Daily oscillation in melatonin synthesis in the Turkey pineal gland and retina: diurnal and circadian rhythms

The aim of the present study was to examine arylalkylamine N-acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light-dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night-time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high-amplitude melatonin rhythms in the turkey.     

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition

Summary Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments.In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study.     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.

Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .     

Daily Oscillation in Melatonin Synthesis in The Turkey Pineal Gland and Retina: Diurnal and Circadian Rhythms

The aim of the present study was to examine arylalkylamine N‐acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light‐dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night‐time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high‐amplitude melatonin rhythms in the turkey.     

The flavonoid myricetin reduces nocturnal melatonin levels in the blood through the inhibition of serotonin N-acetyltransferase

Melatonin is secreted during the hours of darkness and is thought to influence the circadian and seasonal timing of a variety of physiological processes. AANAT, which is expressed in the pineal gland, retina, and various other tissues, catalyzes the conversion of serotonin to N-acetylserotonin and is the rate-limiting enzyme in the biosynthetic pathway of melatonin. The compounds that modulate the activity of AANAT can be used to treat patients with circadian rhythm disorders that are associated with specific circadian rhythm alterations, such as shift work disorder. In the present study, we screened modulators of AANAT activity from the water extracts of medicinal plants. Among the 267 tested medicinal plant extracts, Myricae Cortex (Myrica rubra), Perillae Herba (Perilla sikokiana), and Eriobotryae Folium (Eriobotrya japonica) showed potent inhibition of AANAT activity. Myricetin (5,7,3′,4′,5′-pentahydroxyflavonol), a main component of the Myricae Cortex, strongly inhibited the activity of AANAT and probably block the access to the substrate by docking to the catalytic residues that are important for AANAT activity. Myricetin significantly decreased the nocturnal serum melatonin levels in rats. In addition, the locomotor activity of rats treated with myricetin decreased during the nighttime and slightly increased throughout the day. These results suggest that myricetin could be used as a therapy to increase nighttime alertness by changing the circadian rhythm of serum melatonin and locomotor activity.     

Abnormality of circadian rhythm of serum melatonin and other biochemical parameters in fibromyalgia syndrome

Fibromyalgia syndrome (FMS) is a complex chronic condition causing widespread pain and variety of other symptoms. It produces pain in the soft tissues located around joints throughout the body. FMS has unknown etiology and its pathophysiology is not fully understood. However, abnormality in circadian rhythm of hormonal profiles and cytokines has been observed in this disorder. Moreover, there are reports of deficiency of serotonin, melatonin, cortisol and cytokines in FMS patients, which are fully regulated by circadian rhythm. Melatonin, the primary hormone of the pineal gland regulates the body's circadian rhythm and normally its levels begin to rise in the mid-to-late evening, remain high for most of the night, and then decrease in the early morning. FMS patients have lower melatonin secretion during the hours of darkness than the healthy subjects. This may contribute to impaired sleep at night, fatigue during the day and changed pain perception. Studies have shown blunting of normal diurnal cortisol rhythm, with elevated evening serum cortisol level in patients with FMS. Thus, due to perturbed level of cortisol secretion several symptoms of FMS may occur. Moreover, disturbed cytokine levels have also been reported in FMS patients. Therefore, circadian rhythm can be an important factor in the pathophysiology, diagnosis and treatment of FMS. This article explores the circadian pattern of abnormalities in FMS patients, as this may help in better understanding the role of variation in symptoms of FMS and its possible relationship with circadian variations of melatonin, cortisol, cytokines and serotonin levels.     

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.     

Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR

A second gene encoding a functional tryptophan hydroxylase activity has recently been described (TPH2), which is expressed abundantly in brainstem, the primary site of serotonergic neurons in the CNS. As serotonin (5-HT) has an important role as a precursor of the nocturnal synthesis of the pineal gland hormone, melatonin, it was of interest to determine the relative expression of TPH1 and 2 mRNA in the rat pineal during the light:dark (L:D) cycle using sensitive real-time RT-PCR assays which were developed for each TPH isoform. TPH1 mRNA expression was 105-fold more abundant in rat pineal than TPH2, and showed a significant approximately 4-fold nocturnal increase in expression which may contribute to the previously described nocturnal increase in pineal tryptophan hydroxylase activity. TPH2 expression within the gland showed no significant variation with time of day and was very low (approximately 300 copies/gland) indicating expression in the small proportion of "non-pinealocyte" cells in the gland.     

Arrest of the circadian pacemaker driving the pineal melatonin rhythm in hibernating golden hamsters, Mesocricetus auratus

Pineal melatonin rhythm in golden hamsters was abolished during hibernation. After arousal in darkness, pineal melatonin increased rapidly regardless of whether the arousal was induced during the day or at night. Rapid increase of pineal melatonin after arousal was markedly diminished in animals exposed to light. In hamsters aroused at midnight, the melatonin rhythm in constant darkness ran with the reversed phase relative to hamsters aroused at noon. Since after arousal the melatonin rhythm obviously starts anew from the same phase, we conclude that the circadian pacemaker driving the rhythm might be arrested during hibernation at the day-time phase.     

Expression of melatonin synthesis genes is controlled by a circadian clock in the pike pineal organ but not in the trout

The photosensitive teleost pineal organ exhibits a daily rhythm in melatonin production. In most teleosts, including the pike, this is driven by an endogenous pineal clock. An exception is the trout, in which the pineal melatonin rhythm is a direct response to darkness. This fundamental difference in the regulation of melatonin production in two closely related species provides investigators a novel opportunity to study the molecular mechanisms of vertebrate clock function. We have studied the circadian regulation of mRNA encoding two melatonin synthesis enzymes by Northern blot analysis. These two enzymes are serotonin N-acetyltransferase (AA-NAT), the penultimate enzyme in melatonin synthesis, and tryptophan hydroxylase (TPH), the first enzyme in melatonin synthesis. A clock controls expression of both AA-NAT and TPH mRNAs in the pineal organ of pike, but not that of trout, in which the levels of these mRNAs are tonically elevated. A parsimoneous explanation of this is that a single circadian system regulates the expression of both AA-NAT and TPH genes in most teleosts, and that in trout this system has been disrupted, perhaps by a single mutation.     

褪黑素对大鼠下丘脑薄片视交叉上核神经元放电昼夜节律性的调制

先用持续光照和松果腺切除预处理大鼠,然后制成下丘脑薄片,记录其视交叉上核（ＳＣＮ）神经元自发放电,观察其昼夜变化和褪黑素（ＭＥＬ）对它的影响。实验结果表明：⑴在正常光照（光照：黑暗＝１２：１２）条件下,ＳＣＮ神经元自发放电频率呈现昼夜低的节律性。在昼夜时间（ＣＴ）６－８出现放电高峰,频率约为８．３Ｈｚ;在ＣＴ１８－２０出现低谷,频率约为３．８Ｈｚ。松果腺切除后,ＳＣＮ神经元自发放电的昼夜节律性基本