Nicotine is a drug of abuse that presumably exerts its effects through its interactions with specific nicotine receptors in the brain. Chronic exposure to nicotine results in an increase in the abundance of nicotine binding sites in the brains of both rats and humans. We have discovered, cloned, and sequenced ten members of the gene family that encodes subunits of the neuronal nicotinic receptors. In this application we propose a means of using these clones to study the metabolism and regulation of the nicotine binding sites in the brain. This approach uses our cDNA clones as templates for the synthesis of the extracellular domains of each receptor subunit in bacteria and then uses these bacterially expressed proteins to generate antibodies in rabbits. Affinity chromatography on columns containing subunit specific peptides results in antibodies able to distinguish between the extracellular domains of each receptor subunit. These antibodies and radioactive peptides are used to create quantitative assays sufficiently sensitive to determine amounts of specific receptor subunits in milligram quantities of brain. We will use these assays to explore the metabolism of the nicotinic receptor and the mechanisms that produce the up-regulation of nicotine binding sites following chronic nicotine use. Our goal is to distinguish between the different mechanisms that might generate more nicotine binding sites. We will also use the antibodies in a more qualitative fashion to determine the location on neurons of nicotinic receptors of specific subunit composition. The long-term goal of this research is to understand how the location of the binding sites that are up-regulated during chronic drug treatment relates to the processes that lead to addiction. These experiments will address issues fundamental to nicotine addiction and to the cellular mechanisms that lead to dependence on the drug.