There are a number of human neurobiological disorders that are linked to defects in the circadian timing system. Many of these disorders have been treated effectively with the neuroendocrine hormone melatonin. Circadian effects of melatonin are believed to be mediated by a melatonin receptor subtype (Mel-1a) that is found in the human suprachiasmatic nucleus (SON). In this research proposal, we will use homologous recombination with the mouse Mel-1a gene locus to target nuclear-localized beta-galactosidase (nlacZ) expression to cells within the SON. Unlike Mel-1a mRNA, the nlacZ marker can readily be detected at a cellular level. This will facilitate sensitive double-immunocytochemical labelling to determine if cells that express Mel-1a are neurons and/or glia and will be used to determine what neurotransmitters and neuropeptides colocalize in Mel-1a cells. Most importantly, this knock-in strategy will provide us with the ability to determine, at the level of single cells, if the Mel-1a receptor is expressed in SON neurons that possess an autonomous circadian oscillator. These experiments have the potential of defining and fully characterizing a specific cell-type within the mammalian SON. Additionally, these studies could provide the first molecular tools for accessing the biological clock.
