Project Summary/Abstract Recent analyses of several human populations have identified a number of genetic markers which correlate with aspects of smoking behavior and that correspond to different nAChR subunits. These studies strongly support the contention that genetic differences in receptor subunit structure or expression influence tobacco use. However, it has not been established which of the genetic differences encourage and which discourage tobacco use. Chronic exposure of animals to nicotine, or of humans to tobacco, results in the development of tolerance and dependence. The initial sites of action of nicotine are the nicotinic cholinergic receptors (nAChR), for which the native neurotransmitter is acetylcholine. There are many different types of nAChR in the brain and in the periphery. Nicotine's interaction with these various receptors differs and can lead to different responses to the drug. It is well established that chronic nicotine exposure also changes the number, and perhaps the function, of some nicotinic cholinergic receptors (nAChR). However, chronic nicotine exposure does not affect each nAChR subtype in the same way. Mice will be used as the model organism to examine effects of chronic nicotine treatment. The effects of nicotine in mice are influenced by genotype and mice from which a specific nAChR gene has been deleted (knockout) or mutated (knockin) have been generated. The knockout and knockin mice will be used to investigate some behavioral changes induced by nicotine treatment, primarily tolerance. Furthermore, the effects of chronic nicotine treatment on the expression and function of several different nAChR subtypes will be measured. There are two specific aims. The role of 42*nAChR in modulating response to chronic nicotine will be investigated in Aim 1. 42*- nAChR is the most highly expressed subtype in the brain and chronic nicotine treatment increases the number of these receptors. We plan four types of experiments to study this response in detail a) measure effects in 4 knockouts, where the gene is completely gone; b) measure the response in mice in which either or both 4 and 2 genes have been partially deleted; c) measure response in mice expressing mutations that make these receptors hypersensitive; and d) evaluate the effects of treatment with a drug that interacts selectively with 42-nAChR. The role of the 5 and 4 subunits in modulating responses to chronic nicotine will be investigated in Aim 2. 5 is interesting because it is the nAChR gene that most robustly correlates with tobacco use, while 4 is interesting because nAChR incorporating this subunit affect nicotine withdrawal and may diminish tolerance development. Response of 5 and 4 null mutant and heterozygote mice will be compared to that of wild-type mice. The results of these studies will provide new information on the regulation of the major nicotinic receptor subtype and the contribution of two other subtypes to changes induced by chronic nicotine. The results could also help identify specific behavioral and biochemical responses to chronic nicotine mediated by genes identified as important factors in human smoking.