Sleep and Melatonin in Diurnal Species

Transcription

1 CHAPTER 17 Sleep and Melatonin in Diurnal Species Irina V. Zhdanova Abstract Melatonin secretion, occurring at night in both diurnal and nocturnal species, provides an important circadian signal for initiating different types of behavior. In diurnal vertebrates, e.g., humans, macaques, zebrafish, this pineal hormone also has a pronounced acute effect on sleep. This effect on homeostatic sleep regulation is mediated via specific melatonin receptors and has a distinct dose-dependency, reaching plateau at near-physiological concentrations. Introduction A 24-hour period of rotation of our planet on its axis and its 12-month period of rotation around the Sun determine two major environmental rhythms, the daily and annual variations in ambient light, temperature and solar radiation. Since life on Earth depends on the energy coming from the Sun, these regular variations in energy flow require specific adaptive mechanisms to provide for critical chemical reactions and defend from deadly overheating, freezing or radiation damage. Predicting the major changes in the environment helps to adjust the physiological mechanisms in advance and, thus, increase the probability of the organism s survival. To anticipate changes in the environmental light and temperature, the organisms developed a clock system, which allows to properly time the physiological events. The daily period of the circadian clock is close to 24 hours (circa near; dia day) and can be precisely entrained to 24-hour period by light perceived through the photoreceptors. One of the intrinsic features of the photoreceptors is their ability to synthesize melatonin (N-acetyl-5-methoxytryptamine). The circulating amino acid L-tryptophan is the precursor for melatonin synthesis; it is converted to serotonin (5HT) by a two-step process, catalyzed by the enzymes tryptophan hydroxylase and 5-hydroxytryptophan decarboxylase. This process involves serotonin s N-acetylation, catalysed by N-acetyltransferease (NAT), and then its methylation by hydroxyindole- O-methyltransferase (HIOMT) to produce melatonin. Melatonin production is strictly periodic, occurring only at night, and is acutely suppressed by nighttime light exposure. Apparently, photoreceptors use melatonin for their local purposes, i.e., as a paracrine agent. However, the evolution of one of the major photoreceptory organs, the pineal gland (epiphysis cerebri), led it to produce the excessive amounts of melatonin and release it into the blood stream and cerebrospinal fluid. Due to high lipid solubility and, thus, ability to cross the cell membranes, melatonin can be rapidly distributed throughout the entire organism. As a result, a periodic production of melatonin by the pineal gland became a universal endocrine message of nighttime. Numerous studies on the role of melatonin in different physiological processes suggest that this nocturnal hormone plays an important role in diverse tissues and organs. However, due to different physiological and behavioral processes occurring at daytime and nighttime in diurnal and nocturnal species, melatonin may signal the onset of different activities and can affect similarly or differently the biological events occurring in these two major types of organisms. Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel Cardinali Eurekah.com.

2 2 Melatonin: Biological Basis of Its Function in Health and Disease Figure 1. Sleep efficiency in subjects with age-related insomnia following melatonin or placebo treatment. *p<0.05 (see ref. 9) For example, for diurnal species, increase in melatonin production would signal the onset of their nighttime rest. In contrast, for nocturnal animals, melatonin production would coincide with the onset of their daytime activity. Humans, being diurnal, secrete melatonin during their habitual hours of sleep and the onset of melatonin secretion correlates with the onset of their evening sleepiness. 1-3 The earliest studies, conducted in 1950s by Aaron Lerner and his associates showed that melatonin administration could increase sleepiness in humans and further studies have substantiated this finding (for review 4 ). They also showed that circulating melatonin levels, similar to those observed under physiological conditions, can induce sleepiness, strongly suggesting that melatonin has a physiological role in human sleep regulation. It is now well accepted that melatonin has two major ways of affecting sleep. It can act acutely on the mechanisms involved in sleep homeostasis, thereby making one sleepy, or it can modulate the mechanisms that impart circadian rhythmicity to the temporal pattern of sleep propensity. The contribution of each of these actions to the hormone s net effect depends on the time of its administration, the nocturnal or diurnal lifestyle of the species studied and the individual s sensitivity to melatonin. Melatonin and Circadian Regulation of Sleep The circadian effects of melatonin appear to be almost universal and, largely, similar among the diurnal and nocturnal species. This can be explained by the similarities in the temporal organization of their circadian system. Indeed, in both diurnal and nocturnal animals, the neurons of the major circadian clock, the suprachiasmatic nuclei (SCN) of the hypothalamus, are normally active during the day and slow down at night. The activation of SCN neurons has an inhibitory effect on the pineal gland, defining a nocturnal pattern of melatonin secretion. If SCN neurons are activated at night, e.g., by environmental light perceived by the retina, melatonin production declines. Melatonin, in turn, can acutely attenuate the activity of SCN. This melatonin action is likely to support a normal decline in the activity of the SCN at night, further promoting melatonin secretion and contributing to an overall increase in the amplitude of circadian body rhythms. A temporal and functional interplay between melatonin and SCN, and their response to environmental light, promote a temporal alignment of multiple circadian body rhythms with each other (internal synchronization) and with the periodic changes in the environment (external synchronization). In addition to an acute inhibition of SCN activity, melatonin administration can also produce a shift in the circadian phase of SCN activity, either advancing or delaying its onset. The direction of the phase-shift depends on the time of melatonin treatment, i.e., administration of melatonin in the late afternoon can advance the circadian clock, while early-morning treatment can cause a phase delay. 5 Studies conducted in vitro suggest that a chronobiological effect

3 Sleep and Melatonin in Diurnal Species 3 Figure 2. Melatonin and diazepam affect locomotor activity in zebrafish via specific membrane receptors. Pretreatment with specific antagonist for melatonin receptors, luzindole, blocked the decline in locomotor activity induced by A) melatonin, but not by B) diazepam or C) pentobarbital. Pretreatment with specific benzodiazepine receptor antagonist, flumazenil, blocked reduction in locomotor activity following B) diazepam, but not (a) melatonin or C) pentobarbital treatment. Control solutions are vehicles for each treatment used. Data are expressed as mean ±SEM group changes (%) in daytime locomotor activity, measured for 2-hours after treatment, relative to basal activity. N=30, each group; **p<0.01 (see reference 15). of melatonin, i.e., the induction of circadian phase shift, is likely to be explained by its direct effect on SCN neurons via specific, most likely, MT2 receptor. 6,7 Although the magnitude of the melatonin-induced phase shifts can vary between the species, the overall phenomenon appears to be well conserved. Such phase shifts in the circadian oscillation of SCN activity may change the physiological and behavioral rhythmicity of the entire organism, including the sleep-wake cycle, and can significantly affect the sleep quality in both nocturnal and diurnal species. In humans suffering from circadian sleep disorders, daily melatonin treatment can help to reinforce the circadian synchronization with the environment and entrain the physiological rhythms to a 24-hour cycle. 8 Melatonin and Homeostatic Regulation of Sleep A common phenomenon that the increase in sleepiness is roughly proportional to the duration of prior wakefulness is termed sleep homeostasis, reflecting the need to balance time spent asleep and time spent awake. Currently, the processes involved in homeostatic regulation of sleep wakefulness are poorly understood, reflecting our incomplete knowledge of the physiological significance of the sleep phenomenon per se. Melatonin treatment is typically found to increase sleepiness and to promote sleep in healthy humans, when administered during the day, or to improve overnight sleep in insomniacs 9 (Fig. 1). These effects are observed soon after treatment (within about 30 min) and in response to melatonin doses that induce physiological (i.e., around 100 pg/ml) and pharmacological (over 200 pg/ml) circulating levels of the hormone in human blood. Technical and ethical issues preclude many types of studies that could help to elucidate the mechanisms of melatonin action on human sleep. It is, thus, necessary to conduct such studies using appropriate animal models. However, studies on the effects of melatonin on sleep in nocturnally active rodents produced inconsistent findings when the pharmacological doses were used, with no effect of physiological doses of the hormone detected This is likely to be explained by differences in the predominant timing of sleep relative to the environmental light-dark cycle and synchronized with it circadian physiology in nocturnal and diurnal species. Since both types of species secrete melatonin only at night, the nocturnal animals sleep when their melatonin levels are low, while diurnal species, including humans, sleep when their melatonin production is high. Thus, it appears that nocturnal animals normally are not sensitive to the acute sleep-promoting

4 4 Melatonin: Biological Basis of Its Function in Health and Disease Figure 3. Melatonin and conventional sedatives promote rest behavior in larval zebrafish. Melatonin, diazepam and sodium pentobarbital (barbital) significantly and dose-dependently reduced zebrafish locomotor activity A,C, E) and increased arousal threshold B, D, F). Each data point represents mean ±SEM group changes in a 2-hour locomotor activity relative to basal activity, measured in each treatment or control group for 2 hours prior to treatment administration. Arousal threshold data are expressed as the mean ±SEM group number of stimuli necessary to initiate locomotion in a resting fish. Closed diamond treatment, open square vehicle control; N=20, each group (see reference 15). effects of melatonin, and thus cannot be used to study the homeostatic mechanisms of melatonin action on sleep. An optimal animal for studying the mechanisms of melatonin action on human sleep would be a diurnal vertebrate, phylogenetically close to human, with robust circadian rhythms and consolidated nocturnal sleep episode, with the pineal gland secreting melatonin throughout the night and one being sensitive to the sleep-promoting effects of the hormone. Further significant benefits in elucidating the molecular mechanisms of melatonin action on sleep would also be warranted if the genetic code of such a species were available and its effective manipulation feasible. To have all these features in one laboratory animal species is problematic, however a combination of species can provide a satisfactory answer to a number of questions. So far, the effects of melatonin on sleep initiation and maintenance are being studied in diurnal non-human primates, macaques, 13,14 and in a diurnal teleost, zebrafish. 15 The clear advantages of the non-human primate model is in the phylogenetic proximity of these animals to humans, major similarities in human and macaque brain organization and functioning and, importantly, presence of a characteristic consolidated nocturnal sleep period, with several sleep cycles, consisting of quiet (NREM) and paradoxical (REM) sleep. 17 Although lacking all these important advantages of a non-human primate model, the zebrafish also have robust circadian rhythm of activity and melatonin secretion, 15,16 widely distributed functional melatonin receptors 18 and nighttime rest behavior showing critical behavioral similarities with mammalian sleep. 15 Furthermore, zebrafish model has additional and very significant advantages for deciphering the molecular mechanisms of sleep regulation by melatonin. Those include a small size and

5 Sleep and Melatonin in Diurnal Species 5 Figure 4. Effects of a long-term melatonin treatment with escalating melatonin doses. Mean (SEM) sleep onset times in six monkeys during administration of escalating melatonin doses (5-320 µg/kg, 3 days each dose; black bars), compared to periods of placebo treatment (white bars). PLC1- basal placebo treatment; PLC2- washout placebo treatment. p<0.05 for all melatonin doses, relative to placebo (see reference 14). high reproductive rate, allowing to conduct high throughput behavioral assays. The transparency of zebrafish larvae allows direct non-invasive single-cell recording of neuronal activity in multiple brain areas, using calcium-sensitive dyes. 19 The ability of zebrafish larvae to absorb different substances via skin permits non-invasive and/or long-term administration of pharmacological treatments. Most importantly, an enormous accumulated experience in conducting large-scale genetic screens and multiple mutant zebrafish phenotypes available, 20 with the genetic and physical maps being near completion, make zebrafish a potentially outstanding model for sleep research. Studies conducted in many laboratories demonstrate that melatonin has specific binding sites in various central and peripheral tissues of different animal species. 21 The cloning of a family of G protein-coupled melatonin receptors 22 opened new possibilities for the understanding of melatonin s action in various target cells. These receptors inhibit camp accumulation via a pertussis-toxin sensitive G-protein in most of the tissues tested but, in some tissues, can also affect other signal transduction pathways, e.g., those involving cgmp, diacylglycerol or GABA. 23 Melatonin s high lipid solubility also suggests that this hormone may well have some direct actions inside cells, in addition to its actions on cellular membrane receptors. The exact mechanisms underlying melatonin s effects on homeostatic sleep regulation remain to be elucidated. A zebrafish model showed that acute effect of melatonin on sleep is mediated via specific melatonin receptors and can be blocked by specific melatonin receptor antagonists (Fig. 2). Interestingly, in both primate and teleost animal models, similar to that observed in humans, the effect of melatonin on sleep initiation show a distinct dose dependency, reaching peak activity at high physiological or low pharmacological concentrations (Fig. 3A). A further increase in dose used does not significantly change the magnitude of the effect (Fig. 4). In contrast, other hypnotic agents, none of which are part of a normal neurochemical

6 6 Melatonin: Biological Basis of Its Function in Health and Disease Figure 5. Earlier sleep onset during long-term treatment with a physiological dose of melatonin. Mean (SEM) sleep onset times in four monkeys during six consecutive one-week periods of treatment with placebo (PLC, white bars) or 5 µg/kg melatonin (black bars). PLC1- basal placebo treatment; PLC2- washout placebo treatment. p<0.05 for all melatonin treatment weeks, relative to basal (see reference 14). system of sleep regulation, typically show a gradual dose-dependent increase in their hypnotic property, inducing general anesthesia in both animals and humans, when used at high concentrations (Fig. 3B,C). These differences between the effects of melatonin and those of the commonly used hypnotics might be explained by different mechanisms involved in melatonin s sleep-promoting effects. Unlike most of the existing hypnotics, melatonin does not appear to act via GABA-ergic system, since in humans, 24 macaques or zebrafish a sleep-promoting effect of melatonin is not attenuated by specific benzodiazepine receptor antagonist, flumazenil (Fig. 2). Pharmacological analysis using melatonin receptor ligands with different degree of affinity to MT1 and MT2 specific melatonin receptors suggest that the acute effect of melatonin on sleep is mediated through MT1 receptor (Zhdanova et al, in press). These receptors were documented in multiple brain areas, including those involved in sleep regulation, e.g., hypothalamus or thalamic nuclei. They are saturated at relatively low melatonin levels, close to those observed normally, which might explain why melatonin efficacy reaches plateau at near-physiological concentrations. Other lines of evidence suggest that the effect of melatonin on sleep in humans might also be related to the hormone s ability to induce hypothermia. 25 Indeed, the nocturnal decline in body temperature is concurrent with the increase in melatonin release from the pineal gland. Both of these rhythmic patterns are remarkably stable and maintain their characteristic phase relationships after light-induced circadian shifts. At present, the mechanisms through which melatonin can decrease core body temperature are not clear. The hypothermic effect of melatonin might result from an overall reduction in alertness or may entirely depend on the peripheral action of the hormone, since it has been shown to enhance heat loss, perhaps via peripheral vasodilation. 26 However, the fact that zebrafish, a cold-blooded animal is sensitive to the effect of melatonin on sleep suggest that a thermoregulatory mechanisms might not be the primary one in the ability of melatonin to promote sleep behavior. The use of currently available hypnotics has some drawbacks, including changes they induce in sleep architecture, development of tolerance, requiring increase in the dose used, and manifestation of a post-treatment grogginess in the morning. Human studies show that melatonin treatment does not appear to either significantly affect normal nighttime sleep or increase morning-after sleepiness. The effects of both physiological and pharmacological doses of melatonin are not accompanied by any dramatic changes in electrophysiological sleep architecture, i.e., temporal distribution and duration of different sleep stages. Similarly, we have reported earlier that an increase in circulating melatonin levels within the physiological range is not an imperative signal for sleep, but rather a gentle promoter of general relaxation and seda-

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