?-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.

Public Health Relevance

This proposal requests support to study the composition, expression, and functional roles of nicotinic acetylcholine receptors containing the ?3 and ?2 subunits (?3?2*-nAChR), and discriminate them from those of the closely-related ?6?2*-nAChR subtype. Our preliminary data implicate, for the first time, ?3?2*-nAChR expressed in the medial habenula ? interpeduncular nucleus (MH-IPN) pathway in aspects of nicotine addiction. This proposal will develop and disseminate tools to definitively identify and study both ?3?2*- and ?6?2-nAChR in these two brain regions which contain complex mixes of receptor subtypes, ascertain the two subtypes' respective functional contributions to nicotine addiction, and define the location of the behaviorally- critical nAChR populations in both MH and IPN. Together these data have promise to identify and characterize a novel receptor target (?3?2*-nAChR) for development of compounds that promote smoking cessation with reduced side effects, and provide an essential foundation for future studies examining ?3?2*- and ?6?2*- nAChR roles and functional interactions within the broader addiction network with which MH and IPN are extensively connected.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
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Molecular Neuropharmacology and Signaling Study Section (MNPS)
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Hillery, Paul
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St. Joseph's Hospital and Medical Center
United States
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Morley, Barbara J; Whiteaker, Paul; Elgoyhen, Ana B (2018) Commentary: Nicotinic Acetylcholine Receptor ?9 and ?10 Subunits Are Expressed in the Brain of Mice. Front Cell Neurosci 12:104
Chen, De-Jie; Gao, Fen-Fei; Ma, Xiao-Kuang et al. (2018) Pharmacological and functional comparisons of ?6/?3?2?3-nAChRs and ?4?2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line. Acta Pharmacol Sin 39:1571-1581