Central Melatonin Binding Sites in Rainbow Trout (Onchorhynchus mykiss)

Abstract

A combination of in vitro autoradiography and membrane homogenate receptor assays has been used to localize and characterize 2-[125I]iodomelatonin binding sites in the brain of the rainbow trout (Onchorhynchus mykiss). Specific 2-[125I]iodomelatonin binding, defined as that displaced by 1 μM melatonin, increased linearly with increasing protein concentration in membrane homogenates of whole trout brain. Specific binding was both time and temperature dependent and reversible in the presence of 1 μM melatonin. Binding was saturable at between 100-150 pM 2-[125I]iodomelatonin and Scatchard analysis of saturation isotherms revealed a dissociation constant (Kd) of 15.00 ± 0.95 pM and a maximum receptor number (Bmax) of 42.35 ± 2.70 fm/mg protein (n = 16). Addition of 10-4M GTPγS (an analogue of guanosine triphosphate) to saturation isotherms apparently reduced the Bmax by 75% on average with no apparent change in the affinity of the binding. Scatchard analysis of saturation isotherms generated from whole brain membrane homogenates of trout kept on long days (15 hr light:9 hr dark) and killed either during the midlight or middark phase showed no significant differences in either the Kd or the Bmax of 2-[125I]iodomelatonin binding, although a robust rhythm in melatonin concentration was confirmed in these fish. Displacement of 2-[125I]iodomelatonin binding with increasing concentrations of competing ligands gave an order of potency of 2-iodomelatonin > melatonin ⪢ 5-HT. Localization of specific central 2-[125I]iodomelatonin binding in the rainbow trout showed high levels of binding associated with neuronal areas involved in the processing of visual signals, particularly the optic tectum and nucleus rotundus. Lower levels of binding were present over the hypothalamus but no specific binding was found over the pituitary. Thus 2-[125I]iodomelatonin binding in the rainbow trout brain fulfills the basic criteria for a receptor linked to a mediatory G-protein and is found widely localized within the trout brain.