Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1

Molecular and Cellular Endocrinology 252 (2006) 11–18

Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1 Esther Isorna a,b , Laurence Besseau a , Gilles Boeuf a , Yves Desdevises a , Robin Vuilleumier a , Angel L. Alonso-G´omez b , Mar´ıa J. Delgado b , Jack Falc´on a,∗ a

Laboratoire Arag´o, Universit´e Pierre et Marie Curie and CNRS, UMR 7628, B.P. 44, Avenue du Fontaul´e, F-66651 Banyuls/Mer-Cedex, France b Universidad Complutense de Madrid, Facultad de Biolog´ıa, Departamento de Fisiolog´ıa (Fisiolog´ıa Animal II), 28040 Madrid, Spain

Abstract The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in Teleost fish (Aanat1 in the retina and Aanat2 in the pineal organ) but only Aanat1 is found in tetrapods. This study reports the cloning of Aanat1 from R. perezi. Transcripts were mainly expressed in the retina, diencephalon, intestine and testis. In the retina and pineal organ, Aanat1 expression was in the photoreceptor cells. Expression was also seen in ependymal cells of the 3rd ventricle and discrete cells of the suprachiasmatic area. The expression of Aanat1 in both the retina and pineal organ, and the absence of Aanat2 suggests that green frog resembles more to birds and mammals than to Teleost fish, as far as Aanat is concerned. The significance of Aanat1 in extra-pineal and extra-retinal tissues remains to be elucidated; in the diencephalon, it might be associated to the so-called deep brain photoreceptor cells. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Arylalkylamine N-acetyltransferase; Melatonin; Retina; Pineal organ; Diencephalon; Frog

1. Introduction In vertebrates, melatonin is a major synchronizer of physiological and behavioral processes to the daily and seasonal variations of photoperiod (Zachmann et al., 1992; P´evet, 2003). This signal of darkness is synthesized and released rhythmically by the retina and the pineal gland during the light/dark (LD) cycle. Plasma levels are high at night and low during day in all species investigated so far. This results from the rhythmic activity of the enzyme that converts serotonin to N-acetylserotonin, the arylalkylamine N-acetyltransferase (AANAT) (Klein et al., 1981, 1997; Falc´on, 1999; Falc´on et al., in press). The conversion of N-acetylserotonin to melatonin is catalysed by the hydroxyindole-O-methyltransferase, which activity is constant throughout the light:dark (LD) cycle. In most species, the pineal gland is the main source of plasma melatonin, whereas retinal melatonin is produced and metabolized in situ (Cassone, 1990; Cahill and Besharse, 1995; Falc´on et al., 2003). Frogs are an exception because unlike other vertebrates, the levels of mela-

∗

Corresponding author. Tel.: +33 468 88 73 92; fax: +33 468 88 73 98. E-mail address: [email protected] (J. Falc´on).

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.03.032

tonin and activity of AANAT are higher in the retina than in the pineal gland (Baker et al., 1965; Serino et al., 1993). And, daily and seasonal plasma melatonin profiles are better correlated with ocular than with pineal melatonin profiles (Delgado and Vivien-Roels, 1989). This would suggest that the frog retina provides visual, auto/paracrine and neuroendocrine information as well. Whereas information has accumulated in Teleost fish, birds and mammals, little is known in frogs on the molecular structure and regulation of AANAT. This is important from an evolution standpoint because two Aanat genes are expressed in Teleost fish – Aanat1 specifically in the retina, and Aanat2 preferentially in the pineal organ – whereas birds and mammals express only Aanat1 in both, the retina and pineal gland (Coon et al., 1999; Klein, 2004; Falc´on et al., in press; Iuvone et al., 2005). In addition, the observation that two alleles of AANAT1 seem to be present in Xenopus laevis (accession no. AY316297 and AY316296) adds to this complexity. This study brings original information regarding the cloning, tissue distribution and localization of Aanat1 in the green frog R. perezi. We show that Aanat1 is expressed in photoreceptor cells of the retina and pineal organ as well as in diencephalic

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areas, including cells bordering the 3rd ventricle and cell bodies in suprachiasmatic area. 2. Materials and methods 2.1. Animals Adult frogs (Rana perezi) were from Orense (Spain). They were kept under natural photoperiod and temperature conditions for at least 2 weeks before sacrifice, and fed twice a week with Calliphora sp. Sampling was made in October during the light phase of the photocycle for cloning and RT-PCR studies, and in April at midday and midnight (n = 3 for each) for in situ hybridization (ISH) studies. Some experiments were also performed on R. esculenta a very closed species (http://waterfrogs.csit.fsu.edu/PBhtmls/hybrids). All experiments were performed in accordance with the “Principles of laboratory animal care” (NIH published 86-23, revised 1985) and the French and Spanish national laws. Frogs were anesthetized with cold, decapitated and the encephalon, eyes, gut, heart, spleen and gonads were rapidly dissected out. For cloning and RTPCR, the tissues were immediately frozen in dry ice or liquid nitrogen and stored at −80 ◦ C. For ISH studies, tissues were fixed as described below.

for 1 min, 68 ◦ C for 1 min, and 1 cycle of 68 ◦ C for 10 min) were performed using specific primers designed from the cloned retinal AANAT sequence. First set of primers (RPNATL03 [5′ -GTCCGTCCTCAATGCAATACCATTC3′ ] and RPNATR01 [5′ -GGAATGTGCGATGTACAGCAAGGAC-3′ ]) amplified a 371 bp fragment, and second set of primers (RPNATL01 [5′ ACTGTGCCCAGAGTTGTCACTGGGATGGTT-3′ ] and RPNATR01) amplified a 168 bp fragment. Negative controls included replacement of RT-mRNA by either total RNA or water or RT-water. The PCR products were migrated in agarose gel, from were fragments of the expected size were purified, subcloned and sequenced as indicated above.

2.4. In situ hybridization (ISH) procedure Tissues were fixed overnight in 4% paraformaldehyde (PFA), rinsed in phosphate buffer saline (PBS), cryoprotected, embedded in Tissue Freezing MediumTM (Leica, Rueil Malmaison, France) and frozen, as indicated elsewhere (Eldred et al., 1983). Ten micrometers (retina) and 20 ␮m (encephalon) serial sections were cut on a cryostat, thaw-mounted onto 3-amino-propyltriethoxysilane (APES)-coated slides (Sigma) and kept at −20 ◦ C until used. The sections were then processed for the ISH as detailed elsewhere (Besseau et al., 2006). The sense and anti-sense riboprobes (1 ␮g/ml) contained the 309 bp fragment of the cloned rpAANAT.

2.2. Cloning of arylalkylamine N-acetyltransferase (AANAT) Total RNA was extracted from 4 retinas using the Trizol® method (GibcoBRL ) according to the manufacturer’s instructions, and mRNA was purified from total RNA using olygo(deoxythymidine) beads (DYNAL® ; Biotech). Reverse transcribed (RT) mRNA was used as template in the polymerase chain reaction (PCR). PCR conditions were as described elsewhere (Coon et al., 1999) using Clontech AdvangeTM (BD Bioscience, Clontech) polymerase. The initial conditions used annealing temperatures of, successively, 37 ◦ C (15 cycles) and 43 ◦ C (35 cycles) with the primers, NAT3R (5′ -ARRTA(CT)TG(CT)AARTAXCGCCA-3′ ) and NAT4L (5′ -GA(CT)GCXAT(ACT)AG(CT)GT(AGCT)TT(CT)GA-3′ ). A fragment of the expected size (309 bp) was obtained and used as a template in a semi-nested PCR with primers NAT3R and NAT6L (5′ CT(AGCT)GTXGTXTT(CT)AT(ACT)AT(ACT)GG-3′ ). The amplified product had the expected size of 156 bp. The 309 bp fragment was then purified (Nucleo Spin® , Machery-Nagel) and subcloned in PGEM-T EASY vector (Promega) following commercially available protocols. The vector containing the amplified insert was electroporated into bacteria from which several positive clones were obtained and sequenced. Full-length sequence of rpAANAT was obtained by 5′ -,3′ -RACE after construction of a cDNA library using the SMARTTM RACE cDNA kit (BD Bioscience, Clontech). A first round of PCR (1 cycle of 95 ◦ C for 2 min, 40 cycles of 94 ◦ C for 15 s, 67 ◦ C for 1 min, 68 ◦ C for 3 min, 1 cycle of 68 ◦ C for 10 min) used the kit universal primers and primers designed in the initial 309 bp fragment cloned. The products were submitted to another PCR (same conditions as above) using internal universal and sequence primers. The amplified products were subcloned, amplified and sequenced as indicated above. The full-length rpAANAT sequence obtained was aligned with AANAT1 and AANAT2 DNA sequences gathered from GenBank, from various vertebrate species. The alignment was performed by ClustalX (Thompson et al., 1997) and improved by eye with Se–Al v2.0a11 (Sequence Alignment Editor, available at http://evolve.zoo.ox.ac.uk/; Rambaut et al., 1996). The length of the aligned sequences was 597 base positions. This alignment was used to reconstruct a phylogenetic tree by maximum likelihood using a HKY85 model with a heterogeneous rate of DNA substitution α = 0.58) chosen using the program Modeltest (Posada and Crandall, 1998) through the use of a hierarchical likelihood ratio test. The tree was validated with a bootstrap procedure using 100 replicates. All phylogenetic analyses were performed with PAUP* 4.0d10 (Swofford, 2003).

2.3. RT-PCR studies on different tissues Messenger RNA was extracted as described above for the retina, treated with DNase (Roche) for 1:30 h at 37 ◦ C, extracted again and reverse transcribed. PCR (1 cycle of 95 ◦ C for 2 min, 35 cycles of 94 ◦ C for 20 s, 65 ◦ C

3. Results 3.1. Cloning of arylalkylamine N-acetyltransferase (AANAT) The cloning strategy described in Section 2 allowed obtaining a 819 bp sequence, from which a translated 201 aa sequence was deduced (Fig. 1). This sequence is similar in length to Xenopus laevis AANAT1a1 (AAP576668.1) and AANAT1a2 (AAP57669.1), with which the R. perezi sequence displayed high identity/similarity (94%/98% and 92%/97%, respectively). There was also high similarity/identity (80%/90% and more) with other AANAT1 cloned from birds and fish. In contrast, this ratio was lower (68%/80%) with Teleost fish AANAT2. This was confirmed by the phylogenetic analysis (Fig. 2). Analysis of the amino acids sequence indicates that R. perezi AANAT possesses the conserved A, B, C and D motifs found in the AANAT family. Motifs A, B and C are highly conserved, whereas motif D, close to the C-terminal of the protein, shows more divergence. Such a divergence was also found at the level of the putative phosphorylation sites. Thus, all the putative caseine kinase II (ck2) phosporylation sites were identified except in the C-terminal part. Conversely, the two protein kinase A (pka) phosporylation sites found at each end of vertebrates AANAT as well as the cysteine residues found at positions 33–57–71–154–173, displayed high conservation (Fig. 1). 3.2. Tissue distribution of AANAT Using a PCR approach, with two different sets of specific primers, strong cDNA amplification was obtained with extracts from retina and diencephalon (which included the pineal organ) and, to a lesser degree, with extracts from gut and testis (Fig. 3). A very faint signal was also observed in extracts from telencephalon and rombencephalon (Fig. 3). No amplification was obtained with extracts from the optic tectum, ovary, heart and

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Fig. 1. Complete nucleotide and amino acid sequences of R. perezi Aanat1. The nucleotide above is 819 bp long. The deduced peptid sequence (below) is 201 aa in length. The motifs A–D, characteristic of the superfamily of N-acetyltransferases, are identified (Dyda et al., 2000). Proteine kinase A (pka) phosphorylation sites, involved in binding to 14.3.3. protein (Ganguly et al., 2002) are conserved at the N- and C-terminals. Other conserved potential caseine kinase II (ck2) phosphorylaton sites and cysteine residues are also highlighted.

spleen, or in controls (PCR using RNA, negative RT product, or water, as templates) (Fig. 3). Purification and sequencing of the PCR products confirmed their identity with the corresponding fragment in the AANAT sequence.

labeling (Fig. 5). Their location suggested these cells belong to the suprachiasmatic nuclei (SCN). No labeling was seen when using the sense probe (Fig. 4). 4. Discussion

3.3. Localization of AANAT gene expression in the retina and diencephalon Because AANAT appeared to be expressed in high amounts in the retina and diencephalon, we decided to investigate the cellular localization of AANAT mRNA in these two nervous tissues using ISH. In dark sampled tissues, strong expression was found in the photoreceptor cells layer of the retina with the anti-sense probe (Fig. 4). Some faint labeling was also observed in some cells of the inner part of the inner nuclear layer (Fig. 4). In a similar manner, strong AANAT gene expression was also found in the photoreceptor cells of the pineal organ (Fig. 5) identified by their morphology and position in the pineal epithelium (Falc´on, 1999). In both, the retina and the pineal organ, AANAT1 mRNA concentrated in the apical part of the photoreceptor cells (Figs. 4 and 5). In the diencephalon, ISH positive reactions were also detected in the ependymal cells bordering the lumen of the 3rd ventricle (Fig. 5). Again, the labeling was concentrated in the apical part of the cells. Finally, scattered cells located in the basal part of the diencephalon and above the optic chiasma, exhibited strong

This study reports the cloning of frog Aanat and brings totally new information on its sites of expression. The primary structure of the deduced protein encoded by R. perezi AANAT, displays high homology with the AANAT cloned in other vertebrate species; it contains the four conserved sequence motifs (A–D) typical of the superfamily of N-acteyltransferases (Klein et al., 1997; Sakamoto and Ishida, 1998; Coon et al., 1999; Dyda et al., 2000); motifs A and B are highly conserved among AANATs, and correspond to the AcCoA binding sites. In addition, the peptide sequence possesses important regulatory sites found in mammalian AANAT. These include two PKA phosphorylation sites located at the N- and C-terminals of the protein; in mammals, these sites play a crucial role in the cAMP-dependent phosphorylation of AANAT and binding to 14.3.3. protein (Ganguly et al., 2002). Finally, cysteine residues, which form structurally important disulfide bonds in AANATs from other vertebrates (Klein et al., 1998) have equivalents at positions 34, 58 and 72. Sequence comparison and phylogenetic tree analysis indicate that this enzyme belongs to the AANAT1 family subtype. The fact that no AANAT2 was found in the present study does not mean that this subtype is not expressed in frogs. However,

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Fig. 2. Phylogenetic tree reconstructed from various vertebrate Aanat DNA sequences, including Rana perezi Aanat 1 sequence. This was estimated by maximum likelihood from a 597 bp alignment. Numbers are bootstrap values.

PCR and ISH studies suggest that R. perezi expresses only Aanat1: (1) Sequences analysis indicated that all the products amplified in the different nervous and peripheral tissues corresponded to Aanat1; an observation strengthened by the fact that the fragments were amplified using a forward primer overlapping the start codon, a highly specific part of the Aanat sequences (Coon et al., 1999). (2) Aanat1 was expressed in the retina and the pineal as well; it is noteworthy that in Teleost fish, the

only species were Aanat2 has been found, it is preferentially expressed in the pineal organ, whereas Aanat1 expression is restricted to the retina (Falc´on et al., 2003). Thus, in terms of Aanat expression, the R. perezi would resemble more to birds and mammals than to Teleost fish. This might be an indication that the occurrence of two Aanat genes is a result of a genome duplication that occurred in the Teleost fish linkage (Jaillon et al., 2004).

Fig. 3. Expression of AANAT1 in different tissues of R. perezi. Two rounds of PCR were run as indicated in Section 2. The specific primers used to amplify a fragment of 371 bp (A) or 148 bp (B). H2 O: negative control of water; RET: retina; TEL: telencephalon; OT: optic tectum; DI: diencephalon; RB: rombencephalon; TS: testicle; OV: ovary; HR: heart; SP: spleen. The symbol (+) represents the PCR product using the cDNA as template; the symbol (−) represents the PCR product using total RNA (PCR 1 in A) or RT product (PCR 2 in B) as controls. The PCR products were loaded in a 1.5% (A) or 2% (B) agarose gel in the presence of a DNA size marker (␭X174 DNA/Hinf I marker, Promega). All the products were extracted and sequenced as indicated in Section 2.

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Fig. 4. Cellular localization of AANAT1 mRNA by in situ hybridization in R. Perezi retina. Retinal sections were hybridized using the sense probe (control in B) or the anti-sense probe (A, C, D). The retinas were sampled during day (A) or at night (C, D). The labeling is intense at night and located in the apical part (inner segments) of the photoreceptor cells (P) in the outer nuclear layer (ONL). The outer segments (OS) are not labeled. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelial cells. Bar = 100 ␮m (A–C) and 10 ␮m (D).

This study reports the first demonstration that Aanat1 is expressed in retinal and pineal photoreceptor cells of a frog, and supports previous investigations indicating that the pineal and retinal photoreceptor cells of aquatic vertebrates are analogous and synthesize melatonin (Cahill and Besharse, 1992; Green et al., 1995; Bolliet et al., 1997; Iuvone et al., 2005; Besseau et al., 2006). The labeling was concentrated in the most apical part of the cells, in the so-called inner segment, from which the outer segment arises. The functional significance of this regional distribution of the labeling, if any, is not known. In the retina, there was a clear day/night variation in the abundance of AANAT1 mRNA in the photoreceptor cells layer; substantially, higher levels were found at night than during day; this pattern parallels the diurnal changes in R. perezi AANAT1 activity, and suggests that a transcriptional regulation of AANAT1 exists in addition to the post-transcriptional regulation previously reported (AlonsoG´omez et al., 2000).

Photoreceptors are not the only cells that express Aanat1. In the retina, some (low) expression was also seen in cells of the basal part of the inner nuclear layer. Previous studies have indicated the presence of either a melatonin-like compound (Falc´on and Collin, 1991) or AANAT1 mRNA (Coon et al., 2002; Liu et al., 2004; Garbarino-Pico et al., 2004) in other retinal layers than the photoreceptor cells. In fish, hydroxyindole-Omethyltransferase was also expressed in the basal inner nuclear layer indicating that some amacrine and/or bipolar cells probably synthesize melatonin (Besseau et al., 2006), although Aanat1 might have other functions as suggested recently (Klein, 2004). Most interestingly, we report here the first detailed cellular localization of extra-retinal and extra-pineal sites of Aanat1 expression in brain tissues. In addition, to the pineal organ, we found Aanat1 expression in two diencephalic cell types corresponding, respectively, to cells of the SCN and ependymal cells bordering the 3rd ventricle. Later, expression was found

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Fig. 5. Cellular localization of AANAT1 mRNA by in situ hybridization in R. Perezi diencephalons: (A) Frontal section through the diencephalon showing a labeling in the pineal organ (PO), in cells bordering the 3rd ventricle (3rd V; open arrows), and in two groups of cells located on each side of the basal hypothalamus (closed arrows) above the optic chiasma (OC). (B) High magnification of pineal the organ; the labeled cells are in the apical part of the pineal epithelium (pe) which corresponds to the position of the photoreceptor cells; the labeling concentrates in the inner segment of the cells (arrow), in contact with the cerebrospinal fluid in the pineal lumen (pl). (C, D) High magnifications in the suprachiasmatic area showing the two groups of labeled cells (arrow in C); the labeling is seen in the cytoplasm (D). (E) High magnification of the labeled cells that border the 3rd ventricle in the medial thalamus. Bar = 1 mm (A), 100 ␮m (B–D) and 50 ␮m (E).

predominantly in the apical part of the cells, which bath into the 3rd ventricle in a manner similar to that seen for the pineal photoreceptors. Previous studies using PCR had shown weak extra-pineal and extra-ocular expression of Aanat1 in the brain and/or peripheral tissues of fish and mammals (Coon et al., 1996; Gauer et al., 1999; Hamada et al., 1999; Stefulj et al., 2001; Yu et al., 2002; Shi et al., 2004). These include the SCN, the site of the master biological clock in mammals, where AANAT expression was found to be under circadian control (Hamada et al., 1999; Yu et al., 2002). Thus, expression of Aanat1 in these areas probably reflects an ancestral character. The present results raise the intriguing hypothesis that the Aanat1 expressing cells of the frog diencephalon are linked, in some way, to the deep brain diencephalic photoreceptors. The existence of deep brain diencephalic receptors in a wide range of non mammalian vertebrates has been hypothesized for long (Von Frisch, 1911; Benoit, 1935). In frogs, electrical recording in from non-pineal to non-retinal origin were obtained in the diencephalon and mesencephalon (Cadusseau and Galand, 1980, 1981). Also, opsin- and ␣-transducin-like immunoreactivities have been detected in diencephalic cells bordering the 3rd ventricle in several frog species (Yoshikawa et al., 1994; Okano et al., 2000; Alvarez-Viejo et al., 2004) as well as in Lampreys, fish and lizards (Foster et al., 1994; Garcia-Fernandez et al., 1997; Philp et al., 2000). In some species, the cells were located in the SCN and preoptic areas. Moreover, studies in the lizard

have led to the conclusion that the opsin cells of the brain are circadian in nature (Pasqualetti et al., 2003). The demonstration that diencephalic cells of R. perezi express Aanat1 adds to this puzzling picture and leads to the tempting hypothesis that in addition to the pineal organ and retina, other parts of the brain concentrate photosensitivity, circadian clock function and entrainment of AANAT gene expression in non mammalian vertebrates. In the frog, Aanat1 expression was also found in peripheral tissues, including intestine and testis. To date, some works describe expression of Aanat in peripheral tissues of vertebrates (B´egay et al., 1998; Kato et al., 1999; Stefulj et al., 2001; CarilloVico et al., 2004) whereas others do not (Coon et al., 1996). When reported, the levels of expression are low. Whatever it may be, the questions remain to know whether the extra-retinal and extra-pineal expressions of Aanat are associated with the correspondent protein synthesis and enzymatic activities, and what is the functional significance of such an expression in these tissues (melatonin synthesis or other purposes). In summary, this study reports the cloning and distribution of Aanat1 in the frog R. perezi. The data suggest the existence of only one type of AANAT, as in birds and mammals and in contrast to Teleost fish. The Aanat1 gene is highly expressed in the pineal and retinal photoreceptors. We also report that Aanat1 is expressed in specific cell types of the diencephalon that might be related to the so-called deep brain photoreceptors.

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This brings support to the hypothesis that the diencephalon may contain circadian units participating in the photic entrainment of a neuroendocrine output. Future studies will aim at determining whether the photic (e.g., opsin-like compounds) and neuroendocrine (e.g., Aanat1 expression) components co-localize and what is the functional significance of Aanat1 in extra-pineal and extra-retinal tissues. Acknowledgements This study was supported by the Spanish MCYT (project no. BFI2001-1368), University Pierre et Marie Curie and CNRS. E. Isorna was supported by the Spanish MECD and the European Marie Curie Training sites programs (predoctoral fellowship). References Alonso-G´omez, A.L., Valenciano, A.I., Alonso-Bedate, M., Delgado, M.J., 2000. Melatonin synthesis in the greenfrog retina in culture. I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis. Life Sci. 66, 675–685. Alvarez-Viejo, M., Cernuda-Cernuda, R., Alvarez-Lopez, C., GarciaFernandez, J.M., 2004. Identification of extraretinal photoreceptors in the teleost Phoxinus phoxinus. Histol. Histopathol. 19, 487–494. Baker, P.C., Quay, W., Axelrod, J., 1965. Development of hydroxyindoleO-methyltransferase activity in the eye and brain of amphibian Xenopus laevis. Life Sci. 4, 1981–1987. B´egay, V., Falc´on, J., Cahill, G.M., Klein, D.C., Coon, S.L., 1998. Transcripts encoding two melatonin synthesis enzymes in the teleost pineal organ: circadian regulation in pike and zebrafish, but not in trout. Endocrinology 139, 905–912. Benoit, J., 1935. Stimulation par ma lumi`ere du d´eveloppement testiculaire chez des canards aveugl´es par e´ nucl´eation des globes oculaires. C. R. Soc. Biol. 120, 136–139. Besseau, L., Benyassi, A., Coon, S.L., Weller, J.L., Bœuf, G., Klein, D.C., Falcon, J., 2006. Melatonin synthesis: light increases trout retinal arylalkylamine N-acetyltransferase (AANAT) activity. Exp. Eye Res. 82, 620–627. Bolliet, V., Begay, V., Taragnat, C., Ravault, J.P., Collin, J.P., Falcon, J., 1997. Photoreceptor cells of the pike pineal organ as cellular circadian oscillators. Eur. J. Neurosci. 9, 643–653. Cadusseau, J., Galand, G., 1980. Electrophysiological evidence for white light sensitivity of the encephalon in eyeless and pinealectomized frogs. Exp. Brain Res. 40, 339–341. Cadusseau, J., Galand, G., 1981. Electrophysiological recordings of an extraocular and extrapineal photo-reception in the frog encephalon. Brain Res. 219, 439–444. Cahill, G.M., Besharse, J.C., 1992. Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina. Vis. Neurosci. 8, 487–490. Cahill, G.M., Besharse, J.C., 1995. Circadian rhythmicity in vertebrate retina: regulation by a photoreceptor oscillator. Prog. Retin. Eye Res. 14, 267–291. Cassone, V.M., 1990. Effects of melatonin on vertebrate circadian systems. Trends Neurosci. 13, 457–464. Coon, S.L., Mazuruk, K., Bernard, M., Roseboom, P.H., Klein, D.C., Rodriguez, I.R., 1996. The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): structure, chromosomal localization, and tissue expression. Genomics 34, 76–84. Coon, S.L., B´egay, V., Deurlooo, D., Falc´on, J., Klein, D., 1999. Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish. J. Biol. Chem. 274, 9076–9082. Coon, S.L., Del Olmo, E., Young III, W.S., Klein, D.C., 2002. Melatonin synthesis enzymes in Macaca mulatta: focus on arylalkylamine N-acetyltransferase (EC 2.3.1.87). J. Clin. Endocrinol. Metab. 87, 4699–4706.

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