Tuesday, February 7, 2023
2/7/2023
SUMMARY / ABSTRACT a-Conotoxin MII (a-CtxMII) selectively antagonizes a3ß2*- and a6ß2*-nAChR. We have previously developed a6ß2*-nAChR-selective a-Ctxs to define mesolimbic a6ß2*-nAChR contributions to nicotine and other drug abuse phenotypes. A lack of selective compounds, and lethality in a3 nAChR null mutant mice means virtually no a3ß2*-nAChR studies have been performed. Nor have extra-limbic a6ß2*-nAChR contributions to addiction- relevant behaviors been investigated extensively. Therefore, we will develop a-Ctx ligands to discriminate between, characterize, and define the roles of a3ß2*- and a6ß2*-nAChR. The medial habenula (MH) and interpeduncular nucleus (IPN) contain the densest CNS a3ß2*-nAChR populations (which outnumber MH and IPN a6ß2*-nAChR). a3ß4*- and a5*-nAChR in MH and IPN support nicotine dependence but ganglionic expression of a3ß4*-nAChR and lack of a5*-nAChR in orthosteric binding sites may make these problematic smoking cessation targets. To establish and differentiate MH and IPN a3ß2*- and a6ß2*-nAChR contributions to nicotine abuse and addiction phenotype, three Specific Aims are proposed: 1) To discover further lead a-Ctx ligands with a3ß2*-nAChR selectivity by screening an existing panel of >400 novel peptides, and develop them for enhanced selectivity. We have already identified 2 a-Ctx leads with >10-fold a3ß2*-nAChR selectivity over other subtypes. The most-selective leads will be developed using a novel and streamlined approach to improve a-Ctx selectivity. 2) To elucidate MH and IPN a3ß2*-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 a3ß2*-nAChR composition will be confirmed for the first time using nAChR subunit-null mutant mice. 3) To define the importance of MH and IPN a3ß2*- and a6ß2*-nAChR in nicotine reinforcement and withdrawal. We will test in rats if local infusion of the selective antagonists a-CtxMII (a3ß2* & a6ß2*), a- CtxPIA (a6ß2*-only), or a novel a3ß2*-only a-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 a3ß2*-nAChR-selective a-Ctxs. This proposal's new screening and peptide-development features will radically advance future utilization of the invaluable a-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 a-Ctx selectivity in combination with refined behavioral testing across male and female subjects greatly enhances translational impact. This proposal promises to identify and characterize a3ß2*-nAChR as novel, druggable, smoking therapeutic targets, and confirm a6ß2*-nAChR as a viable tobacco cessation target. These advances may produce a major impact on public health by promoting smoking cessation.
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