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.

Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

Melatonin inhibits GnRH-1, GnRH-3 and GnRH receptor expression in the brain of the European Sea Bass, Dicentrarchus labrax.

Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day-night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day-night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.

The retina is a target for GnRH-3 system in the European sea bass, Dicentrarchus labrax.

The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass.

Afferent and efferent connections of the pineal organ in the European sea bass Dicentrarchus labrax: a carbocyanine dye tract-tracing study.

The pineal organ of fish is a photosensitive structure that receives light information from the environment and transduces it into hormonal (rhythmic melatonin secretion) and neural (efferent projections/neurotransmitters) signals. In this study, we focused on this neural output. Thus, we performed a tract-tracing study using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a fluorescent carbocyanine dye, in order to elucidate the efferent and afferent connections of the pineal organ in the European sea bass. The axonal transport of DiI revealed extensive bilateral projections in the sea bass brain. The efferent projections of the sea bass pineal organ reach the habenula, ventral thalamus, periventricular pretectum, central pretectal area, posterior tubercle and medial and dorsal tegmental areas. In addition, in this study we also examined the pinealopetal system in sea bass. This analysis demonstrated that the sea bass pineal organ receives central projections from neurons located, to a large extent, in brain areas innervated by pineal efferent projections, i.e. the thalamic eminence, habenula, ventral thalamus, dorsal thalamus, periventricular pretectum, posterior commissure, posterior tubercle and medial tegmental area. This study is the first description of pinealofugal projections in a representative of Perciformes, which constitutes a derived order within teleosts. Moreover, it represents the first evidence for the presence of pinealopetal neurons in the brain of a teleost species. Our findings, together with the analysis of retinal connections, represent a step forward in the understanding of the integration of photoperiodic signals into the central nervous system of sea bass.

Melatonin receptors in the brain of the European sea bass: An in situ hybridization and autoradiographic study.

Melatonin is synthesized in the pineal organ and retina of vertebrates and exhibits a clear nocturnal rhythm of secretion. This hormone influences a number of important physiological processes acting through specific transmembrane G-protein-coupled receptors. Recently, we have cloned three different melatonin receptors in sea bass belonging to the MT1, MT2, and Mel1c subtypes. In this paper, we have analyzed the central expression of the MT1 gene by in situ hybridization and compared its distribution with the localization of 2-[(125)I]-iodomelatonin binding sites. In situ hybridization and autoradiographic studies provided consistent results. Melatonin receptors were mainly expressed in visually related areas of the sea bass brain, such as the pretectal area, glomerular complex, optic tectum, torus longitudinalis, and thalamus. A conspicuous expression was also detected in neuroendocrine regions including the ventral telencephalon, preoptic area, and hypothalamus. Furthermore, melatonin receptors were evident in the ganglionic cell layer of the cerebellum. The presence of iodomelatonin binding and/or MT1 mRNA-expressing cells was also observed in the hindbrain, in particular in the oculomotor and trigeminal nuclei and in the reticular formation. Our results suggest an important role of MT1 in the mediation of melatonin actions in visual/light integration, mechanoreception, somatosensation, eye-body motor coordination, and integrative and neuroendocrine functions. Remarkable differences in the number and distribution of brain nuclei expressing MT1 mRNAs in sea bass and trout, the only fish species analyzed to date, represent another piece of evidence for differences in the organization of the visual and circadian systems observed between salmoniform and perciform teleosts.

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax.

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.