This project tests two hypotheses: (A) Most of the acute effects of nicotine, and most of the addictive effects of chronic application, arise from actions on nicotinic receptors containing the alpha4 subunit (termed alpha4* receptors). (B). Addiction arises in part from changes in the number and distribution of alpha4* receptors. A series of knock-in mouse strains serve as appropriate tools to test these hypotheses. Some strains harbor hypersensitive alpha4 receptor subunits, which isolate and amplify nicotine responses evoked by alpha4* receptors. The most promising strain is the neo-deleted Leu9'Ala strain. This project characterizes cellular and circuit aspects of responses to nicotine, primarily in the VTA-nucleus accumbens system and forebrain of the hypersensitive mice. Most experiments are performed with acute application of nicotine as well as with tissue from animals chronically exposed to nicotine. Leu9'Ala and WT strains are characterized neurochemically, including epibatidine binding in brain homogenates, nicotine-induced release of dopamine, GABA, and glutamate, and tyrosine hydroxylase staining in midbrain. Time-resolved cellular aspects of nicotinic responses and of synaptic transmission are studied, in midbrain, forebrain, and hindbrain. These studies are enhanced by crossing the hypersensitive mice with strains that have GFP-labeled interneurons and dopaminergic neurons: whole-cell patch in transfected cells and cultures; electrode arrays in slices; single-unit and field potential recording in awake, behaving animals; and multi-tetrode single-unit recording. The project characterizes nicotine-induced immediate early genes, primarily fos, in baseline and nicotine-dependent states, using unbiased stereology. Gene expression is analyzed with microarrays, to determine differences between mutant WT mice and nicotine-induced changes. The project also characterizes knock-in mice that have fluorescently labeled alpha4 receptors, both normally sensitive and hypersensitive. Receptor assembly and localization is studied, including FRET, in naive and nicotine-treated states.