Viral vectors for desensitization-resistant nAChRs

PROJECT SUMMARY Tobacco dependence represents a major public health challenge, and novel treatment approaches are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are known to mediate the addictive action of nicotine, but it has been difficult to know which aspects of nAChR function are involved in the behavioral and physiological response to nicotine. In particular, the contribution of nAChR activation vs. desensitization to the establishment and maintenance of nicotine reinforcement has remained unclear. We will leverage our unique experience with nAChR molecular pharmacology and self-administration assays to address this issue. In Aim 1, we will design, produce, and validate AAV vectors for expression of desensitization-resistant β2 and β4 nAChR subunits. We will test several β2 and β4 variants with specific mutations at a conserved leucine residue in the 2nd transmembrane ⍺-helix (i.e. the “pore-forming” ⍺-helix). Patch clamp electrophysiology will be used to characterize the biophysical change induced by the mutation, and 2-photon laser scanning microscopy and nicotine uncaging will verify proper trafficking of variant subunits in neurons. In Aim 2, we will study how removal of β2 desensitization in VTA impacts nicotine self-administration and dopamine release. We will test the hypothesis that a lack of desensitization will fundamentally alter nicotine's action as a reinforcer. We will also determine how removal of desensitization from VTA β2* nAChRs impacts evoked dopamine release using electrochemical methods. Together, these Aims will 1) produce/validate novel tools for nAChR research and 2) help us understand how nAChR desensitization normally modulates nicotine reinforcement and associated DA release. These vectors and their characterization in behavioral assays will lay the groundwork for future projects related to nAChR physiology and pharmacology, and may also facilitate the development of novel tobacco cessation treatments.