SUMMARY / ABSTRACT ?-Conotoxin MII (?-CtxMII) selectively antagonizes ?3?2*- and ?6?2*-nAChR. We have previously developed ?6?2*-nAChR-selective ?-Ctxs to define mesolimbic ?6?2*-nAChR contributions to nicotine and other drug abuse phenotypes. A lack of selective compounds, and lethality in ?3 nAChR null mutant mice means virtually no ?3?2*-nAChR studies have been performed. Nor have extra-limbic ?6?2*-nAChR contributions to addiction- relevant behaviors been investigated extensively. Therefore, we will develop ?-Ctx ligands to discriminate between, characterize, and define the roles of ?3?2*- and ?6?2*-nAChR. The medial habenula (MH) and interpeduncular nucleus (IPN) contain the densest CNS ?3?2*-nAChR populations (which outnumber MH and IPN ?6?2*-nAChR). ?3?4*- and ?5*-nAChR in MH and IPN support nicotine dependence but ganglionic expression of ?3?4*-nAChR and lack of ?5*-nAChR in orthosteric binding sites may make these problematic smoking cessation targets. To establish and differentiate MH and IPN ?3?2*- and ?6?2*-nAChR contributions to nicotine abuse and addiction phenotype, three Specific Aims are proposed: 1) To discover further lead ?-Ctx ligands with ?3?2*-nAChR selectivity by screening an existing panel of >400 novel peptides, and develop them for enhanced selectivity. We have already identified 2 ?-Ctx leads with >10-fold ?3?2*-nAChR selectivity over other subtypes. The most-selective leads will be developed using a novel and streamlined approach to improve ?-Ctx selectivity. 2) To elucidate MH and IPN ?3?2*-nAChR subunit composition. We will make radio- and fluorescence-labeled derivatives of the highly-selective peptides developed in Aim 1. Using these labeled peptides, detailed ?3?2*-nAChR composition will be confirmed for the first time using nAChR subunit-null mutant mice. 3) To define the importance of MH and IPN ?3?2*- and ?6?2*-nAChR in nicotine reinforcement and withdrawal. We will test in rats if local infusion of the selective antagonists ?-CtxMII (?3?2* & ?6?2*), ?- CtxPIA (?6?2*-only), or a novel ?3?2*-only ?-Ctx into MH, IPN or 2 more control regions a) affects motivation to work for nicotine (under a progressive ratio schedule) and b) affects spontaneous somatic and behavioral withdrawal symptoms. For Aims 2 & 3, we describe how to proceed using existing compounds in the extremely unlikely case that Aim 1 does not yield suitably ?3?2*-nAChR-selective ?-Ctxs. This proposal's new screening and peptide-development features will radically advance future utilization of the invaluable ?-Ctx resource. The resources developed in this proposal will be vital to enable future studies probing nAChR function within the addiction-related network to which MH and IPN are extensively connected. Using our novel and potent method for engineering ?-Ctx selectivity in combination with refined behavioral testing across male and female subjects greatly enhances translational impact. This proposal promises to identify and characterize ?3?2*-nAChR as novel, druggable, smoking therapeutic targets, and confirm ?6?2*-nAChR as a viable tobacco cessation target. These advances may produce a major impact on public health by promoting smoking cessation.