Theme : Dreams

Behavioral Effects of Melatonin Treatment in Non-Human Primates

Irina V. Zhdanova, Margo L. Cantor, Ojingwa U. Leclair, Alex I. Kartashov and Richard J. Wurtman
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Abstract
Melatonin treatment has been shown to induce sleepiness and promote sleep in humans. In order to understand the mechanisms by which melatonin acts on human sleep and behavior, it would be useful to have an animal model in which the physiological nocturnal increase in melatonin secretion correlated with nocturnal sleep, i.e., a diurnal species. In this pilot study the oral administration of melatonin to two Pigtail macaques (Macaca Nemestrina) at different times of the day significantly decreased motor activity and promoted earlier sleep onset, as measured actigraphically. The decline in the animals' motor activity occurred within 25-40 min after melatonin ingestion. The duration of motor inhibition was dose dependent. Administration of a 0.05 mg dose induced serum melatonin levels comparable to the peak physiologic concentrations reported in untreated humans and the non-human primates. These data suggest that melatonin may modulate motor activity and sleep pattern in certain diurnally-active primates.

Current Claim: Oral melatonin treatment significantly diminishes actigraphically recorded motor activity and promotes earlier sleep onset in a diurnal non-human primate, Macaca Nemestrina.

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Oral doses of melatonin causing either physiologic or vastly elevated serum melatonin concentrations promote sleep in humans (Anton-Tay et al., 1971; Cramer et al., 1974; Tzischinsky and Lavie, 1994; Dollins et al., 1994; Zhdanova et al., 1995, 1996). This effect is typically achieved within 30-60 minutes of hormone administration (Nave et al., 1995; Zhdanova et al., 1996). In young healthy human volunteers doses of 0.1-0.3 mg of melatonin typically induce circulating melatonin levels comparable to those that occur nocturnally in untreated individuals, i.e., less than 200 pg/ml (Dollins et al., 1994). These observations suggest that melatonin may have a physiologic role in sleep initiation and maintenance in humans and, perhaps, in other diurnal species.
While there is an extensive body of data regarding effects of the pineal hormone on circadian organization in diurnal or nocturnal animals, there have been few animal studies on melatonin's acute effects. Implantation of crystalline melatonin (15-30 µg) into cats' subcortical structures caused, soon thereafter, initiation of sleep, with an increase in amplitude and a slowing of electrical activity in the structures tested (Marczynski et al., 1964). Intraventricular injection of 1-100 ng of melatonin (Goldstein and Pavel, 1981) induced slow wave sleep in cats, a 3 hour suppression of REM sleep, and a subsequent rebound of this sleep stage. A somnogenic or sleep-potentiating effect of intraperitoneal injections of melatonin (2.5-10 mg/kg) or its analogues has been described in rats (Sugden, 1995); however, in other studies on this species similar or lower doses of the hormone were either ineffective (Tobler et al., 1994), or they even tended to reduce sleep (Mendelson et al., 1980). Such inconsistent findings may be due to the extremely high pharmacological concentrations of melatonin in the blood, cerebrospinal fluid and brain tissue that were, no doubt, induced by the very high doses of the hormone that were used. Another major factor to be considered in an attempt to interpret such results is differences among various species in the temporal organization of sleep-wake behavior and melatonin secretory rhythms. In nocturnally active rats and in crepuscular cats, melatonin is principally secreted at nighttime, as in humans. This suggests that melatonin's relation to sleep in these species must differ from its relation to sleep in people. Until now, there has not been a suitable animal model identified for the study of the "sleep promoting effect" of melatonin in diurnal species.

In order to evaluate possible effects of melatonin on sleep and behavior in diurnal primates, we conducted a pilot study using continuous actigraphic recordings of motor activity in two non-human primates, Pigtail Macaques (Macaca Nemestrina). In a double-blind, placebo controlled study the animals received a range of melatonin doses at different times of the day, and their responses were monitored. Our observations indicate that melatonin dose-dependently diminishes spontaneous motor activity in Macaca Nemestrina and promotes earlier sleep onset.

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Two naive male macaques, one 4 years old (7.5 kg) and the other 7 years old (11.7 kg) were housed in individual 2m x 2m primate runs. They were maintained in a 12:12 light:dark cycle with lights off at 6:00 p.m. No other monkeys were present in the experimental room. Interaction with humans was limited to short periods of exposure for food presentation and the administration of the treatment, scheduled between 8 a.m. and 3 p.m. Actigraphic monitors, worn on the animals' collars, were changed between test trials, on Mondays. Water was available ad libitum and a diet of monkey chow, fruits, vegetables, and nuts was provided three times a day.
A continuous recording of the monkeys' locomotor activity was obtained using an actigraph ("Actiwatch", Mini-Mitter Co., OR), attached to the animals' neck collars. A sampling rate of 32 Hz, with an epoch length of 1 min was obtained. The actigraphic data were evaluated for motor activity and total sleep period (TSP) using the Sleepwatch Software Program (Mini-Mitter Co., OR).

Two-hour video tape recordings of the monkeys' behavior were made automatically, in the absence of the experimenter, starting at 3 p.m., the time at which melatonin (0.025 or 0.1 mg) or a placebo capsule was administered. Thus, the video recording of the animals' movements complemented the quantitative measures of their activity registered by the actigraph.

The study was double-blind and placebo controlled. The animals received a gelatin capsule containing either a melatonin dose mixed with microcellulose, or microcellulose alone (placebo). In order to test for the acute effect of melatonin treatment and for the effect of repeated administration of the hormone, two different treatment schedules were used. The first schedule involved alternating daily administration of the placebo, or one of three melatonin doses (0.025, 0.05 or 0.1 mg), at 3 p.m. for four consecutive days (Tuesday through Friday). Each monkey participated in two such trials, separated by a washout week of no treatment, with each melatonin dose. In order to test the effects of repeated treatment, the placebo was administered daily at 8 a.m. for four consecutive days (Tuesday-Friday), followed by three days of no treatment (Sunday-Monday), and then by four consecutive days of treatment at 8 a.m. (Tuesday-Friday) using one of two different melatonin doses (0.1 mg or 3.0 mg). Each monkey participated in one such trial at each dose. Regardless of the treatment schedule or dose used, different trials were separated by a washout period of one week with no treatment.

When the data were analyzed, each melatonin-treatment day had a paired placebo day chosen either according to the day sequence (placebo day always preceding melatonin day in the first type of schedule) or the corresponding day of the week, if recordings were conducted for two consecutive weeks (second type of schedule). Paired placebo/melatonin days were always recorded using the same activity monitor in order to avoid a possible effects of inter-monitor variation.

Daytime blood melatonin levels were determined an hour after the administration of placebo, and after a 0.05 mg dose of melatonin, at 10 a.m. Blood samples (3 ml each) were withdrawn from the vena saphena under general anesthesia (ketamine, 10 mg/kg), and stored protected from light at room temperature for 30 min; serum was separated by centrifugation, stored frozen at -20°C, and assayed for melatonin. Melatonin concentrations were measured in 0.5 ml aliquots of serum using a radioimmunoassay (RIA) kit (Buehlmann Laboratories, Allschwil, Switzerland). The limit of sensitivity of this assay was 0.5 pg/ml in serum. Intra-assay coefficients of variation for control samples were 6.1% at 6 pg/ml and 6.4% at 122 pg/ml; the corresponding inter-assay coefficients of variation were 8.5% and 11.9%.

Data collected from the two monkeys were analyzed using repeated measures ANOVA on mean values of motor activity per certain period after the treatment administration (as specified in the Results), and on the sleep onset time. p-value less than 0.05 was considered significant.

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Figure 1

Figure 2

Actigraphic recordings showed that the administration of a 0.025 mg dose of melatonin at 3 p.m. did not significantly change the monkeys' motor activity between 4 p.m. and 6 p.m. compared to the corresponding placebo treatment (mean ± SD: 105.8 ± 136.8 vs. 108.7 ± 148.9 movements/min on melatonin). The motor activity measured between 4 p.m. and 6 p.m. was substantially diminished by the 0.05 mg, or the 0.1 mg dose melatonin treatment at 3 p.m. in both monkeys (Table 1, Fig. 1), although the difference between the two doses and placebo did not reach statistical significance, perhaps due to a small number of observations. After treatment with either melatonin dose the animals appeared to fall asleep (mean ± SD 16.49 ± 0.36 h) significantly earlier (p = 0.0012), according to the analysis of the actigraphic recording, than after placebo treatment (18.24 ± 0.08 h). Motor activity during the habitual hours of sleep (6 p.m.-6 a.m.), after the 0.05 or 0.1 mg melatonin doses, ingested at 3 p.m., did not differ from that after the placebo treatment. Wake-up time was concurrent with the time of lights-on (i.e., 6 a.m.) and did not significantly change after repeated treatment with either melatonin dose. The monkey's behavior on the morning after melatonin treatment was similar to that observed after placebo treatment.
Early morning (8 a.m.) treatment with a low (0.1 mg) dose of melatonin for four consecutive days significantly (p = 0.036) decreased the animals' motor activity compared to placebo, as measured from 9 a.m. to 12 noon (mean ± SD: 243.3 ± 35.0 vs. 117.5 ± 18.4 movements/min). Melatonin treatment, administered also at 8 a.m. for four consecutive days, with a higher, 3 mg dose (Fig. 2), markedly (p = 0.028) decreased motor activity during the day (6 a.m.-6 p.m.): 118.5 ± 20.3 vs. 31.9 ± 13.7 movements/min. Neither treatment significantly affected nocturnal motor activity (3.87 ± 2.49 vs. 0.51 ± 0.19 movements/min after a 3 mg dose). Although the monkeys' spontaneous daytime motor activity was significantly diminished by melatonin treatment in the absence of the experimenter, both animals seemed to be very eager to interact with their human observer when she was present. Their motor reactions (for example, reaching for toys or food), however, were notably slower in the presence of the investigator.

Withdrawal from melatonin after treatment with the 0.1 mg dose did not alter daytime motor activity nor the time of sleep onset. Upon withdrawal from the course of treatment with the 3 mg dose, the monkeys continued to fall asleep earlier for at least two days (Fig. 2). Withdrawal from neither melatonin dose significantly changed the time of awakening, which was synchronized to the "lights on" schedule.

At 10 a.m., an hour after the administration of placebo, serum melatonin levels in the two monkeys measured 2.5 and 3.6 pg/ml. An hour after administration of a 0.05 mg dose (also at 10 a.m.), serum levels of the hormone were increased to 88 and 126 pg/ml in the two animals.

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This preliminary study in two Macaca Nemestrina shows that the administration of a range of oral doses of melatonin decreases the monkeys' motor activity, and may facilitate sleep onset. A dose of 0.05 mg induced circulating concentrations of the hormone comparable to those that have been reported to occur nocturnally in non-human primates (Wilson and Gordon, 1989; Webley et al., 1991), and that we have observed in humans (Dollins et al., 1994; Zhdanova et al., 1995, 1996). A dose dependency of this behavior-altering effect was detected. The lowest melatonin dose tested, 0.025 mg, did not significantly change the animals' motor activity. The duration of the motor activity suppression was significantly longer after the 3 mg treatment when it was administered in the morning than after the administration of a 0.1 mg dose at the same time of the day. Remarkably, the animals' motivation to interact with the experimenter was not suppressed when the high pharmacologic dose (3 mg) of melatonin was administered, and, in fact, seemed to be even more pronounced, compared with what was observed after placebo treatment. This response might represent a mild anxiolitic property of the pineal hormone.
We were not able to detect a phase-shift in our animals' motor activity after melatonin treatment was interrupted. Their sleep pattern, according to the actigraph data, was not changed after a four-day course of treatment using the 0.1 mg dose, administered at 8 a.m. Neither did administration of a 3 mg dose of the pineal hormone at 8 a.m. cause a delay in the subjects' daytime motor activity, as might have been expected on the basis of the reported human phase-response curve for melatonin (Lewy and Sack, 1993). The timing of the monkeys' sleep onset remained advanced after treatment with the 3 mg dose ended. However, the timing of the sleep offset remained the same as it was during the pretreatment period (Fig. 2). It is very likely that the fixed environmental light-dark schedule prevented us from uncovering endogenous periodicities in the animals' behavior. However, actigraphic recording indicated that the monkeys were falling asleep while the lights were still on, thus, even after the treatment was withdrawn, their sleep onset occurred earlier than it did habitually, in spite of the environmental light. Another possible explanation for this phenomenon, observed after the treatment was interrupted, might be that, even after the treatment is over, direct or indirect effects of melatonin on motor activity and sleep initiation remain present. A direct effect could be due to the accumulation of melatonin in the animal's tissues, especially in lipid-containing tissues, which might thereby extend its acute sleep-promoting effect. The indirect effect may be conveyed by a sustained modulatory effect of melatonin on structures involved in the regulation of sleep propensity, causing its facilitation. Although these hypotheses remain to be tested, the overall decrease in the animals' motor activity for several days following withdrawal of treatment is remarkable.

This study, involving a diurnal species that is phylogenetically close to man, demonstrates that melatonin has behavioral effects in another diurnal primate similar to those repeatedly observed in nocturnal actigraphic recordings in humans. Our data suggest that certain non-human primates might serve as useful models in the study of the complex sleep promoting and behavioral effects of melatonin and related compounds.

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This work was supported in part by grants from the National Institutes of Health (1RO1 AG13667-01, MO1 RR 02172 and MO1 RR 00088-34) and the Center for Brain Sciences and Metabolism Charitable Trust.

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