Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular nucleus induces daytime secretion of melatonin

Despite a pronounced inhibitory effect of light on pineal melatonin synthesis, usually the daily melatonin rhythm is not a passive response to the surrounding world. In mammals, and almost every other vertebrate species studied so far, the melatonin rhythm is coupled to an endogenous pacemaker, i.e. a circadian clock. In mammals the principal circadian pacemaker is located in the suprachiasmatic nuclei (SCN), a bilateral cluster of neurons in the anterior hypothalamus. In the present paper we show in the rat that bilateral abolition of gamma-aminobutyric acid (GABA), but not vasopressin, neurotransmission in an SCN target area, i.e. the paraventricular nucleus of the hypothalamus, during (subjective) daytime results in increased pineal melatonin levels. The fact that complete removal of the SCN results in a pronounced increase of daytime pineal mRNA levels for arylalkylamine N-acetyltransferase (AA-NAT), i.e. the rate-limiting enzyme of melatonin synthesis, further substantiates the existence of a major inhibitory SCN output controlling the circadian melatonin rhythm.     

Encoding time of day and time of year by the avian circadian system

The most important zeitgeber for seasonal rhythmicity of physiology and behaviour in birds is the annual cycle of photoperiod. Regulatory mechanisms are less well understood in birds than in mammals since photic information can be perceived by photoreceptors in the retina and the pineal gland, as well as in the brain, and photoperiodic time measurement might be performed with reference to at least three autonomous circadian systems, the retina, the pineal gland and a hypothalamic oscillator. In many bird species, the pineal melatonin rhythm plays a central role in circadian organization. Durations of elevated melatonin in the blood reflect night length when animals are kept under natural photoperiodic conditions, as well as under different light/dark schedules in the laboratory. In the house sparrow, time of year is encoded in a particular melatonin signal, being short in duration and high in amplitude in long photoperiods and being long in duration and low in amplitude in short photoperiods, independent of whether the light zeitgeber is natural or artificial or varies in strength. Specific features of the melatonin signal are retained in vivo as well as in vitro when birds or isolated pineal glands are transferred to constant conditions. To regulate daily and seasonal changes of behaviour and physiology, melatonin may act at various target sites, including a complex hypothalamic oscillator that, unlike that in mammals, is not confined to a single cell group in the house sparrow. There is increasing evidence that interactions between two or more components of the songbird circadian pacemaking system are essential to encode and store biologically meaningful information about time, and thus provide the basis for photoperiodic time measurements and after effects in birds.     

Daily rhythms of serotonin metabolism in the medial hypothalamus of the chicken: effects of pinealectomy and exogenous melatonin

Indoleamine levels in punches of the medial hypothalamus containing the suprachiasmatic nuclei (SCN) of 4-week-old cockerels were determined by HPLC-EC. Melatonin levels in punches were determined by radioimmunoassay (RIA). Daily rhythms of serotonin (5-HT) and of its metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were observed; levels were higher at midnight than at midday. A daily rhythm with the same phase in punch melatonin content was also observed. Pinealectomy at 1 week after hatching abolished the 5-HIAA and melatonin rhythm in 4-week-old birds but did not abolish the 5-HT rhythm. Injections of melatonin (0.5 mg/kg) increased 5-HT, 5-HIAA and melatonin levels in the hypothalamic punches. These results indicate that circulating melatonin of pineal origin may act to increase 5-HT turnover and/or release in the SCN. They suggest a link between the circadian secretion of pineal melatonin and the regulation of 5-HT projections to the hypothalamus from the raphe nuclei in the brainstem of the chicken. We have previously shown that the rhythmic secretion of melatonin by the pineal is influenced by oscillators in the brain via the superior cervical ganglia. The results reported here indicate that melatonin in turn may regulate brain oscillators, suggesting a neuroendocrine loop within the avian circadian system.     

Melatonin directly resets the rat suprachiasmatic circadian clock in vitro.

The environmental photoperiod regulates the synthesis of melatonin by the pineal gland, which in turn induces daily and seasonal adjustments in behavioral and physiological state. The mechanisms by which melatonin mediates these effects are not known, but accumulating data suggest that melatonin modulates a circadian biological clock, either directly or indirectly via neural inputs. The hypothesis that melatonin acts directly at the level of the suprachiasmatic nucleus (SCN), a central mammalian circadian pacemaker, was tested in a rat brain slice preparation maintained in vitro for 2-3 days. Exposure of the SCN to melatonin for 1 h late in the subjective day or early subjective night induced a significant advance in the SCN electrical activity rhythm; at other times melatonin was without apparent effect. These results demonstrate that melatonin can directly reset this circadian clock during the period surrounding the day-night transition.     

Circadian rhythm of melatonin release from the photoreceptive pineal organ of a teleost, ayu (Plecoglossus altivelis) in flow-thorough culture

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow-through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free-running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six-hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.     

The Management of Sleep and Circadian Disturbance in Patients with Dementia

Sleep and circadian disturbances are common among patients with dementia. Symptomatic manifestations vary according to dementia subtype, with one commonly shared pattern—the irregular sleep-wake rhythm (ISWR), a circadian disorder characterized by an absence of the sleep-wake cycle’s circadian synchronization. Hypothesized mechanisms of circadian rhythm disturbance include suprachiasmatic nucleus (SCN) circadian pacemaker damage, pineal gland and melatonin secretion alterations, and reduced zeitbeigers and decreased input to the SCN. Management options include prescribed sleep/wake scheduling, light therapy, melatonin, physical and social activity, and mixed modality. The mixed-modality approach is the most effective method in treating ISWR. Pharmacologic interventions are controversial, with no evidence supporting their effectiveness while associated with multiple side effects. They should be used with caution and only be considered as short-term therapy. All treatment strategies should be individualized to achieve the best outcomes.     

Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day- and night-time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N-acetyltransferase (AA-NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h-pattern of melatonin secretion in SCN-lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN-lesioned and ganglionectomised rats, AA-NAT levels and pineal melatonin content were low (i.e. 12% of night-time control levels) for both day- and night-time periods. In SCN-lesioned rats, AA-NAT levels were intermediate (i.e. 30% of night-time control levels) and the 48-h secretion of melatonin presented constant levels not exceeding 20% of night-time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day-length (i.e. adaptation to seasons).     

The brain's master circadian clock: implications and opportunities for therapy of sleep disorders

The suprachiasmatic nuclei (SCN) residing in the anterior hypothalamus maintains a near-24-h rhythm of electrical activity, even in the absence of environmental cues. This circadian rhythm is generated by intrinsic molecular mechanisms in the neurons of the SCN; however, the circadian clock is modulated by a wide variety of influences, including glutamate and pituitary adenylate cyclase-activating peptide (PACAP) from the retinohypothalamic tract, melatonin from the pineal gland, and neuropeptide Y from the intergeniculate leaflet. By virtue of these and other inputs, the SCN responds to environmental cues such as light, social and physical activities. In turn, the SCN controls or influences a wide variety of physiologic and behavioral functions, including attention, endocrine cycles, body temperature, melatonin secretion, and the sleep-wake cycle. Regulation of the sleep-wake cycle by the SCN has important implications for development of therapies for sleep disorders, including those involving desynchronization of circadian rhythms and insomnia.     

Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release

The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 μM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.     

Portacaval anastomosis disrupts circadian locomotor activity and pineal melatonin rhythms in rats

To determine whether hepatic encephalopathy may be associated with a disruption of circadian function, the circadian rhythms of locomotor activity and pineal melatonin content were examined in an animal model of complete portal-systemic shunting, rats with a portacaval anastomosis (PCA). The locomotor activity rhythm of all sham-operated animals entrained normally to a light/dark cycle and exhibited a normal free-running period during exposure to constant light. In contrast, PCA led to a dampening of the locomotor activity rhythm in all animals and the abolishment of a circadian periodicity in the activity rhythm of approximately 50% of rats during exposure to either a light/dark cycle or constant light. While normal diurnal variations of pineal melatonin content were seen in sham-operated rats, the amplitude of this variation appeared to be decreased in PCA animals. The similar effects of PCA on both a behavioral and an endocrine circadian rhythm, known to be regulated by a common neural pacemaker, coupled with studies indicating that a variety of other circadian rhythms may be disrupted in both animals and humans with hepatic dysfunction, suggests that this circadian disturbance originates within the pacemaker or on one of its afferent/efferent pathways.     

Disturbance and strategies for reactivation of the circadian rhythm system in aging and Alzheimer's disease

Circadian rhythm disturbances, such as sleep disorders, are frequently seen in aging and are even more pronounced in Alzheimer's disease (AD). Alterations in the biological clock, the suprachiasmatic nucleus (SCN), and the pineal gland during aging and AD are considered to be the biological basis for these circadian rhythm disturbances. Recently, our group found that pineal melatonin secretion and pineal clock gene oscillation were disrupted in AD patients, and surprisingly even in non-demented controls with the earliest signs of AD neuropathology (neuropathological Braak stages I-II), in contrast to non-demented controls without AD neuropathology. Furthermore, a functional disruption of the SCN was observed from the earliest AD stages onwards, as shown by decreased vasopressin mRNA, a clock-controlled major output of the SCN. The observed functional disconnection between the SCN and the pineal from the earliest AD stage onwards seems to account for the pineal clock gene and melatonin changes and underlies circadian rhythm disturbances in AD. This paper further discusses potential therapeutic strategies for reactivation of the circadian timing system, including melatonin and bright light therapy. As the presence of melatonin MT1 receptor in the SCN is extremely decreased in late AD patients, supplementary melatonin in the late AD stages may not lead to clear effects on circadian rhythm disorders.     

Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats

Chronic treatment of methamphetamine (MAP) in rats desynchronized the locomotor activity rhythm from the light-dark cycle. When the activity rhythm was completely phase-reversed with respect to a light dark-cycle, 24 h profiles were examined for the clock gene (rPer1, rPer2, rBMAL1, rClock) expressions in several brain structures by in situ hybridization, and for the pineal as well as plasma melatonin levels. In the MAP-treated rats, the rPer1 expression in the suprachiasmatic nucleus (SCN) showed a robust circadian rhythm which was essentially identical to that in the control rats. Circadian rhythms in pineal as well as plasma melatonin were not changed significantly in the MAP-treated rats. However, robust circadian rhythms in the rPer1, rPer2 and rBMAL1 expressions detected in the caudate-putamen (CPU) and parietal cortex were completely phase-reversed in the MAP-treated rats, compared with those in the control rats, indicating desynchronization from the SCN rhythm. Such desynchronization was not observed in the circadian rhythms of clock gene expression in the nucleus accumbens and cingulate cortex. The circadian rClock expression rhythm in the MAP-treated rats was not phase-reversed in the CPU and parietal cortex. These findings indicate that the locomotor activity rhythm in rats is directly driven by the pacemaker outside the SCN, in which rPer1, rPer2 and rBMAL1 in the CPU and parietal cortex are involved.     

Tetrodotoxin administration in the suprachiasmatic nucleus prevents NMDA-induced reductions in pineal melatonin without influencing Per1 and Per2 mRNA levels

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a light-entrainable circadian pacemaker. Neurons in the SCN are part of a circuit that conveys light information from retinal efferents to the pineal gland. Light presented during the night acutely increases mRNA levels of the circadian clock genes Per1 and Per2 in the SCN, and acutely suppresses melatonin levels in the pineal gland. The present study investigated whether the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels requires sodium-dependent action potentials in the SCN. Per1 and Per2 mRNA levels in the SCN and pineal melatonin levels were measured in Syrian hamsters injected with tetrodotoxin (TTX) prior to light exposure or injection of N-methyl-D-aspartate (NMDA). TTX inhibited the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels. TTX did not, however, influence the ability of NMDA to increase Per1 and Per2 mRNA levels, though it did inhibit the ability of NMDA to suppress pineal melatonin levels. These results demonstrate that action potentials in the SCN are not necessary for NMDA receptor activation to increase Per1 and Per2 mRNA levels, but are necessary for NMDA receptor activation to decrease pineal melatonin levels. Taken together, these data support the hypothesis that the mechanism through which light information is conveyed to the pacemaker in the SCN is separate from and independent of the mechanism through which light information is conveyed to the SCN cells whose efferents suppress pineal melatonin levels.     

The bidirectional phase-shifting effects of melatonin on the arginine vasopressin secretion rhythm in rat suprachiasmatic nuclei in vitro

In vivo melatonin serves as a feedback signal to the circadian pacemaker located in the suprachiasmatic nuclei (SCN) and in vitro it phase advances the circadian rhythm of electrical activity in pacemaker cells. However, the occurrence and nature of phase shifting in secretion by cultured SCN neurons has not yet been established. Here we studied the effects of melatonin on the pattern of spontaneous arginine vasopressin (AVP) release in organotypic SCN slices. This culture mimicked the in vivo circadian AVP secretory rhythm, with low release during the subjective night and with peaks in secretion during the middle of subjective day. The endogenous period of the AVP secretory rhythm in organotypic culture ranged between 23 and 26 h, with the mean period of 24.1 +/- 0.3 h. Melatonin (10 nM) had variable effects on the pattern of AVP secretion depending on time of its application directly to the medium with organotypic SCN slices. When introduced at circadian time 22, 2 and 6 (the times corresponding to the late night and early day), melatonin delayed the AVP secretory rhythm by 1-4 h. When applied at circadian time 10 (late day), however, melatonin advanced the AVP secretory rhythm by about 2 h. At other circadian times, melatonin was ineffective. These results indicate that melatonin exhibits the bidirectional phase-shifting effects on circadian secretory rhythm clock, which depends on the time-window of its application.     

Two mechanisms of photoendocrine transduction in cultured chick pineal cells: pertussis toxin blocks the acute but not the phase-shifting effects of light on the melatonin rhythm

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin release. Light has two apparent effects on this melatonin rhythm: the first is an acute inhibition of melatonin output, the second is entrainment of the underlying pacemaker. These two effects could be mediated by the same or different mechanisms. Pertussis toxin, which acts to block the function of transducin and certain other G-proteins, blocked the acute effects of light on chick pineal cells, but not the ability of light pulses to induce phase-dependent phase shifts of the rhythm. There must, therefore, be at least two mechanistic pathways by which light affects chick pineal melatonin production. Transducin or other pertussis toxin-sensitive G-proteins would appear to be involved in the acute effects of light on the melatonin-synthesizing apparatus, but not in the effects of light on the circadian pacemaker which generates the melatonin rhythm. Some plausible pertussis toxin-sensitive mechanisms are discussed.     

持续光照或摘除松果腺对仓鼠视交叉上核神经元褪黑素敏感性的影响

为探讨视交叉上核 (SCN)神经元对褪黑素敏感性的昼夜节律改变的机制 ,先对仓鼠进行持续光照或摘除松果腺 ,然后制成下丘脑薄片 ,记录昼夜周期中 SCN神经元的自发单位放电 ,并观察其对褪黑素的反应。结果表明 ,取自正常光照动物的脑薄片 ,SCN对外源性褪黑素产生抑制反应的单位数量有昼多夜少的节律性。取自持续光照条件下或摘除松果腺动物的 SCN,对外源性褪黑素反应的昼夜节律性消失。持续光照条件下 ,起抑制反应的单位数量增加 ,引起反应的阈值无明显改变 ;在摘除松果腺后 ,起抑制反应的单位数量减少 ,引起反应的阈值升高。实验结果提示 ,SCN神经元对外源性褪黑素敏感性 ,与内源性褪黑素水平和褪黑素受体的适应性调制有关 ,还可能与松果腺和内源性褪黑素的其他神经生化作用有关。     

In vitro photic entrainment of the circadian rhythm in melatonin release from the pineal organ of a teleost,ayu (Plecoglossus altivelis) in flow-through culture

The effects of light on the circadian rhythm in melatonin release from the pineal organ of a teleost, ayu (Plecoglossus altivelis) were investigated in flow-through culture. Under the reversed light-dark (LD) cycle, the melatonin rhythm phase shifted as compared with those under the normal LD cycle. This phase shift persisted even under constant darkness (DD). Single 6-h light pulses starting at six different circadian phases under DD acutely suppressed melatonin release. Phase-dependent phase shifts in the circadian rhythm of melatonin release were also observed. The phase response curve to light pulses in the ayu pineal organ is typical of that found in many circadian systems. Thus, the ayu pineal organ should provide a useful model for analyzing the physiological and molecular basis of the entrainment mechanism of vertebrate circadian system.     

Circadian clock in cell culture: I. Oscillation of melatonin release from dissociated chick pineal cells in flow-through microcarrier culture

The avian pineal gland contains circadian oscillators that regulate the rhythmic release of melatonin. We have developed a dissociated chick pineal cell culture system in order to begin a cellular analysis of this vertebrate circadian oscillator. Dissociated pineal cells maintained in cyclic light conditions (LD 12:12) released melatonin rhythmically. The release of melatonin was elevated during the dark and low during the light. A circadian oscillation of melatonin release persisted for at least 5 cycles in constant darkness with a period close to 24 hr; however, there was a gradual damping of the amplitude. Analysis of the rhythm revealed that the observed damping was consistent with either desynchronization of multiple oscillators within the cultures or damping of individual oscillators. The circadian oscillation of melatonin release persisted for up to 4 cycles under conditions of constant light; however, the oscillation was strongly damped and the period of the oscillation was lengthened significantly. Thus, dissociated pineal cells express a persistent circadian oscillation of melatonin release in constant darkness, and properties of this oscillation are modulated by light treatment in vitro. This flow-through cell culture method for dissociated chick pineal cells should provide a useful model for the analysis of a vertebrate circadian system at the cellular level.     

Melatonin: a clock-output,a clock-input

In mammals, the circadian system is comprised of three major components: the lateral eyes, the hypothalamic suprachiasmatic nucleus (SCN) and the pineal gland. The SCN harbours the endogenous oscillator that is entrained every day to the ambient lighting conditions via retinal input. Among the many circadian rhythms in the body that are driven by SCN output, the synthesis of melatonin in the pineal gland functions as a hormonal message encoding for the duration of darkness. Dissemination of this circadian information relies on the activation of melatonin receptors, which are most prominently expressed in the SCN, and the hypophyseal pars tuberalis (PT), but also in many other tissues. A deficiency in melatonin, or a lack in melatonin receptors should therefore have effects on circadian biology. However, our investigations of mice that are melatonin-proficient with mice that do not make melatonin, or alternatively cannot interpret the melatonin message, revealed that melatonin has only minor effects on signal transduction processes within the SCN and sets, at most, the gain for clock error signals mediated via the retino-hypothalamic tract. Melatonin deficiency has no effect on the rhythm generation, or on the maintenance of the oscillation. By contrast, melatonin is essential for rhythmic signalling in the PT. Here, melatonin acts in concert with adenosine to elicit rhythms in clock gene expression. By sensitizing adenylyl cyclase, melatonin opens a temporally-restricted gate and thus lowers the threshold for adenosine to induce cAMP-sensitive genes. This interaction, which determines a temporally precise regulation of gene expression, and by endocrine-endocrine interactions possibly also pituitary output, may reflect a general mechanism by which the master clock in the brain synchronizes clock cells in peripheral tissues that require unique phasing of output signals.     

The superior cervical ganglia are not necessary for entrainment or persistence of the pineal melatonin rhythm in Japanese quail

The avian pineal exhibits a daily rhythm in the synthesis and secretion of the hormone, melatonin, which is involved in maintaining temporal order within the circadian system of some species. The pineal is richly innervated by sympathetic nerves which originate in the superior cervical ganglia (SCG) and, in the chicken, these nerves play a role in generating the melatonin rhythm. In the Japanese quail, the pineal melatonin rhythm can be entrained by light perceived directly by the pineal or by light perceived by the eyes. The role of the sympathetic innervation of the pineal was investigated in the Japanese quail by subjecting birds to bilateral superior cervical ganglionectomy (SCGX) and determining if SCGX either abolished the ability of retinally perceived light to entrain the pineal melatonin rhythm or if it disrupted the rhythm under constant darkness (DD). The results show that SCGX neither prevented entrainment of the pineal melatonin rhythm by retinally perceived light nor affected the rhythm expressed in DD. An entrainment pathway between the eyes and pineal exists in quail which does not involve the SCG.