Some Perturbations That Disturb the Circadian Melatonin Rhythm

The circadian melatonin rhythm is highly reproducible and generally not easily altered. The few perturbations that are capable of significantly changing either the amplitude or the pattern of the 24-h melatonin rhythm are summarized herein. Aging alters cyclic melatonin production by decreasing the amplitude of the nocturnal melatonin peak in all species in which it has been studied. The best known acute suppressor of nocturnal melatonin is light exposure. The brightness of light required to acutely depress pineal melatonin production is species dependent; of the visible wavelengths, those in the blue range (∼500-520 nm) seem most effective in suppressing melatonin production. Nonvisible, nonionizing radiation in the extremely low frequency range (e.g., 60 Hz) seems also capable of altering pineal melatonin synthesis. Hormones have relatively little influence on the circadian production of melatonin, although either adrenalectomy or hypo-physectomy does attenuate the amplitude of the melatonin cycle. Exercise at the time of high melatonin production rapidly depresses pineal concentrations of the indole without influencing its synthesis; the mechanism of this suppression remains unknown.     

Circadian rhythm in the number of granulated vesicles in the pinealocytes of mice

Summary Adult, Charles River CD-1, male mice were housed in an environmental control chamber under strict conditions of controlled light (12D/12L) and temperature. The mice were sacrificed at various times throughout the twenty-four hour clock and their pineals prepared routinely for electron microscopy. The number of dense-cored or granulated vesicles present in the polar terminals of pinealocytes were quantitated in thin cross sections through pericapillary areas. A distinct circadian rhythm was observed in the number of granulated vesicles with a three- to four-fold difference between late photoperiod maximum and late dark period minimum. The rhythm was abolished by bilateral superior cervical ganglionectomy. These results are consistent with the hypothesis that the granulated vesicles are synthesized and stored in the pinealocytic cytoplasm during the photoperiod under the tropic influence of norepinephrine, and are released during the dark period when melatonin synthesis is greatest. Melatonin, administered as daily intraperitoneal doses of 50 g over a period of five days, was observed to increase markedly the number of pinealocytic granulated vesicles during the light period, but led during the dark period to a decrease in their numbers to levels below that of diluent-treated controls. It may be that melatonin stimulates the synthesis and/or release of granulated vesicles which represent the packaged form of a major secretory product.Supported in part by N.I.H. Grant No. HD 08795     

高原鼠兔松果腺褪黑激素含量昼夜节律的研究

自然光照条件下,高原鼠兔（Ｏｃｈｏｔｏｎａｃｕｒｚｏｎｉａｅ）松果腺褪黑激素（Ｍｅｌａｔｏｎｉｎ,ＭＬＴ）含量呈现明显的昼夜节律（Ｐ＜０．００１,夜间组含量均值与白天组含量均值差异显著性比较）。在２月份的实验中,对１８只鼠兔（体重１２２—１６４克）松果腺的采样时间分别为０２．００,０９．００,１２．００,１８．００,２２．００和２４．００时。白天ＭＬＴ含量波动为５６—６４微微克／松果腺,夜间波动为１１３—１７０微微克／松果腺。夜间ＭＬＴ含量高峰值出现在２４．００时。在１０月份的实验中,对６０只鼠兔（体重１０２—１５３克）松果腺的采样时间分别为０３．００,０６．００,０９．００,１２．００,１５．００,１８．００,２１．００和２４．００时。白天ＭＬＴ含量波动为７７—１１９微微克／松果腺,夜间波动为１３９—５０５微微克／松果腺。夜间ＭＬＴ含量高峰值出现在０３．００时。将２月份和１０月份高原鼠兔松果腺ＭＬＴ含量进行差异显著性比较,１０月组显著高于２月组（Ｐ＜０．０５）。结果表明,该动物的松果腺本身对光周期具有敏感性,它能够感知环境光周期的变化,成功地完成神经内分泌的转换。     

鳜肝脏和心脏生物钟基因表达节律分析

在12h光照、12h黑暗交替(Light-Dark; LD)光制下,研究分析了褪黑素和皮质醇水平在鳜血清中的昼夜变化规律,以及13个生物钟基因(Arntl1、Clock、Cry1a、Cry3、Cry-dash、Npas2、Npas4、Nr1d1、Nr1d2、Per1、Per3、Rora和Tim)在鳜(Siniperca chuatsi)肝脏和心脏中的昼夜表达规律。试验在一昼夜内的ZT0(06:00)、ZT3(09:00),ZT6(12:00),ZT9(15:00),ZT12(18:00),ZT15(21:00),ZT18(24:00),ZT21(03:00,2nd d),ZT24(06:00,2nd d) (Zone time,ZT) 9个时间点随机抽取3尾鳜采集其血清、肝脏和心脏。经SPSS 单因素方差分析和Matlab余弦分析,结果显示: 鳜血清中褪黑素和皮质醇含量均呈现出昼夜节律性振荡,褪黑素含量白天显著降低(P0.05),夜间显著上升,皮质醇含量白天缓慢降低,夜间ZT15(21:00)-ZT18(24:00)显著升高,随后开始缓慢降低; 两种激素最低相位都为ZT15(21:00)。在13个生物钟基因中,Cry-dash、Npas4、Nr1d1、Per1和Tim 5个基因在鳜肝脏内具有明显的昼夜节律性,其中Npas4、Nr1d1、Per1、Tim的表达规律相似,皆呈现出光照阶段表达降低,黑暗阶段表达升高的趋势; 但Cry-dash则表现出光照阶段先升高后降低,黑暗阶段先降低后升高的规律。在鳜心脏中,Arntl1、Clock、Cry1a、Npas2、Nr1d1、Nr1d2、Per3、Rora和Tim 9个基因都表现出明显的昼夜节律,表达趋势分为两种: Arntl1、Clock、Nr1d2的表达量在光照阶段降低,黑暗阶段升高; 而Cry1a、Npas2、Nr1d1、Per3、Rora和Tim的表达量在ZT0(06:00)-ZT15(21:00)持续低水平,ZT15(21:00)-ZT18(24:00)表达量显著上升,ZT18(24:00)-ZT21(03:00)表达量降低。研究结果表明: 生物钟基因在鳜肝脏和心脏中所表达的昼夜节律不同。     

AGING AND THE CIRCADIAN RHYTHM OF MELATONIN: A CROSS-SECTIONAL STUDY OF CHINESE SUBJECTS 30–110 YR OF AGE

Although previous reports indicate that nocturnal plasma melatonin secretion declines with age, some recent findings do not support this point. In the present cross-sectional study, we documented serum melatonin concentrations at two time points, 02:00 and 08:00h, in 144 persons aged 30–110 yr and found a significant age-related decline. It began around the age of 60 and reached a very significantly lower level in subjects in their 70s and over 80 yr of age (P<0.01, when compared with age <60 yr). Nocturnal melatonin levels were higher among (post-menopausal only) women than men overall (P<0.05). In the older age-groups, nocturnal melatonin levels did not differ between healthy controls and subjects with high blood pressure or ischemic heart disease. To further check these results, we also assessed the circadian pattern of serum melatonin in four subgroups of healthy men, aged 30–39, 40–49, 50–59, and 60–69 yr: blood samples were taken at 2h intervals from 08:00 to 22:00h and hourly from 22:00 to 08:00h. Our results showed generally similar circadian melatonin patterns that peaked at night with very low levels during the daytime. No significant difference was found among the three younger groups, but nocturnal melatonin levels were significantly lower in the men in their 60s.     

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle

Summary The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied.In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed.Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only.Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.Abbreviations NAT N-acetyltransferase - LD cycle light-dark cycle - CT circadian time - LD xy light dark cycle comprising x h of light and y h of darkness     

动物内源褪黑素调控生物节律的研究进展

内源褪黑素对人类和其他哺乳动物的节律行为具有调控功能。生物节律是自然进化赋予生命的基本特征之一,生物体的生命活动受到生物节律的控制与影响。在哺乳动物中,节律调控中心是松果体,其主要功能是合成和分泌褪黑素。褪黑素广泛参与生物体节律行为的调节,本文从褪黑素的产生和作用机制,分别阐述褪黑素对昼夜节律行为和多种年节律行为的调控作用,同时明确褪黑素与生物钟及神经内分泌系统的直接作用和反馈互动的复杂集合,进一步揭示褪黑素调控生物节律的重要作用,以期为褪黑素的基础研究以及未来探究生物体的生物钟内源性发生机制提供参考。     

Book of Abstracts

A widespread occurrence of melatonin (MEL) in plant kingdom has been reported. MEL is a highly conserved molecule occurring in evolutionary distant organisms. Its role in plants seems to be similar to that in animals. Although MEL function in plants is not well known, yet a hypothesis can be put forward that it probably functions as a night signal, coordinating responses to diurnal and photoperiodic environmental cues. It has also been suggested that MEL is an independent plant growth regulator, probably its action is analogous to IAA and it may mediate the actions of other plant growth regulators. Due to its antioxidant properties MEL may also stabilize cell red-ox status and protect them against reactive oxygen species (ROS) and other harmful environmental influence.     

Melatonin modulates the ERG circadian rhythm in crayfish

One of the most important functions modulated by melatonin is the synchronization of circadian rhythms. In crayfish (Procambarus clarkii), we have obtained evidence that the amplitude of the electrical response to light of the retinal photoreceptors the receptor potential, is modified by the action of melatonin and that the magnitude of this action depends on the circadian time of melatonin application. In contrast, the electroretinogram (ERG) circadian rhythm can be synchronized by either single or periodic melatonin application. In this work we hypothesized that, in crayfish, melatonin acts on effectors and on pacemaker of ERG circadian rhythm as a non-photic synchronizer. Melatonin could be a hormone that sends a signal of darkness to the ERG circadian system.     

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.Abbreviations CT circadian time - LD light-dark - LL constant light - SCN suprachiasmatic nucleus - LLdim constant dim light - NE norepinephrine - SCG superior cervical ganglia - WB whole body - E eye - EX extraretina - C control     

Are G Protein‐Coupled Receptor Heterodimers of Physiological Relevance?—Focus on Melatonin Receptors

In mammals, the circadian hormone melatonin targets two seven‐transmembrane–spanning receptors, MT₁ and MT₂, of the G protein‐coupled receptor (GPCR) super‐family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT₁ and MT₂ homodimers and MT₁/MT₂ heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT₁/MT₂ heterodimers remains to be shown in native tissues but is suggested by the documented co‐expression of MT₁ and MT₂ in many melatonin‐sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.     

The Relationship Between Lifestyle Regularity and Subjective Sleep Quality

In previous work we have developed a diary instrument—the Social Rhythm Metric (SRM), which allows the assessment of lifestyle regularity—and a questionnaire instrument—the Pittsburgh Sleep Quality Index (PSQI), which allows the assessment of subjective sleep quality. The aim of the present study was to explore the relationship between lifestyle regularity and subjective sleep quality. Lifestyle regularity was assessed by both standard (SRM-17) and shortened (SRM-5) metrics; subjective sleep quality was assessed by the PSQI. We hypothesized that high lifestyle regularity would be conducive to better sleep. Both instruments were given to a sample of 100 healthy subjects who were studied as part of a variety of different experiments spanning a 9-yr time frame. Ages ranged from 19 to 49 yr (mean age: 31.2 yr, s.d.: 7.8 yr); there were 48 women and 52 men. SRM scores were derived from a two-week diary. The hypothesis was confirmed. There was a significant (rho=?0.4, p<0.001) correlation between SRM (both metrics) and PSQI, indicating that subjects with higher levels of lifestyle regularity reported fewer sleep problems. This relationship was also supported by a categorical analysis, where the proportion of “poor sleepers” was doubled in the “irregular types” group as compared with the “non-irregular types” group. Thus, there appears to be an association between lifestyle regularity and good sleep, though the direction of causality remains to be tested.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

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.     

LIGHT INTENSITY EXPOSURE,SLEEP DURATION,PHYSICAL ACTIVITY,AND BIOMARKERS OF MELATONIN AMONG ROTATING SHIFT NURSES

Long-term, night shiftwork has been identified as a potential carcinogenic risk factor. It is hypothesized that increased light at night exposure during shiftwork reduces melatonin production, which is associated with increased cancer risk. Sleep duration has been hypothesized to influence both melatonin levels and cancer risk, and it has been suggested that sleep duration could be used as a proxy for melatonin production. Finally, physical activity has been shown to reduce cancer risk, and laboratory studies indicate it may influence melatonin levels. A cross-sectional study of light exposure, sleep duration, physical activity, and melatonin levels was conducted among 61 female rotating shift nurses (work schedule: two 12?h days, two 12?h nights, five days off). Light intensity was measured using a light-intensity data logger, and sleep duration and physical activity were self-reported in a study diary and questionnaire. Melatonin concentrations were measured from urine and saliva samples. The characteristics of nurses working day and night shifts were similar. Light intensity was significantly higher during sleep for those working at night (p<?0.0001), while urinary melatonin levels following sleep were significantly higher among those working days (p?=?0.0003). Mean sleep duration for nurses working during the day (8.27?h) was significantly longer than for those working at night (4.78?h, p<?0.0001). An inverse association (p?=?0.002) between light exposure and urinary melatonin levels was observed; however, this was not significant when stratified by shift group. There was no significant correlation between sleep duration and melatonin, and no consistent relationship between physical activity and melatonin. Analysis of salivary melatonin levels indicated that the circadian rhythms of night workers were not altered, meaning peak melatonin production occurred at night. This study indicates that two nights of rotating shift work may not change the timing of melatonin production to the day among those working at night. Additionally, in this study, sleep duration was not correlated with urinary melatonin levels, suggesting it may not be a good proxy for melatonin production. (Author correspondence: anne.grundy@queensu.ca)