Localization and Partial Characterization of Melatonin Receptors in Amphioxus, Hagfish, Lamprey, and Skate

Through its secretion of melatonin, the pineal complex of vertebrates exerts a range of physiological effects including regulation of circadian rhythms, seasonal reproduction, metamorphosis, and body color change. Little is known about phylogenetic differences in the distribution and characteristics of melatonin binding sites in fishes. We usedin vitroautoradiography to examine binding of [2-125I]iodomelatonin (IMEL) in 20-μm frozen sections of amphioxus (Branchiostoma lanceolatum), Atlantic hagfish (Myxine glutinosa), larval and adult lamprey (Petromyzon marinus), little skate (Raja erinacea), and rainbow trout (Oncorhynchus mykiss). Tissue was incubated with IMEL in the presence or absence of unlabeled melatonin (1 μM, in order to assess nonspecific binding). A concentration of 32 pM IMEL was used for single point assays and competition studies. No specific binding was found in hagfish or amphioxus, which lack a pineal complex. In the optic tecta of lamprey, skate, and trout, IMEL binding is highly specific (melatonin⪢N-acetylserotonin>5- methoxytryptophol⪢serotonin). Scatchard analysis revealed that the tectal binding sites are of high affinity (Kd=36, 38, and 50 pM) and low capacity (Bmax=8.1, 19.8, and 21.8 fmol/mg protein) in lamprey, skate, and trout, respectively. In adult lampreys, intense specific IMEL binding is found in the optic tectum (layer I>II>III) and preoptic nucleus (pars parvocellularis>magnocellularis). Binding was less intense and consistent in the same areas of ammocoete brain. In skates and trout, intense specific binding is found in optic tectum, lateral geniculate body, diencephalic preoptic and suprachiasmatic nuclei, basal hypothalamus, and the medial pallium. These results indicate that specific melatonin binding sites are present in all craniate taxa examined except in hagfish. Although we cannot rule out the possibility that melatonin receptors are secondarily lost in hagfish, their absence in amphioxus makes this unlikely. We speculate that melatonin actions in early vertebrates may have included regulation of visual and endocrine responses to light.