Melatonin Phase Research Articles

Abstract P364: Altered Circadian Rhythm in Blood Pressure in Obstructive Sleep Apnea

Background: In people with obstructive sleep apnea (OSA), adverse cardiovascular (CV) events occur most commonly during the night, about 6 hours earlier than the morning peak of events observed in the general population. In healthy individuals, circadian rhythms in many CV risk markers peak in the morning, around the common time of occurrence of adverse CV events. We therefore tested whether circadian rhythms are advanced in those with OSA, which might explain the earlier time of adverse CV events. Methods: Eleven volunteers (4 healthy controls and 7 OSA; 55 ± 5 years of age) completed a 5-day laboratory protocol with sleep and all other behaviors evenly spaced across the 24 h circadian period. To achieve this, after a baseline night, participants completed 10 consecutive short-day cycles of 2 h 40 min each of wakefulness and sleep opportunity. Blood pressure and heart rate were recorded 10 min after each wake time. Salivary melatonin and cortisol concentration were measured at 1-1.5h intervals during wakefulness. Cosinor analyses were performed to determine the rhythmicity of the all variables. We also compared OSA data with larger groups of historical healthy controls from our laboratory. Results: The circadian times of peak systolic and diastolic blood pressure were significantly advanced (up to 7 hours) in OSA as compared to healthy controls, p<0.05. Circadian peaks in all other measured variables were not different between OSA and controls. Conclusions: These results suggest that OSA is associated with a change in the timing of endogenous circadian blood pressure rhythms. This may be specific to the cardiovascular system as there was no evidence for altered phase of melatonin or cortisol secretion patterns. The relatively advanced blood pressure rhythms in OSA is similar to the advancement of the time of adverse CV events in OSA and should be further investigated.

Correcting delayed circadian phase with bright light therapy predicts improvement in ADHD symptoms: A pilot study

Attention-deficit/hyperactivity disorder (ADHD) is a common condition with comorbid insomnia reported in >70% of children and adults. These patients demonstrate delays in sleep-wake rhythms, nocturnal rise in melatonin, and early morning rise in cortisol. Given that standard psychopharmacologic treatments for ADHD often do not completely control symptoms in participants with circadian rhythm delay, we sought to test whether bright light therapy (BLT) advances circadian rhythms and further reduces ADHD symptoms over standard treatments. In addition to standard of care, participants with ADHD diagnosis underwent 1 week of baseline assessment followed by 2-weeks of 30-min morning 10,000-lux BLT beginning 3 h after mid-sleep time. Participants minimized overhead light after 4 p.m., wore an actigraphy watch, and recorded BLT time on daily sleep logs. Dim Light Melatonin Onset (DLMO) was assessed at baseline and after 2-week treatment. ADHD symptoms were measured by the ADHD-Rating Scales (ADHD-RS). BLT significantly advanced the phase of DLMO by 31 min [mean time (SEM), 20:36 (0:21) advanced to 20:05 (0:20)] and mid-sleep time by 57 min [4:37 (0:22) advanced to 3:40 (0:16); paired t-tests, p = 0.002 and 0.004, respectively). Phase advances (in DLMO or mid-sleep time) were significantly correlated with decreased ADHD-RS total scores (p = 0.027 and 0.044) and Hyperactive-Impulsive sub-scores (p = 0.014 and 0.013, respectively). Actigraphy analysis for a subset of 8 participants with significant DLMO phase advance revealed no significant changes in total sleep time, sleep efficiency, wake after sleep onset, or percent wake during sleep interval. This is the first successful use of BLT for advancing melatonin phase and improving ADHD symptoms in adults. BLT may be a complementary treatment for both delayed sleep timing and ADHD symptoms in adults.

Blood transcriptome based biomarkers for human circadian phase

Diagnosis and treatment of circadian rhythm sleep-wake disorders both require assessment of circadian phase of the brain's circadian pacemaker. The gold-standard univariate method is based on collection of a 24-hr time series of plasma melatonin, a suprachiasmatic nucleus-driven pineal hormone. We developed and validated a multivariate whole-blood mRNA-based predictor of melatonin phase which requires few samples. Transcriptome data were collected under normal, sleep-deprivation and abnormal sleep-timing conditions to assess robustness of the predictor. Partial least square regression (PLSR), applied to the transcriptome, identified a set of 100 biomarkers primarily related to glucocorticoid signaling and immune function. Validation showed that PLSR-based predictors outperform published blood-derived circadian phase predictors. When given one sample as input, the R2 of predicted vs observed phase was 0.74, whereas for two samples taken 12 hr apart, R2 was 0.90. This blood transcriptome-based model enables assessment of circadian phase from a few samples.

Late circadian phase in adults and children is correlated with use of high color temperature light at home at night

ABSTRACTLight is the strongest synchronizer of human circadian rhythms, and exposure to residential light at night reportedly causes a delay of circadian rhythms. The present study was conducted to investigate the association between color temperature of light at home and circadian phase of salivary melatonin in adults and children. Twenty healthy children (mean age: 9.7 year) and 17 of their parents (mean age: 41.9 years) participated in the experiment. Circadian phase assessments were made with dim light melatonin onset (DLMO). There were large individual variations in DLMO both in adults and children. The average DLMO in adults and in children were 21:50 ± 1:12 and 20:55 ± 0:44, respectively. The average illuminance and color temperature of light at eye level were 139.6 ± 82.7 lx and 3862.0 ± 965.6 K, respectively. There were significant correlations between color temperature of light and DLMO in adults (r = 0.735, p < 0.01) and children (r = 0.479, p < 0.05), although no significant correlations were found between illuminance level and DLMO. The results suggest that high color temperature light at home might be a cause of the delay of circadian phase in adults and children.

Association between Melatonin Secretion and Nocturia in Elderly Individuals: a Cross-Sectional Study of the HEIJO-KYO Cohort

Nocturnal voids are regulated by circadian biological rhythms, including decreased urine production and increased bladder storage capacity at night. A previous experimental study suggested that exogenous melatonin decreases urine production and increases bladder capacity in rats. However, little is known about whether melatonin secretion, which is considerably lower than exogenous melatonin, is associated with nocturia in humans. We evaluated the association between melatonin secretion and nocturia. In this cross-sectional study we examined 861 community based elderly individuals with a mean age of 72.1 years. We measured nocturnal void frequency and overnight urinary 6-sulfatoxymelatonin excretion as an index of melatonin secretion. Nocturia was defined as 2 or more nocturnal voids. Univariate comparisons between the 261 and 600 study participants with and without nocturia, respectively, showed marginal to significant associations of nocturia with age, gender, body mass index, estimated glomerular filtration rate, calcium channel blockers, benign prostatic hyperplasia, total voided urine volume, urinary 6-sulfatoxymelatonin, duration in bed, day length, and daytime and nighttime physical activity. The multivariate logistic regression model adjusted for the former confounding factors revealed that higher urinary 6-sulfatoxymelatonin was significantly associated with a lower nocturia OR (adjusted OR 0.73, 95% CI 0.56-0.96, p=0.023). After adjustment for age, gender and duration in bed the mean volume of a single voided urine significantly increased with tertiles of increasing urinary 6-sulfatoxymelatonin (p for trend=0.022). Melatonin secretion is significantly and inversely associated with nocturia in a general elderly population.

Dissonance Between Parent‐Selected Bedtimes and Young Children's Circadian Physiology Influences Nighttime Settling Difficulties

Nighttime settling difficulties (i.e., bedtime resistance, sleep-onset delay) occur in about 25% of young children and are associated with attentional, behavioral, and emotional problems. We examined whether the timing of internal (endogenous) circadian melatonin phase (i.e., dim light melatonin onset; DLMO) and its relationship with parent-selected bedtimes were related to nighttime settling behaviors. Fourteen regularly napping preschoolers (8 females; 30-36 months) participated in a 6-day protocol (parent-report of nighttime settling, actigraphic assessment of sleep onset latency, evening salivary DLMO). Average DLMO clock time was 07:40 p.m. ± 00:48 minutes, occurring 29 minutes ± 32 minutes prior to bedtime (lights-out). Children with later DLMOs had longer sleep-onset latencies (r = .62) and poorer success in falling asleep (r = -.59). Children whose bedtimes were closer to their DLMO had longer sleep-onset latencies (r = .72) and increased bedtime resistance (r = -.54). We conclude that dissonance between parent-selected bedtimes and children's circadian physiology may contribute to the development of nighttime settling difficulties in early childhood.

Impact of insufficient sleep on total daily energy expenditure, food intake, and weight gain

Insufficient sleep is associated with obesity, yet little is known about how repeated nights of insufficient sleep influence energy expenditure and balance. We studied 16 adults in a 14- to 15-d-long inpatient study and quantified effects of 5 d of insufficient sleep, equivalent to a work week, on energy expenditure and energy intake compared with adequate sleep. We found that insufficient sleep increased total daily energy expenditure by ∼5%; however, energy intake--especially at night after dinner--was in excess of energy needed to maintain energy balance. Insufficient sleep led to 0.82 ± 0.47 kg (±SD) weight gain despite changes in hunger and satiety hormones ghrelin and leptin, and peptide YY, which signaled excess energy stores. Insufficient sleep delayed circadian melatonin phase and also led to an earlier circadian phase of wake time. Sex differences showed women, not men, maintained weight during adequate sleep, whereas insufficient sleep reduced dietary restraint and led to weight gain in women. Our findings suggest that increased food intake during insufficient sleep is a physiological adaptation to provide energy needed to sustain additional wakefulness; yet when food is easily accessible, intake surpasses that needed. We also found that transitioning from an insufficient to adequate/recovery sleep schedule decreased energy intake, especially of fats and carbohydrates, and led to -0.03 ± 0.50 kg weight loss. These findings provide evidence that sleep plays a key role in energy metabolism. Importantly, they demonstrate physiological and behavioral mechanisms by which insufficient sleep may contribute to overweight and obesity.

Melatonin and clock genes expression in the cardiovascular system

Generation of circadian oscillations is based on rhythmic expression of clock genes and subsequent post-transcriptional and post-translational modifications. In addition to the central circadian oscillator - the suprachiasmatic nucleus (SCN), peripheral oscillators have been demonstrated in many tissues, including the heart and blood vessels. Melatonin mediates cyclic lighting conditions to rhythmic endocrine signal and is able to synchronize neuronal firing in the SCN via membrane receptors. Clock gene expression is melatonin sensitive in the pars tuberalis, genes cry1 and tim1 respond to single injection while neurod1 and npas4 are influenced via long lasting mechanisms. In the rat heart, melatonin phase advanced expression of per2 and bmal1 independently from its effects on the SCN. Melatonin is an important endogenous signal able to synchronize circadian oscillations in the cardiovascular system. It may be effective especially in situations when the circadian control is weakened or organism must adapt to rapid changes in rhythmic environmental conditions.

An Improved Method for Estimating Human Circadian Phase Derived From Multichannel Ambulatory Monitoring and Artificial Neural Networks

Recently, we developed a novel method for estimating human circadian phase with noninvasive ambulatory measurements combined with subject-independent multiple regression models and a curve-fitting approach. With this, we were able to estimate circadian phase under real-life conditions with low subject burden, i.e., without need of constant routine (CR) laboratory conditions, and without measuring standard circadian markers, such as core body temperature (CBT) or pineal hormone melatonin rhythms. The precision of ambulatory-derived estimated circadian phase was within an error of 12 ± 41 min (mean ± SD) in comparison to melatonin phase during a CR protocol. The physiological measures could be reduced to a triple combination: skin temperatures, irradiance in the blue spectral band of ambient light, and motion acceleration. Here, we present a nonlinear regression model approach based on artificial neural networks for a larger data set (25 healthy young males), including both the original data and additional data collected in the same protocol and using the same equipment. Throughout our validation study, subjects wore multichannel ambulatory monitoring devices and went about their daily routine for 1 wk. The devices collected a large number of physiological, behavioral, and environmental variables, including CBT, skin temperatures, cardiovascular and respiratory functions, movement/posture, ambient temperature, spectral composition and intensity of light perceived at eye level, and sleep logs. After the ambulatory phase, study volunteers underwent a 32-h CR protocol in the laboratory for measuring unmasked circadian phase (i.e., “midpoint” of the nighttime melatonin rhythm). To overcome the complex masking effects of many different confounding variables during ambulatory measurements, neural network–based nonlinear regression techniques were applied in combination with the cross-validation approach to subject-independent prediction of circadian phase. The most accurate estimate of circadian phase with a prediction error of −3 ± 23 min (mean ± SD) was achieved using only two types of the measured variables: skin temperatures and irradiance for ambient light in the blue spectral band. Compared to our previous linear multiple regression modeling approach, motion acceleration data can be excluded and prediction accuracy, nevertheless, improved. Neural network regression showed statistically significant improvement of variance of prediction error over traditional approaches in determining circadian phase based on single predictors (CBT, motion acceleration, or sleep logs), even though none of these variables was included as predictor. We, therefore, have identified two sets of noninvasive measures that, combined with the prediction model, can provide researchers and clinicians with a precise measure of internal time, in spite of the masking effects of daily behavior. This method, here validated in healthy young men, requires testing in a clinical or shiftwork population suffering from circadian sleep-wake disorders. (Author correspondence: vitaliy.kolodyazhniy@sbg.ac.at)

Analysis Method and Experimental Conditions Affect Computed Circadian Phase from Melatonin Data

Accurate determination of circadian phase is necessary for research and clinical purposes because of the influence of the master circadian pacemaker on multiple physiologic functions. Melatonin is presently the most accurate marker of the activity of the human circadian pacemaker. Current methods of analyzing the plasma melatonin rhythm can be grouped into three categories: curve-fitting, threshold-based and physiologically-based linear differential equations. To determine which method provides the most accurate assessment of circadian phase, we compared the ability to fit the data and the variability of phase estimates for seventeen different markers of melatonin phase derived from these methodological categories. We used data from three experimental conditions under which circadian rhythms - and therefore calculated melatonin phase - were expected to remain constant or progress uniformly. Melatonin profiles from older subjects and subjects with lower melatonin amplitude were less likely to be fit by all analysis methods. When circadian drift over multiple study days was algebraically removed, there were no significant differences between analysis methods of melatonin onsets (P = 0.57), but there were significant differences between those of melatonin offsets (P<0.0001). For a subset of phase assessment methods, we also examined the effects of data loss on variability of phase estimates by systematically removing data in 2-hour segments. Data loss near onset of melatonin secretion differentially affected phase estimates from the methods, with some methods incorrectly assigning phases too early while other methods assigning phases too late; missing data at other times did not affect analyses of the melatonin profile. We conclude that melatonin data set characteristics, including amplitude and completeness of data collection, differentially affect the results depending on the melatonin analysis method used.

Morning and nocturnal serum melatonin rhythm levels in patients with major depressive disorder: an analytical cross-sectional study.

The pineal gland is an adaptive organ that precisely regulates the biological rhythms of melatonin brain hemostasis. Variation in the regulation of melatonin rhythms is a likely cause of depressive disorder. The purpose of this study was to measure serum melatonin levels in patients with major depressive disorder (MDD) and normal control subjects. Analytical cross-sectional study at the industrial medical unit of the Iron Smelting Company of Isfahan, Iran. The morning and nocturnal serum melatonin levels of patients and controls were measured using the enzyme-linked immunosorbent assay (ELISA) method. All data were assessed using variance analysis. The morning and nocturnal serum melatonin levels of depressed and healthy subjects differed (P < 0.05). The nocturnal serum melatonin levels of depressed women were lower than those of depressed men (P < 0.05). The findings of this study showed that the nocturnal serum melatonin levels in the depressed patients were lower than in the controls. Thus, the peak melatonin phase in the depressed patients was reached with delay.

Impact of Sleepiness and Sleep Deficiency on Public Health—Utility of Biomarkers

Sleep disorders and sleep deficiency are important causes of adverse health effects and increased mortality in the United States and worldwide. Sleep deficiency can also result in myriad adverse behavioral consequences, including profound sleepiness, cognitive slowing, automatic behavior, attentional failures and performance degradation, errors, and accidents. It is important to recognize that sleepiness and sleep deficiency are not synonymous.

Clinical Implications of the Melatonin Phase Response Curve

In this issue of JCEM, the Burgess et al. report (1) on the melatonin phase response curve (PRC) has many clinical implications worthy of further discussion. A PRC is a mathematical tool established by convention because of its heuristic value for describing the direction and magnitude of phase shifts in the endogenous circadian pacemaker and its driven rhythms that result from applying a stimulus, such as melatonin or bright light. One of these driven rhythms results in a melatonin curve or profile; melatonin production by the pineal gland occurs almost exclusively at night in both diurnal and nocturnal species, resulting in a profile shaped like a square wave (2). Thus, the timing of melatonin production is governed by, and feeds back time-of-day information to, the pacemaker—which is primarily entrained to the light/dark cycle. [However, there is no negative feedback inhibition limiting the amount of melatonin produced (3).] Endocrinologists, as well as other clinicians, are primarily interested in: 1) the function of melatonin in humans as a neurohormone; 2) pathologies related to melatonin production; and 3) therapies resulting from melatonin supplementation. The jury is out as to whether or not there are significant melatonin disorders that are simply a matter of hyper- or hyposecretion. The medical implications of melatonin are best understood in a circadian context. Circadian disorders are numerous and important, and their treatment is informed by the melatonin (and light) PRCs.

Ischemic stroke destabilizes circadian rhythms

BackgroundThe central circadian pacemaker is a remarkably robust regulator of daily rhythmic variations of cardiovascular, endocrine, and neural physiology. Environmental lighting conditions are powerful modulators of circadian rhythms, but regulation of circadian rhythms by disease states is less clear. Here, we examine the effect of ischemic stroke on circadian rhythms in rats using high-resolution pineal microdialysis.MethodsRats were housed in LD 12:12 h conditions and monitored by pineal microdialysis to determine baseline melatonin timing profiles. After demonstration that the circadian expression of melatonin was at steady state, rats were subjected to experimental stroke using two-hour intralumenal filament occlusion of the middle cerebral artery. The animals were returned to their cages, and melatonin monitoring was resumed. The timing of onset, offset, and duration of melatonin secretion were calculated before and after stroke to determine changes in circadian rhythms of melatonin secretion. At the end of the monitoring period, brains were analyzed to determine infarct volume.ResultsRats demonstrated immediate shifts in melatonin timing after stroke. We observed a broad range of perturbations in melatonin timing in subsequent days, with rats exhibiting onset/offset patterns which included: advance/advance, advance/delay, delay/advance, and delay/delay. Melatonin rhythms displayed prolonged instability several days after stroke, with a majority of rats showing a day-to-day alternation between advance and delay in melatonin onset and duration. Duration of melatonin secretion changed in response to stroke, and this change was strongly determined by the shift in melatonin onset time. There was no correlation between infarct size and the direction or amplitude of melatonin phase shifting.ConclusionThis is the first demonstration that stroke induces immediate changes in the timing of pineal melatonin secretion, indicating that cortical and basal ganglia infarction impacts the timing of melatonin rhythms. The heterogeneous direction and amplitude of melatonin shifts suggests that the upstream regulation of hypothalamic timekeeping is likely anatomically diffuse and mechanistically complex. Finally, our study exemplifies the use of pineal microdialysis to evaluate the effect of neurological diseases on circadian function.

Inter-Individual Differences In Habitual Sleep Timing and Entrained Phase of Endogenous Circadian Rhythms of BMAL1, PER2 and PER3 mRNA in Human Leukocytes

Individual sleep timing differs and is governed partly by circadian oscillators, which may be assessed by hormonal markers, or by clock gene expression. Clock gene expression oscillates in peripheral tissues, including leukocytes. The study objective was to determine whether the endogenous phase of these rhythms, assessed in the absence of the sleep-wake and light-dark cycle, correlates with habitual sleep-wake timing. Observational, cross-sectional. Home environment and Clinical Research Center. 24 healthy subjects aged 25.0 +/- 3.5 (SD) years. Actigraphy and sleep diaries were used to characterize sleep timing. Circadian rhythm phase and amplitude of plasma melatonin, cortisol, and BMAL1, PER2, and PER3 expression were assessed during a constant routine. Circadian oscillations were more robust for PER3 than for BMAL1 or PER2. Average peak timings were 6:05 for PER3, 8:06 for PER2, 15:06 for BMAL1, 4:20 for melatonin, and 10:49 for cortisol. Individual sleep-wake timing correlated with the phases of melatonin and cortisol. Individual PER3 rhythms correlated significantly with sleep-wake timing and the timing of melatonin and cortisol, but those of PER2 and BMAL1 did not reach significance. The correlation between sleep timing and PER3 expression was stronger in individuals homozygous for the variant of the PER3 polymorphism that is associated with morningness. Individual phase differences in PER3 expression during a constant routine correlate with sleep timing during entrainment. PER3 expression in leukocytes represents a useful molecular marker of the circadian processes governing sleep-wake timing.

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light‐dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights‐off and declined around lights‐on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights‐on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights‐on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights‐on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase‐shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.

Phase-Dependent Treatment of Delayed Sleep Phase Syndrome with Melatonin

Delayed sleep phase syndrome (DSPS) is a circadian-rhythm sleep disorder characterized by abnormally late sleep and wake times. Melatonin, taken in the evening, advances sleep and circadian phase in patients with DSPS. However, little is known about the most effective dose or time of administration. In the present study, we tested the effectiveness of melatonin to advance the timing of sleep and circadian phase in individuals with DSPS. Following baseline assessment of sleep and circadian phase, subjects were randomly assigned to 1 of 3 treatment groups. The administration of melatonin (0.3 or 3.0 mg) or placebo was double-blinded. All procedures were conducted on an outpatient basis. Thirteen subjects with DSPS, recruited via flyers, advertisements, and referrals from the Sleep Clinic, completed this study. Melatonin (0.3 or 3.0 mg) or placebo was administered between 1.5 and 6.5 hours prior to dim light melatonin onset for a 4-week period. Both doses of melatonin advanced the circadian phase of endogenous melatonin. The magnitude of phase advance in dim-light melatonin onset correlated strongly with the time of melatonin administration, with earlier times being more effective (r2 = 0.94, P < .0001). Similar, though weaker, relationships were obtained between the timing of melatonin administration and changes in sleep time. These results indicate that melatonin advances the circadian clock and sleep in patients with DSPS in a phase-dependent manner. This is the first study that reports a relationship between timing of melatonin administration and phase changes in patients with DSPS.

Stability of Melatonin and Temperature as Circadian Phase Markers and Their Relation to Sleep Times in Humans

Circadian rhythms of core body temperature and melatonin are commonly used as phase markers of the circadian clock. Melatonin is a more stable marker of circadian phase when measured under constant routine conditions. However, little is known about the variability of these phase markers under less controlled conditions. Moreover, there is little consensus about the preferred method of analysis. The objective of this study was to assess various methods of calculating melatonin and temperature phase in subjects with regular sleep schedules living in their natural environment. Baseline data were analyzed from 42 healthy young subjects who were studied on at least two occasions. Each hospital admission was separated by at least 3 weeks. Subjects were instructed to maintain a regular sleep schedule, which was monitored for 1 week before admission by sleep logs and actigraphy. Subjects spent one habituation night under controlled conditions prior to collecting baseline temperature and melatonin measurements. The phase of the melatonin rhythm was assessed by 9 different methods. The temperature nadir (Tmin) was estimated using both Cleveland and Cosine curve fitting procedures, with and without demasking. Variability between admissions was assessed by correlation analysis and by the mean absolute difference in timing of the phase estimates. The relationship to sleep times was assessed by correlation of sleep onset or sleep offset with the various phase markers. Melatonin phase markers were more stable and more highly correlated with the timing of sleep than estimates of Tmin. Of the methods for estimating Tmin, simple cosine analysis was the least variable. In addition, sleep offset was more strongly correlated with the various phase markers than sleep onset. The relative measures of melatonin offset had the highest correlation coefficients, the lowest study-to-study variability, and were more strongly associated with sleep timing than melatonin onsets. Concordance of the methods of analysis suggests a tendency for the declining phase of the melatonin profile to be more stable and reliable than either markers of melatonin onset or measures of the termination of melatonin synthesis.

Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin

The hormone melatonin phase shifts circadian rhythms generated by the mammalian biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, through activation of G protein-coupled MT2 melatonin receptors. This study demonstrated that pretreatment with physiological concentrations of melatonin (30-300 pM or 7-70 pg/mL) decreased the number of hMT2 melatonin receptors heterologously expressed in mammalian cells in a time and concentration-dependent manner. Furthermore, hMT2-GFP melatonin receptors heterologously expressed in immortalized SCN2.2 cells or in non-neuronal mammalian cells were internalized upon pretreatment with both physiological (300 pM or 70 pg/mL) and supraphysiological (10 nM or 2.3 ng/mL) concentrations of melatonin. The decrease in MT2 melatonin receptor number induced by melatonin (300 pM for 1 h) was reversible and reached almost full recovery after 8 h; however, after treatment with 10 nM melatonin full recovery was not attained even after 24 h. This recovery process was partially protein synthesis dependent. Furthermore, exposure to physiological concentrations of melatonin (300 pM) for a time mimicking the nocturnal surge (8 h) desensitized functional responses mediated through melatonin activation of endogenous MT2 receptors, i.e., stimulation of protein kinase C (PKC) in immortalized SCN2.2 cells and phase shifts of circadian rhythms of neuronal firing in the rat SCN brain slice. We conclude that in vivo the nightly secretion of melatonin desensitizes endogenous MT2 melatonin receptors in the mammalian SCN thereby providing a temporally integrated profile of sensitivity of the mammalian biological clock to a melatonin signal.

The actions of a charged melatonin receptor ligand, TMEPI, and an irreversible MT2 receptor agonist, BMNEP, on mouse hippocampal evoked potentials in vitro

We have previously determined that melatonin modulates hippocampal synaptic transmission in a biphasic way: an initial depression was followed by a recovery/amplification phase. Here we describe the influence of two novel melatonin receptor ligands, BMNEP (N-bromoacetyl-2-iodo-5-methoxytryptamine) and TMPEI (N-[2-(2-Trimethylammoniumethyleneoxy-7-methoxy)ethyl]propionamide iodide), on the population spike (PS) and excitatory postsynaptic potentials (EPSP) recorded from mouse hippocampal slices. BMNEP, which specifically alkylates and constitutively activates the MT2 melatonin receptor, mimicked the first phase of melatonin's action by irreversibly depressing both the PS and EPSP. TMPEI, a charged ligand of plasma membrane melatonin receptors, amplified those potentials in a manner similar to the effect of melatonin observed during the second, recovery phase. Melatonin had no influence on the potentials amplified by the action of TMPEI. Our results suggest that the biphasic, receptor-dependent action of melatonin and its analogs modulates the efficiency of the hippocampal glutamergic synapse and is most likely mediated through two different, sequentially occurring mechanisms.