each day. For feeding activity, all birds were clearly synchronized to the photoperiodic cycle. In DD, the laying birds showed a free-running rhythm of oviposition with a period longer than 24 h for both profiles but the delayed profile females had a longer period than stable profile females. In comparison, the free-running period of feeding rhythm of the same birds was shorter than 24 h. A cyclic administration of melatonin had no effect on laying rhythm, which continued to free-run in DD, whereas feeding activity was synchronized as soon as the first cycle of melatonin was administered. From these results, it seems that two different circadian systems drive each of the two types of behavior separately. Melatonin could be the main synchronizer for the temporal control of feeding behavior, but it does not play a part in the control of oviposition in Japanese quail.     

Cyclic AMP Resets the Circadian Clock in Cultured Xenopus Retinal Photoreceptor Layers

Abstract: The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180° out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

Increased photic sensitivity for phase resetting but not melatonin suppression in Siberian hamsters under short photoperiods

Light regulates a variety of behavioral and physiological processes, including activity rhythms and hormone secretory patterns. Seasonal changes in the proportion of light in a day (photoperiod) further modulate those functions. Recently, short (SP) versus long days (LP) were found to markedly increase light sensitivity for phase shifting in Syrian hamsters. To our knowledge, photoperiod effects on light sensitivity have not been studied in other rodents, nor is it known if they generalize to other circadian responses. We tested whether photic phase shifting and melatonin suppression vary in Siberian hamsters maintained under LP or SP. Select irradiances of light were administered, and shifts in activity were determined. Photic sensitivity for melatonin suppression was examined in a separate group of animals via pulses of light across a 4 log-unit photon density range, with post-pulse plasma melatonin levels determined via RIA. Phase shifting and melatonin suppression were greater at higher irradiances for both LP and SP. The lower irradiance condition was below threshold for phase shifts in LP but not SP. Melatonin suppression did not vary by photoperiod, and the half saturation constant for fitted sigmoid curves was similar under LP and SP. Thus, the photoperiodic modulation of light sensitivity for phase shifting is conserved across two hamster genera. The dissociation of photoperiod effects on photic phase shifting and melatonin suppression suggests that the modulation of sensitivity occurs downstream of the common retinal input pathway. Understanding the mechanistic basis for this plasticity may yield therapeutic targets for optimizing light therapy practices.     

Effects of twilights on circadian entrainment patterns and reentrainment rates in squirrel monkeys

Entrainment patterns of the circadian rhythms of body temperature and locomotor activity were compared in 6 squirrel monkeys (Saimiri sciureus) exposed to daily illumination cycles with abrupt transitions between light and darkness (LD-rectangular) or with gradual dawn and dusk transitions simulating natural twilights at the equator (LD-twilight). Daytime light intensity was 500 lux, and the total amount of light emitted per day was the same in the two conditions. Mean daytime body temperature levels were stable in LD-rectangular but increased gradually in LD-twilight, reaching peak levels during the dusk twilight. Locomotor activity showed a similar pattern, but with an additional, secondary peak near the end of dawn. Activity duration was about 0.5 h longer in LD-twilight than in LD-rectangular, but the time of activity midpoint was similar in the two conditions. Reentrainment of the body temperature rhythm was faster following an 8-h advance of the LD cycle than following an 8-h delay, but did not differ significantly between the two LD conditions. These results provide no evidence that the inclusion of twilight transitions affected the strength of the LD Zeitgeber, and suggest that the observed differences in the daily patterns reflected direct effects of light intensity on locomotor activity and body temperature rather than an effect of twilights on circadian entrainment mechanisms.Abbreviation LD light-dark     

Food availability but not melatonin affects nocturnal restlessness in a wild migrating passerine

A large number of passerine species migrate at night, although most of them are diurnal outside the migratory seasons. This diurnal-to-nocturnal transition is a major life-history event, yet little is known about its physiological control. Previous work showed that during the migratory periods captive birds showing nocturnal migratory restlessness (Zugunruhe) have reduced concentrations of circulating melatonin at night compared to non-migratory periods. This suggested that the hormone melatonin, a main component of the avian circadian system, is involved in the expression of Zugunruhe. Other studies demonstrated that the relationship between low melatonin levels and Zugunruhe is not a seasonal correlation. When Zugunruhe was interrupted by exposing birds to a fasting-and-refeeding protocol, melatonin levels increased. Here we studied whether melatonin and food availability influence the intensity of Zugunruhe in wild migrating garden warblers (Sylvia borin) at a stopover site. Birds were held in recording cages overnight, with or without food available, and either bled to determine melatonin concentrations or treated transdermally with melatonin. We found that melatonin levels at night were correlated with the intensity of diurnal locomotor activity and with condition, but were not correlated with Zugunruhe. Similarly, the melatonin treatment did not have effects on Zugunruhe, whereas food availability increased it. Our study shows that the nocturnal melatonin levels in migrating warblers depend on food availability and are correlated with condition. In addition, it suggests that melatonin does not control Zugunruhe and might rather be involved in energy conservation and/or clock synchronization during migration.     

Thermoregulatory effects of melatonin in relation to sleepiness

Thermoregulatory processes have long been implicated in the initiation of human sleep. In this paper, we review our own studies conducted over the last decade showing a crucial role for melatonin as a mediator between the thermoregulatory and arousal system in humans. Distal heat loss, via increased skin temperature, seems to be intimately coupled with increased sleepiness and sleep induction. Exogenous melatonin administration during the day when melatonin is essentially absent mimics the endogenous thermophysiological processes occurring in the evening and induces sleepiness. Using a cold thermic challenge test, it was shown that melatonin‐induced sleepiness occurs in parallel with reduction in the thermoregulatory set‐point (threshold); thus, melatonin may act as a circadian modulator of the thermoregulatory set‐point. In addition, an orthostatic challenge can partially block the melatonin‐induced effects, suggesting an important role of the sympathetic nervous system as a link between the thermoregulatory and arousal systems. A topographical analysis of finger skin temperature with infrared thermometry revealed that the most distal parts of the fingers, i.e., fingertips, represent the important skin regions for heat loss regulation, most probably via opening the arteriovenous anastomoses, and this is clearly potentiated by melatonin. Taken together, melatonin is involved in the fine‐tuning of vascular tone in selective vascular beds, as circulating melatonin levels rise and fall throughout the night. Besides the role of melatonin as “nature's soporific”, it can also serve as nature's nocturnal vascular modulator.     

Daily and circadian variation in the electroretinogram of the domestic fowl: effects of melatonin

Visual and circadian function are integrally related in birds, but the precise nature of their interaction is unknown. The present study determined whether visual sensitivity measured electroretinographically (ERG) in 7-week-old cockerels varies over the time of day, whether this rhythm persists in constant darkness (DD) and whether exogenous melatonin affects this ERG rhythmicity. ERG b-wave amplitude was rhythmic in LD and persisted in DD with peak amplitude during mid- to late afternoon in LD and mid-subjective day in DD, indicating that the ERG rhythm is endogenously generated. No daily or circadian variation in a-wave amplitude was observed, and ERG component latency and durations were not rhythmic. Intramuscular injection of 10 g/kg melatonin at ZT10 in LD significantly decreased b-wave amplitude but had no effect on a-wave. Intraocular injection of 600 pg melatonin, however, had no effect on any aspect of the ERG. These data indicate that a circadian clock regulates ocular sensitivity to light and that melatonin may mediate some or all of this effect. The level at which melatonin modulates retinal sensitivity is not known, but the present data suggest a central site rather than a direct effect of the hormone in the eye.Abbreviations DD constant darkness - ERG electroretinography - EW Edinger-Westphal nuclei - IMEL iodomelatonin - IO isthmooptic nucleus - LD light-dark cycle - SCG superior cervical ganglion - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

Stable individual profiles of daily timing of migratory restlessness in European quail

Temporal characteristics of migratory behavior in birds are usually studied at the species and population levels, and rarely at the individual level. Variations among species and populations of the seasonal onset of migratory behavior have been widely investigated, but very little is known about its daily organization or whether birds are conservative in their behavior. The determination of intra- and inter-individual variability is important for the study of genetic variations and can reveal the existence of different adaptation capacities within populations. This laboratory study analyzed intra- and inter-individual variability of daily initiation and time course of nocturnal restlessness in partial-migrant European quail (Coturnix coturnix coturnix). Thirty-five quail were selected randomly from a captive stock, and their spring activity was recorded under natural daylenghs. Eighteen of the thirty-five quail presented behavioral profiles of migrant birds. Migrant birds initiated their nocturnal activity punctually, and the time courses of the nocturnal activity of 88% of them revealed intra-individual stability over six consecutive nights. All birds initiated their nocturnal activity after sunset and civil twilight, and they were more active at the beginning than the middle or end of the night, suggesting that their drive to migrate could be synchronized with particular skylight conditions. For the first time, stable individual profiles in the daily time course of migratory restlessness are shown. These results support previous findings concerning biological rhythms of quail and raise questions concerning the timing of migratory behavior.     

Size and accumulation of fuel reserves at stopover predict nocturnal restlessness in a migratory bird

Early arrival at the breeding site positively affects the breeding success of migratory birds. During migration, birds spend most of their time at stopovers. Therefore, determining which factors shape stopover duration is essential to our understanding of avian migration. Because the main purpose of stopover is to accumulate fat as fuel for the next flight bout, fuel reserves at arrival and the accumulation of fuel are both expected to affect stopover departure decisions. Here, we determined whether fuel reserves and fuel accumulation predict a bird''s motivation to depart, as quantified by nocturnal migratory restlessness (Zugunruhe), using northern wheatears (Oenanthe oenanthe) that were captured and temporarily contained at spring stopover. We found that fuel reserves at capture were positively correlated with Zugunruhe, and negatively correlated with fuel accumulation. This indicates that fat birds were motivated to depart, whereas lean birds were set on staying and accumulating fuel. Moreover, the change in fuel reserves was positively correlated with the concurrent change in Zugunruhe, providing the first empirical evidence for a direct link between fuel accumulation and Zugunruhe during stopover. Our study indicates that, together with innate rhythms and weather, the size and accumulation of fuel reserves shape stopover duration, and hence overall migration time.     

Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

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 daytime 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. Accepted: 10 December 1999