Serotonin synthesis and its light–dark variation in the rat retina Evelyne Chanut,* Jeanine Nguyen-Legros, Benoı ˆt Labarthe,* Jean-Hugues Trouvin* and Claudine Versaux-Botteri  , à *Laboratoire de Pharmacologie, Faculte ´ de Pharmacie, Cha ˆtenay-Malabry, France  Laboratoire de De ´veloppement, Vieillissement et Pathologie de la Re ´tine, INSERM U450, Paris, France à Laboratoire d’Anatomie Compare ´e, Museum National dÕHistoire Naturelle, Paris, France Abstract Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by pho- toreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To dem- onstrate local synthesis of serotonin in the rat retina, sub- strates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas a-methyl- tryptophan, a competitive substrate inhibitor, was hydroxylated into a-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT–PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis. Keywords: retina, rhythms, serotonin, tryptophan, trypto- phan hydroxylase. J. Neurochem. (2002) 83, 863–869. Retinal physiology is subject to circadian rhythms driven by an intrinsic oscillator in a number of vertebrates including rats (Terstein et al. 1980; Reme ´ et al. 1991). One of the chemical supports of this oscillator is the nocturnal secretion of melatonin by photoreceptor cells. Melatonin is synthesized from serotonin (5-HT) through the successive enzymes serotonin (arylalkylamine) N-acetyltransferase (AANAT; EC 2.3.1.87) and hydroxyindole O-methyltrans- ferase (HIOMT; EC 2.1.1.4), the activities of which have been demonstrated in the rat retina (Nagle et al. 1973; Pang et al. 1980; Reiter et al. 1983). AANAT mRNA expression exhibits a circadian rhythm, and is localized to photore- ceptor cells in rat retina (Niki et al. 1998; Sakamoto and Ishida 1998). However, although the enzymatic pathway has now been identified in the rat retina, the major question of the origin(s) of 5-HT, as precursor of melatonin, is still unresolved. 5-HT can neither be consistently detected within the retina of adult rat (Lima and Urbina 1994), nor can its presence be demonstrated using immunohistochem- ical techniques (Tornqvist et al. 1983), whereas N-acetyl serotonin, which is the product of AANAT, the substrate of HIOMT and precursor of melatonin, has been detected (Pang et al. 1981). Unlike non-mammalian retina, in which a clear light–dark regulation of 5-HT occurs and a subpopulation of 5-HT- synthesizing amacrine cells exists (Huang and Moody 1997; Dkhissi et al. 1998), the mammalian retina does not possess such cells. Serotonin-accumulating amacrine cells, devoid of biosynthetic enzymes, have been demonstrated in some mammals, but they do not exist in rats (Osborne 1984; for review). Moreover, tryptophan hydroxylase (TPH; EC 1.14.16.4), the rate-limiting enzyme in the synthesis of 5-HT, has not to date been detected by immunohistochemical methods in rat retina, even at night time or after prolonged dark adaptation (Nguyen-Legros J. and Versaux-Botteri C., Received April 25, 2002; accepted June 23, 2002. Address correspondence and reprint requests to Dr C. Versaux-Botteri, INSERM U450, 15 rue de l’Ecole de Me ´decine, 75006 Paris Cedex 06, France. E-mail: claudine.botteri@bhdc.jussieu.fr Abbreviations used: AADC, aromatic amino acid decarboxylase; AANAT, serotonin (arylalkylamine) N-acetyltransferase; AM-5-HT, a-methyl-serotonin; AM-5-HTP, a-methyl-5-hydroxytryptophan; AM-Trp, a-methyl-DL-tryptophan; HIOMT, hydroxyindole O-methyl- transferase; 5-HT, 5-hydroxytryptamine or serotonin; 5-HTP, 5-hydroxy- tryptophan; NSD 1015, m-hydroxybenzylhydrazine; TPH, tryptophan hydroxylase; Trp, tryptophan. Journal of Neurochemistry , 2002, 83, 863–869 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 83, 863–869 863