Neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to related to several neurological disease and nicotine addiction. In order to study the function of the nAChRs, gene knockout approach is adopted. Two receptor genes, beta3 and alpha3, will be knocked out. For beta3, IRES-tau-lacZ-neo fragment will be inserted into exon V of the gene in order to create the blue knockout mice. For alpha3, inducible gene knockout will be created by using Cre/loxp system. In situ hybridization and immunohistochemistry will be performed in order to make sure the absence of beta 3 and alpha3 in knockout mice. Dopamine release will be studied by synaptosome preparation from knockout mice to assess the possible roles of these receptors in regulation of the release. To test what role these receptor play in motor coordination, learning and memory, various behavior assays, including open field, rotarod, ring test and water maze, will be used. Electrophysiology recording of brain slices will reveal the contribution of alpha3 and beta3 to nicotine response in brain. The function of nicotine modulated dopamine release in EPSC depression will be studied. To find out the function of alpha3 and beta3 in nicotine addiction, the knockout mice will be studied by using self-administration method.
