Rhythmic Production Of Melatonin Research Articles

From the Pineal Gland to the Central Clock in the Brain: Beginning of Studies of the Mammalian Biological Rhythms in the Institute of Physiology of the Czech Academy of Sciences.

The Institute of Physiology of the Czech Academy of Sciences (CAS) has been involved in the field of chronobiology, i.e., in research on temporal regulation of physiological processes, since 1970. The review describes the first 35 years of the research mostly on the effect of light and daylength, i.e., photoperiod, on entrainment or resetting of the pineal rhythm in melatonin production and of intrinsic rhythms in the central biological clock. This clock controls pineal and other circadian rhythms and is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. During the early chronobiological research, many original findings have been reported, e.g. on mechanisms of resetting of the pineal rhythm in melatonin production by short light pulses or by long exposures of animals to light at night, on modulation of the nocturnal melatonin production by the photoperiod or on the presence of high affinity melatonin binding sites in the SCN. The first evidence was given that the photoperiod modulates functional properties of the SCN and hence the SCN not only controls the daily programme of the organism but it may serve also as a calendar measuring the time of a year. During all the years, the chronobiological community has started to talk about "the Czech school of chronobiology". At present, the today´s Laboratory of Biological Rhythms of the Institute of Physiology CAS continues in the chronobiological research and the studies have been extended to the entire circadian timekeeping system in mammals with focus on its ontogenesis, entrainment mechanisms and circadian regulation of physiological functions. Key words: Pineal, Melatonin, AA-NAT rhythm, Light entrainment, Photoperiod, SCN clock.

Immune-pineal-ocular axis in amphibians: unveiling a novel connection?

Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in the ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3h over 24h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10h) or at night (22h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retina light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group.

Effect of blue and red monochromatic light during incubation on the early post-embryonic development of immune responses in broiler chicken

ABSTRACT 1. The light regime during incubation can influence embryonic and post-embryonic life and its effects can be mediated by rhythmic melatonin production in the embryonic pineal gland. 2. This study explored whether the incubation of chick embryos under red or blue monochromatic light, which induces maximum and minimum melatonin production, respectively, can influence the development and reactivity of the immune system in chicks. 3. In hatchlings, basal expression of immune genes (AvBD-1, PSEN-1, and IL-6) was evaluated in the duodenum using real-time PCR. The expression of these genes was measured weekly for three weeks after hatching, 3 h after intraperitoneal lipopolysaccharide (LPS) injection. At these times, the heterophil/lymphocyte ratio (He/Ly) was evaluated on blood smears, plasma immunoglobulin Y (IgY) concentrations by ELISA and IL-6 gene expression in the spleen by real-time PCR were determined. 4. During development, the He/Ly ratio and plasma IgY concentration were not significantly influenced by the light quality during incubation. Red light increased gene expression of AvBD-1 in hatchlings and IL-6 in two-week-old chickens compared to birds incubated under blue light. The expression of IL-6 after LPS stimulation increased in an age-dependent manner, both in the duodenum and the spleen, reflecting the maturation of the immune system. 5. The results suggested that red light may increase the local immune response in the gut immediately after hatching, but this effect was not apparent during later development.

The effect of a complex of melatonin, aluminum oxide and polymethylsiloxane on the cellular composition of the mice spleen kept in round-the-clock lighting conditions

It is known that the circadian rhythm of melatonin production depends on the intensity of illumination. Violation of the light regime leads to suppression of melatonin synthesis and the development of desynchronosis, which increases the risk of developing a number of pathologies. In this regard, it is relevant to search for opportunities to restore disturbed circadian rhythms and, especially, to correct immune dysfunctions that occur in these situations.The aim of this study was to examine the effect of a complex of melatonin, aluminum oxide and polymethylsiloxane on the lymphocytes of the spleen of mice kept under round-the-clock lighting.Materials and methods. Mice of the C57Bl/6J line were kept under round-the-clock lighting for 14 days, against which they were intragastrically injected with distilled water, aluminum oxide with polydimethylsiloxane, melatonin and a complex of melatonin, aluminum oxide and polymethylsiloxane (a new drug developed by the Research Institute of Clinical and Experimental Lymphology – Branch of the Federal Research Center Institute of Cytology and Genetics SB RAS; Patent of Russian Federation No. 2577580, 2016), represented by a complex of porous material (aluminum oxide with polydimethylsiloxane) and melatonin, immobilized in the pores, from which it is gradually released in a liquid medium. Intact animals kept under the light regime of ST 12/12 and under round-the-clock lighting served as a control. Immunophenotyping of spleen B- and T-lymphocytes was performed on a flow cytofluorimeter with monoclonal antibodies APC CD3 and FITC CD19. For studying the distribution of cells by stages of the cell cycle in splenocytes, the amount of intracellular DNA was measured by the level of inclusion of propidium iodide.Results. Flow cytometry of the distribution of B- and T-lymphocytes of the spleen in male mice of the C57Bl/6J line kept under round-the-clock lighting conditions (KO 24/0 h) revealed a decrease in the percentage of B-lymphocytes and an increase in the number of T-lymphocytes, compared with animals kept under standard lighting conditions (the light/dark photoperiod – 14/10 hours). The ratio of CD19+/CD3+ lymphocytes of the spleen in mice under the conditions of KO significantly decreases (1.5 times) compared to intact animals (p ≤ 0.001). The administration of pure and modified melatonin (Complex M) to animals kept under round-the-clock lighting conditions has an equally pronounced normalizing effect on the cellular composition of B- (CD19) and T- (CD3) lymphocytes of the spleen, bringing the values of the studied parameters to the control values of the intact animals (p ≤ 0.001) Round-the-clock lighting affects the proliferative potential of splenocytes, reducing the number of cells in the G2/M phase, compared with animals treated with melatonin (p ≤ 0.050). The introduction of melatonin leads to an increase in the percentage of cells in the G2/M phase relative to the placebo group (p ≤ 0.050). In the group of mice treated with Complex M, the greatest increase in cells at the S + G2/M phases and the highest percentage of cells at the G2/M phase were revealed compared to the placebo control group (p ≤ 0.050).Conclusion. The complex of melatonin, aluminum oxide and polymethylsiloxane has additional immunotropic properties in relation to the modifier molecule, which, apparently, are due to the joint immunostimulating effect of melatonin and the lymphostimulating effect of the sorbent. Melatonin in the composition of the complex shows its properties more stably.

NEW DEFINITIONS IN THE MANAGEMENT OF NEONATES WITH OXIDATIVE STRESS DISEASES

Introduction. The problem of premature infants’ management remains extremely relevant and has acute medical, social and economic significance. Tissue damage due to oxidative stress is an important pathogenetic factor in the leading diseases among premature infants. Сorrection of the antioxidant status of a newborn is one of the promising areas in the therapeutic approach to oxidative stress diseases. While searching for some safe and effective medications, researchers pay great attention to melatonin, a neurohormone secreted by the pineal gland, which has an outstanding antioxidant and anti-inflammatory effect. The aim of the work is to analyze current data on the ontogenesis of the pineal gland, the world experience of melatonin therapy for the etiopathogenetic justification of the use of melatonin in the management of newborns with perinatal pathology and to highlight problems and prospects. Results. Reactivity and adaptability of the pineal gland is an important link in the compensation of perinatal pathology. Formation of neuroendocrine functional activity of the pineal gland occurs postnatally, so the fetus totally depends on transplacental melatonin. Premature infants have a significant delay in the rhythmic production of melatonin and its significant deficiency compared with fullterm newborns, in addition, the existence of desynchronosis in neonates with perinatal pathology and premature babies is a proven fact. Current scientific data on the use of exogenous melatonin as an antioxidant replacement therapy for oxidative stress diseases in premature infants is quite reasonable and promising. Numerous studies have proven the effectiveness of melatonin in the treatment of perinatal pathology such as hypoxic brain injury, respiratory distress syndrome, bronchopulmonary dysplasia, retinopathy of prematurity, neonatal sepsis and usage of melatonin as an antenatal neuroprotective agent. However, there is no single view on the target therapeutic concentration and dosage of melatonin in neonatal practice. Controversial pharmacokinetic data is one of the limitations of the widespread use of melatonin in the management of premature infants. For the etiopathogenetic substantiation of the optimal therapeutic dose of melatonin, further studies of the adaptive and compensatory capabilities of the pineal gland in premature infants are needed. Conclusions. The direction of future clinical research on the expansion of the melatonin usage panel in premature infants, the establishment of a therapeutic range and the possibility of dynamic adjustment of the dose depending on pineal function is promising.

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.

Intense blue light therapy during the night-time does not suppress the rhythmic melatonin biosynthesis in a young boy.

Melatonin is a hormone predominantly synthesized and secreted during the night by the pineal gland. Artificial light at night, especially its blue part, acutely suppresses the melatonin production. Th e aim of the present study was to find out, whether an intense blue light phototherapy of severe hyperbilirubinemia, may suppress the melatonin production during the night when the eyes will be properly protected by a sleep mask. The main melatonin metabolite, 6-sulphatoxymelatonin was measured in urine in a nine-year old boy suffering from the Crigler-Najjar syndrome type I. The boy was treated during the sleep period with an intense blue light (to 1800 lx) 10 h/day, since his birth. During the phototherapy, his eyes were protected with a sleep mask. The concentration of 6-sulphatoxymelatonin was determined in the first morning urine and urine collected afternoon during the six days. The patient was exposed to phototherapy for three nights, two nights without and the last one with the treatment. The control urine samples were obtained from 8 healthy nine-year old boys. The level of 6-sulphatoxymelatonin was measured by radioimmunoassay and the data were normalized to urinary creatinine. A distinct melatonin production rhythm was found and 6-sulphatoxymelatonin concentration in urine of the patient was comparable with the values obtained by the control group. No differences in 6-sulphatoxymelatonin levels were found between the nights with and without the phototherapy applied. We conclude that the whole night treatment of hyperbilirubinemia with intense blue light has negligible side effect on the rhythmic melatonin production, when the eyes are sufficiently protected by the sleep mask.

Peculiarities of the psychic state and quality of life in patients with duodenal ulcer in the context of chronomedicine

Most clinicians consider duodenum ulcer as a psychosomatic disease. Objective: To show the interdependence of this condition and mental disorders and their relation to disturbances of melatonin production . Materials and Methods: 15 patients with seasonal DU and 15 healthy subjects of the control group were examined during 3 years using laboratory, endoscopic, and standard psychodiagnostic methods. Results. It was found that all patients with exacerbation of DU experienced enhanced anxiety, reduced background mood, and impaired quality of life based on general health and mental health scoring scales. The circadian rhythm of melatonin production was markedly distorted throughout the observation period but especially during exacerbations of the disease. Conclusions. The results indicate a high degree of correlation between DU and mental disorders caused by impaired production of melatonin. It suggests common etiological mechanisms of DU and psychosomatic symptom complex.

Subfunctionalization of arylalkylamine N-acetyltransferases in the sea bass Dicentrarchus labrax: two-ones for one two.

Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N-acetyltransferase (Aanat: serotonin→N-acetylserotonin) and acetylserotonin-O-methyltransferase (Asmt: N-acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost-specific whole-genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co-expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N-acetylserotonin and N-acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.

The adrenergic-regulated CRTC1 and CRTC2 phosphorylation and cellular distribution is independent of endogenous SIK1 in the male rat pinealocyte

Salt inducible kinase 1 (SIK1) has been reported to repress cAMP-response element binding protein (CREB)-mediated gene transcription by causing the nuclear export of CREB-regulated transcription coactivators (CRTCs) through phosphorylation. Although the repressor role of SIK1 in suppressing the expression of arylalkylamine N-acetyltransferase, the enzyme that controls the daily rhythm in melatonin production in the rat pineal gland, has been established, whether SIK1 regulates the phosphorylation and localization of CRTC1 and CRTC2 in this tissue remains unclear. The present study found that overexpressing SIK1 in NE-stimulated rat pinealocytes could increase the phosphorylation of CRTC1 and CRTC2, reduced selectively the nuclear level of CRTC2 (but not that of CRTC1), and elevated the cytosolic levels of both CRTC1 and CRTC2. In contrast, transient knockdown of endogenous SIK1 had no effect on the phosphorylation or distribution of CRTC1 and CRTC2 in norepinephrine (NE)-stimulated pinealocytes. Our results also showed that adrenergic blockade during NE stimulation led to a rapid rephosphorylation and decline in the nucleus levels of CRTC1 and CRTC2; however SIK1 knockdown had no effect on this rapid rephosphorylation. Moreover, studies with kinase inhibitors revealed that kinase(s) sensitive to KT5823 appeared to be involved in this rapid rephosphorylation. Together, these results indicate that although overexpressing SIK1 can phosphorylate CRTC1 and CRTC2 in the NE-stimulated pinealocyte, the endogenous SIK1, in spite of its induction by NE, does not appear to be the main regulator of the phosphorylation and intracellular localization of these two coactivators.

Comparative study of pineal clock gene and AANAT2 expression in relation to melatonin synthesis in Atlantic salmon (Salmo salar) and European seabass (Dicentrarchus labrax)

The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 &2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway.

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya

Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.

Circadian rhythms of melatonin secretion during nocturnal sleep deprivation

The circadian rhythm of melatonin secretion was studied by measuring the amount of its decomposition product (6-sulfatoxymelatonin) in discrete urine sample collected during daytime and night-time periods. The study included a total of 29 subjects ten of whom suffered nocturnal sleep deprivation. 6-sulfatoxymelatonin in the patients of the latter group was measured twice throughout the study: during the 24-hour period with normal sleep and during the 24-hour period with nocturnal sleep deprivation. It was shown that nocturnal sleep deprivation resulted in the disturbances of the circadian rhythms of melatonin production. It sharply decreased at night-time to the levels characteristic of daytime secretion. In the subjects with the normal sleep regimen (i.e. without nocturnal deprivation) the daytime adiponectin levels amounted to 63-75% of the average daily value.

RGS2 is a feedback inhibitor of melatonin production in the pineal gland

The 24-h rhythmic production of melatonin by the pineal gland is essential for coordinating circadian physiology. Melatonin production increases at night in response to the release of norepinephrine from sympathetic nerve processes which innervate the pineal gland. This signal is transduced through G-protein-coupled adrenergic receptors. Here, we found that the abundance of regulator of G-protein signaling 2 (RGS2) increases at night, that expression is increased by norepinephrine and that this protein has a negative feedback effect on melatonin production. These data are consistent with the conclusion that RGS2 functions on a daily basis to negatively modulate melatonin production.

Functional diversity of Teleost arylalkylamine N‐acetyltransferase‐2: is the timezyme evolution driven by habitat temperature?

Arylalkylamine N-acetyltransferase-2 (AANAT2) is the enzyme responsible for the rhythmic production of the time-keeping hormone melatonin. It plays a crucial role in the synchronization of biological functions with changes in the environment. Annual and daily fluctuations in light are known to be key environmental factors involved in such synchronization. Previous studies have demonstrated that AANAT2 activity is also markedly influenced by temperature but the mechanisms through which it impacts the enzyme activity need to be further deciphered. We investigated AANAT2 primary to tertiary structures (3D models) and kinetics in relation to temperature for a variety of Teleost species from tropical to Arctic environments. The results extend our knowledge on the catalytic mechanisms of AANAT enzymes and bring strong support to the idea that AANAT2 diversification was limited by stabilizing selection conferring to the enzyme well conserved secondary and tertiary structures. Only a few changes in amino acids appeared sufficient to induce different enzyme activity patterns. It is concluded that AANAT2 evolution is mainly driven by phylogenetic relationships although catalytic properties (enzyme turnover and substrate affinity) are also under the influence of the respective species normal habitat temperature.

Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day-night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.

Peptide Geroprotector from the Pituitary Gland Inhibits Rapid Aging of Elderly People: Results of 15-Year Follow-Up

The paper presents the results of randomized comparative study of the efficiency of peptide geroprotector from the pituitary gland in elderly patients with rapidly aging cardiovascular system. Over three years 39 coronary patients received, in addition to basic therapy, regular courses of epithalamin (peptide drug), while 40 coronary patients (control group) received basic therapy alone. Long-term treatment with epithalamin (6 courses over 3 years) decelerated aging of the cardiovascular system, prevented age-associated impairment of physical endurance, normalized circadian rhythm of melatonin production and carbohydrate and lipid metabolism. A significantly lower mortality in the group of patients treated with epithalamin in parallel with basic therapy also indicated a geroprotective effect of the peptide preparation from the pineal gland.

Melatonin in octopus (Octopus vulgaris): tissue distribution, daily changes and relation with serotonin and its acid metabolite

Information regarding melatonin production in molluscs is very limited. In this study the presence and daily fluctuations of melatonin levels were investigated in hemolymph, retina and nervous system-related structures in the cephalopod Octopus vulgaris. Adult animals were maintained in captivity under natural photoperiod and killed at different times in a regular daily cycle. Levels of melatonin, serotonin (5-HT) and its acid metabolite (5-hydroxyindole acetic acid, 5-HIAA) in the hemolymph, retina, optic lobe, and cerebral ganglion were assayed by HPLC. Melatonin content fluctuated rhythmically in the retina and hemolymph, peaking at night. In the retina, but not in the other neural tissues, the rhythm was opposite to that of 5-HT, which displayed basal levels at night. Also, 5-HIAA levels in the retina were higher during the night, supporting that rhythmic melatonin production could be linked to diurnal changes in 5-HT degradation. The high levels of melatonin found in the retina point to it as the major source of melatonin in octopus; in addition, a large variation of melatonin content was found in the optic lobe with maximal values at night. All these data suggest that melatonin might play a role in the transduction of the light-dark cycle information for adjustment of rhythmic physiological events in cephalopods.

Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny

Circadian rhythms of many body functions in mammals are controlled by a master pacemaker, residing in the hypothalamic suprachiasmatic nucleus (SCN), which synchronises peripheral oscillators. The SCN and peripheral oscillators share several components of the molecular clockwork and comprise transcriptional activators (BMAL1 and CLOCK/NPAS2) and inhibitors (mPER1/2 and mCRY1/2). Here we compared the ontogenetic maturation of the clockwork in the SCN and pars tuberalis (PT). The PT is a peripheral oscillator that strongly depends on rhythmic melatonin signals. Immunoreactions for clock gene proteins were determined in the SCN and PT at four different timepoints during four differential stages of mouse ontogeny: foetal (embryonic day 18), newborn (2-day-old), infantile (10-day-old), and adult. In the foetal SCN, levels of immunoreactions of all clock proteins were significantly lower than adult levels except for BMAL1. In the newborn SCN the clock protein immunoreactions had not yet reached adult levels, but the infantile SCN showed similar levels of immunoreactions as the adult. In contrast, immunoreactions for all clock gene proteins in the foetal PT were as intense as in newborn, infantile and adult, and showed the same phase. As the foetal pineal gland is not yet capable of rhythmic melatonin production, the rhythms in clock gene proteins in the foetal PT are presumably dependent on the maternal melatonin signal. Thus, our data provide the first evidence that maternal melatonin is important for establishing and maintaining circadian rhythms in a foetal peripheral oscillator.

Cloning and expression of arylalkylamine N-acetyltranferase-2 during early development and metamorphosis in the sole Solea senegalensis

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in teleost fish, one retinal specific, Aanat1, and the other one pineal specific, Aanat2, being the latter the main enzyme responsible of the plasma nocturnal melatonin increase in fish. In anurans melatonin has been involved in metamorphosis through antagonizing thyroid hormone function; however, no available data reports a relationship between melatonin system and metamorphosis in fish. In this study, we have cloned the AANAT2 ( SsAanat2) in a flatfish, Solea senegalensis, and studied its sites of expression and developmental expression pattern by in situ hybridization and Real Time PCR. These studies allowed us to demonstrate a specific signal in the pineal gland of sole larvae from 2 days post-fertilization (dpf), which was evident until post-metamorphosis. Immunohistochemical analysis on the hybridized slides showed that the sole pineal Aanat2 expressing cells corresponded to pineal photoreceptor cells. Real Time PCR was performed in animals kept under natural photoperiod and sampled at different stages from 0 to 21 dpf (including pre-, early-, middle- and late-metamorphic stages) and at midlight (ML) and middark (MD) daytimes. Sole Aanat2 expression was higher at MD than at ML from 2 dpf and at most developmental stages analyzed. The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf. A significant 60-fold reduction in Aanat2 expression was seen just before metamorphosis demonstrating, for the first time in a vertebrate species, that the expression of pineal AANAT and thyroid hormones levels exhibit an inverse pattern during metamorphosis.