Melatonin is the main synchronizer of circadian and seasonal functions and is also involved in numerous others physiological processes. In mammals melatonin binds two high affinity G-protein coupled receptors, MT1 and MT2. These 2 receptors have been cloned but to date, the paucity of mRNA for these receptors hampered the use of in situ hybridization to locate and identify MT1 or MT2 expressing cells. Many attempts have also been carried out to develop specific primary antibodies against these receptors without success. A second hurdle stands in the lack of selectivity of the available pharmacological drugs to study MT1 and MT2 properties.

Therefore the aim of the present study was to develop new fluorescent agonists designed by linking melatonin linked to a fluorescent molecule (Bodipy, cyanin or NBD [nitrobenzoxadiazole]). After synthesis, the fluorescent agonists were first pharmacologically characterized and only those having affinities < 2.10^-6 M were further tested on cell lines expressing human MT1 (hMT1) or MT2 (hMT2). Tests were performed on HEK cells as they display very scarce autofluorescence. All selected ligands were incubated at 12°C with hMT1 or hMT2 expressing cells. Cells were then rinsed, fixed and subsequently observed under a confocal microscope. Location of labeling and its intensity on hMT1 and hMT2 cells were compared with observations performed on wild type (WT) cells incubated with the same amount of ligand. Association of fluorochrome alone (not linked with melatonin) with hMT1, hMT2 and WT cells was also tested to quantify basal background. Promising data were obtained for NBD associated ligands while Bodipy ligands provided mainly non specific results.

Validation of synthetized ligands on cell cultures and on brain tissue is still underway and newly designed ligands are currently synthesized.

Financial support from Région Centre.