SUMMARY / ABSTRACT α-Conotoxin MII (α-CtxMII) selectively antagonizes α3β2*- and α6β2*-nAChR. We have previously developed α6β2*-nAChR-selective α-Ctxs to define mesolimbic α6β2*-nAChR contributions to nicotine and other drug abuse phenotypes. A lack of selective compounds, and lethality in α3 nAChR null mutant mice means virtually no α3β2*-nAChR studies have been performed. Nor have extra-limbic α6β2*-nAChR contributions to addiction- relevant behaviors been investigated extensively. Therefore, we will develop α-Ctx ligands to discriminate between, characterize, and define the roles of α3β2*- and α6β2*-nAChR. The medial habenula (MH) and interpeduncular nucleus (IPN) contain the densest CNS α3β2*-nAChR populations (which outnumber MH and IPN α6β2*-nAChR). α3β4*- and α5*-nAChR in MH and IPN support nicotine dependence but ganglionic expression of α3β4*-nAChR and lack of α5*-nAChR in orthosteric binding sites may make these problematic smoking cessation targets. To establish and differentiate MH and IPN α3β2*- and α6β2*-nAChR contributions to nicotine abuse and addiction phenotype, three Specific Aims are proposed: 1) To discover further lead α-Ctx ligands with α3β2*-nAChR selectivity by screening an existing panel of >400 novel peptides, and develop them for enhanced selectivity. We have already identified 2 α-Ctx leads with >10-fold α3β2*-nAChR selectivity over other subtypes. The most-selective leads will be developed using a novel and streamlined approach to improve α-Ctx selectivity. 2) To elucidate MH and IPN α3β2*-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 α3β2*-nAChR composition will be confirmed for the first time using nAChR subunit-null mutant mice. 3) To define the importance of MH and IPN α3β2*- and α6β2*-nAChR in nicotine reinforcement and withdrawal. We will test in rats if local infusion of the selective antagonists α-CtxMII (α3β2* & α6β2*), α- CtxPIA (α6β2*-only), or a novel α3β2*-only α-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 α3β2*-nAChR-selective α-Ctxs. This proposal's new screening and peptide-development features will radically advance future utilization of the invaluable α-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 α-Ctx selectivity in combination with refined behavioral testing across male and female subjects greatly enhances translational impact. This proposal promises to identify and characterize α3β2*-nA...