Susan Benloucif

Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms

This study demonstrates the involvement of the MT2 (Mel1b) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin‐labeled oligonucleotide probes revealed for the first time the expression of mt1 and MT2 melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 μg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose‐dependent manner [EC50 = 0.72 μg/mouse; 0.98 ± 0.08 h (n = 15) maximal advance at 9 μg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P‐ADOT and 4P‐PDOT (90 μ/mouse, s.c.) nor luzindole (300 μg/mouse, s.c.), which shows 25‐fold higher affinity for the MT2 than the mt1 subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose‐response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 mg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane‐bound melatonin receptor and strongly suggests that this effect is mediated through the MT2 melatonin receptor subtype within the circadian timing system. We conclude that the MT2 melatonin receptor subtype is a novel therapeutic target for the development of subtype‐selective analogs for the treatment of circadian sleep and mood‐related disorders.—Dubocovich, M. L., Yun, K., Al‐Ghoul, W. M., Benloucif, S., Masana, M. I. Selective MT2 melatonin receptor antagonists block melatonin‐mediated phase advances of circadian rhythms. FASEB J. 12, 1211–1220 (1998)

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 instructedto 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.

Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse

This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2‐[125I]‐Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3±1.7 fmol/mg protein, n=5, at ZT2) or at the beginning of the subjective day (48.6±2.1 fmol/mg protein, n=6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35 S‐labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CT10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions.

Melatonin and Light Induce Phase Shifts of Circadian Activity Rhythms in the C3H/HeN Mouse

This study examines the effect of light pulses and administration of the pineal hormone melatonin on the circadian activity rhythm of C3H/HeN mice. Mice were housed in constant dark in cages equipped with running wheels. Phase shifts in the circadian rhythm of wheel-running activity were measured following treatment with a 15-min pulse of light (300 lux) or administration of vehicle (ethanol/saline) or melatonin (90 μg, sc). Light treatment induced phase changes in circadian activity rhythms; specifically, delays during early subjective night (circadian time [CT] 12.5 to CT 18.5) and advances during late subjective night (CT 0.5). A single dose of melatonin administered at various CTs had no consistent effect on free-running circadian activity rhythms. By contrast, melatonin administration for 3 consecutive days at the same clock time induced advances in circadian activity rhythms by more than 1 h when the first dose was administered at CT 10 and induced delays in circadian activity rhythms by up to 1 h when the first dose was administered between CT 24 and CT 2. With the caveat that multi- ple melatonin treatments are required to induce phase shifts, the results suggest that the circadian timing system controlling the rhythm of wheel-running activity in the C3H/HeN mouse is responsive to both light and melatonin.

Melatonin receptors in the mammalian suprachiasmatic nucleus

The SCN of the hypothalamus, the site of the circadian pacemaker in mammals, is endowed with melatonin receptors of the ML-1 subtype. Here, we present evidence suggesting that activation of melatonin receptors in the SCN regulates circadian rhythms of behavior in the mouse. In a paradigm simulating a eastbound transmeridian flight, timed administration of melatonin may either accelerate or decrease the rate of reentrainment. Moreover, under constant environmental conditions, exogenous melatonin phase shifts circadian rhythms only during times when the production of the hormone is inhibited. Similarly, light shows periods of circadian sensitivity only at times when light is not present in a natural photoperiod. The maximal phase shifts elicited by melatonin and light coincide with the subjective light-dark (dusk) and subjective dark-light (dawn) transitions. The periods of sensitivity for melatonin, occur at the same circadian times in mouse and in man. Under a short photoperiod the duration of the nocturnal melatonin production may overlap with periods of sensitivity for the hormone, and therefore, melatonin, may be important in synchronizing circadian rhythms to changes in the natural photoperiod. It follows that the identification of periods of circadian sensitivity to melatonin in mammals is important for the development of effective treatments with melatonin and related analogues for sleep disorders characterized by alterations of circadian rhythmicity.

Molecular Pharmacology and Function Ofmelatonin Receptor Subtypes

The secretion of melatonin primarily from the vertebrate retina and pineal gland with high levels at night is controlled by circadian clocks locally within the retina and the hypothalamic suprachiasmatic nucleus and is synchronized by environmental light (1,2). The first biological activity of melatonin can be traced back to 1917 when McCord and Allan (3) discovered that extracts of bovine pineal gland caused blanching of Rana pipiens tadpole skin. This bioassay was used to isolate melatonin from pineal extracts which led to the elucidation of its chemical structure (4). The property of melatonin to aggregate pigment granules (melanosomes) of amphibian dermal melanophores was used 1) to postulate the presence of melatonin receptors and to propose that Nacetyltryptamine is a melatonin receptor antagonist (5), 2) to establish the first structure-activity relationships of melatonin analogues (5), and 3) to demonstrate, in cultured Xenopus laevis melanophores, that activation of melatonin receptors inhibits cAMP formation through coupling to a pertussis toxin-sensitive G-protein (6). Subsequently, expression cloning led to the isolation of the first cDNA encoding a melatonin receptor from the Xenopus laevis melanophore (7). This landmark discovery facilitated the cloning and characterization of mammalian melatonin receptor subtypes, belonging to a novel subfamily of seven transmembrane domain G-protein coupled receptors

Light-induced phase shifts of circadian activity rhythms and immediate early gene expression in the suprachiasmatic nucleus are attenuated in old C3H/HeN mice.

Alterations in the mechanisms of entrainment and/or response of the circadian pacemaker to zeitgebers may contribute to age related changes in sleep/wake rhythms. This study examined the effect of age on light-induced phase shifts of circadian activity rhythms and on the expression of the immediate early genes c-fos and jun-B in the suprachiasmatic nucleus (SCN) of young and old C3H/HeN mice. Mice (4 months or 16 months at the beginning of the experiment) were housed in constant darkness with circadian rhythms assessed by running wheel activity. Mice were exposed to light pulses of 30, 100, 300 or 1000 lux and steady state phase shifts of circadian activity rhythms determined. In young mice exposed to light at circadian time (CT) 14, light pulses of 30, 100, 300 or 1000 lux induced phase delays of circadian activity rhythms of similar magnitude (averaging 2.8 h). Phase delays following photic stimulation were reduced in the old mice at all light levels (averaging 1.1 h, P < 0.001). Following behavioral testing, mice were exposed to light (1000 lux) at CT 14 for determination of the light-induced expression of c-fos and jun-B mRNA in the SCN by in situ hybridization histochemistry. Immediate early gene expression following light exposure was reduced by 42% (c-fos) and 48% (jun-B) in the SCN of old mice compared to young controls (P < 0.001). Together, these results suggest an age related reduction in responsiveness to light by the circadian pacemaker.

Responsiveness to melatonin and its receptor expression in the aging circadian clock of mice

This study determined the effect of age on the efficacy of melatonin treatment to phase shift circadian activity rhythms and on melatonin receptor expression in the suprachiasmatic nucleus (SCN) and paraventricular nucleus of the thalamus (PVNT) of C3H/HeN mice. The circadian rhythm of 2-[125I]iodomelatonin binding, assessed at three times of the day [circadian times (CT) 2, 10, and 18], showed a modest age-related decrease in the SCN but not the PVNT of old C3H/HeN mice (24 mo). There was a tendency for age to reduce Mel1a melatonin receptor mRNA expression in the suprachiasmatic nucleus during the day, but not during the night. The magnitude of phase shifts of circadian activity rhythms (advances or delays) induced by administration of melatonin at CT 10 or CT 2 was identical in young and old C3H/HeN mice. Together, these results suggest that the decrease in melatonin receptor expression in the SCN had little effect on melatonin-induced phase shifts of circadian activity rhythms. We conclude that the responsiveness of the circadian timing system to melatonin administration does not decrease with age.This study determined the effect of age on the efficacy of melatonin treatment to phase shift circadian activity rhythms and on melatonin receptor expression in the suprachiasmatic nucleus (SCN) and paraventricular nucleus of the thalamus (PVNT) of C3H/HeN mice. The circadian rhythm of 2-[125I]iodomelatonin binding, assessed at three times of the day [circadian times (CT) 2, 10, and 18], showed a modest age-related decrease in the SCN but not the PVNT of old C3H/HeN mice (24 mo). There was a tendency for age to reduce Mel1a melatonin receptor mRNA expression in the suprachiasmatic nucleus during the day, but not during the night. The magnitude of phase shifts of circadian activity rhythms (advances or delays) induced by administration of melatonin at CT 10 or CT 2 was identical in young and old C3H/HeN mice. Together, these results suggest that the decrease in melatonin receptor expression in the SCN had little effect on melatonin-induced phase shifts of circadian activity rhythms. We conclude that the responsiveness of the circadian timing system to melatonin administration does not decrease with age.

Responsiveness of the aging circadian clock to light.

The present study assessed whether advances in sleep times and circadian phase in older adults might be due to decreased responsiveness of the aging circadian clock to light. Sixteen young (29.3+/-5.6 years) and 14 older adults (67.1+/-7.4 years) were exposed to 4h of control dim (10lux) or bright light (3500lux) during the night. Phase shifts of the melatonin rhythm were assessed from the nights before and after the light exposure. Bright light delayed the melatonin midpoint in both young and older adults (p<0.001). Phase delays for the older subjects were not significantly different from those of the young subjects for either the bright or dim light conditions. The magnitude of phase delays was correlated with both sleep offset and phase angle in the older, but not the younger subjects. The present results indicate that at light intensities commonly used in research as well as clinical practice older adults are able to phase delay to the same extent as younger subjects.

Light-induced c-fos mRNA expression in the suprachiasmatic nucleus and the retina of C3H/HeN mice

Light-induced expression of c-fos mRNA was studied over a circadian period (approximately 24 h) in C3H/HeN mice maintained in constant dark. This mouse strain expresses an rd mutation (retinal degeneration) which does not affect light-induced phase shifts of circadian rhythms. c-fos mRNA expression in the retina and the suprachiasmatic nucleus (SCN) after a light pulse (300 lux) was determined by in-situ hybridization autoradiography using a 35S-labeled c-fos riboprobe. Light induced the expression of c-fos mRNA in retino-recipient areas of the SCN. This response was dependent on the circadian time (CT) and was observed only during the subjective night (CT14-CT22) and early subjective day (CT2). However, the period of photosensitivity for c-fos induction extended 1 h over the period of photosensitivity for phase shifts in circadian behavior. In the retina of C3H/HeN mice, light-induced c-fos mRNA expression was observed in a small number of cells in the ganglion cell layer (approximately 0.2%) which may represent ganglion cells projecting to the SCN. A dependence of c-fos expression with the circadian time was observed in retinal ganglion cells, suggesting that retinal photosensitivity may also be controlled by a circadian oscillator. In conclusion, we demonstrated light-induced expression of the immediate early gene c-fos mRNA in both the retina and SCN of C3H/HeN mice expressing the rd mutation.