Regulation and Function of Ventral Tegmental Area Nicotinic Acetylcholine Receptors at the Interface of Alcohol and Nicotine Reward

Abstract

Alcohol and nicotine are the most commonly abused substances worldwide, and comorbid alcohol and nicotine addiction is highly prevalent. Drugs of abuse act within neural reward circuitry to enhance dopamine (DA) release and drive drug-related behaviors. DA neurons originate in the ventral tegmental area (VTA), a critical brain region for reward. Nicotinic acetylcholine receptors (nAChRs) are widely expressed pentameric cation channels that are implicated in both nicotine and alcohol reward mechanisms. Nicotine acts as a potent and selective agonist and neuronal nAChRs, while alcohol potentiates nAChR currents through allosteric modulation. nAChRs are diverse with 11 different subunits that can form a variety of channels with distinct pharmacological properties. Both DA and non-DA neurons in the VTA express a variety of nAChR subtypes, which directly influence DA release and reward behaviors. Notably, VTA nAChRs containing the high affinity α6 subunits have been implicated in both alcohol and nicotine consumption and reward using a variety of genetic and pharmacological tools. The goal of this thesis is to provide mechanistic insight into the way VTA nAChRs influence alcohol and nicotine reward using a genes-to-behavior approach. The work in this thesis provides novel information on the role of α6 nAChRs in the VTA at the cellular, circuit, and behavioral levels in the context of nicotine and alcohol reward. In chapter 2 I investigated the role of α4 and α6 nAChRs in the VTA in mediating alcohol consumption using a novel preclinical drug, sazetidine-A (SAZ-A), which acts as a partial agonist at a subset of high-affinity nAChRs. I found that SAZ-A infusion directly into the VTA is sufficient to decrease alcohol consumption. In a series of conditioning experiments, I demonstrated that SAZ-A treatment did not impact the subjective rewarding properties of alcohol, but instead enhanced conditioned alcohol aversion through non-α4 nAChRs. These data provide mechanistic insight into the ability of drugs like SAZ-A to reduce alcohol consumption and suggest that non-α4 nAChRs, and likely nAChRs containing the α6 subunit, in the VTA may be a promising target for novel therapeutics to treat alcohol use disorders. In chapter 3 I investigated the cellular regulation of α6 nAChRs in the ventral midbrain (VMB) by the signaling molecule protein kinase C epsilon (PKCε). PKCε impacts the transcription of nAChRs, as male PKCε-/- mice have reduced expression of α6 and β3 transcripts in the VMB. Because previous work on PKCε was completed in male animals, I sought to determine if PKCε played a similar role in female rodents. We found female PKCε-/- mice had substantially higher expression levels of α6 and β3 mRNA in the VMB compared with wild-type (WT) littermates, an effect opposite of previously observed data in males. Further characterization of these animals revealed that female PKCε-/- mice show behavioral phenotypes consistent with increased signaling through α6-containing nAChRs, including locomotor activity in response to low-dose nicotine. These data reinforce the hypothesis that PKCε regulates the expression of α6 nAChRs and reveals a bidirectional effect of sex in transcriptional regulation of these genes by PKCε. Finally, I sought to study the role of nAChRs in specific VTA cell types, which have not been well described due to limited genetic and pharmacological tools. In chapter 4, I developed and characterized a viral gene approach that results in selective knockdown of α6 and β3 nAChR mRNA in specific cell types using Cre-dependent RNA interference. I show that this tool is effective in vivo and can be used to further delineate cell-specific roles of nAChR subtypes in physiology and behavior. Taken together, the work in this thesis provides novel insight into the way nAChRs in the VTA influence both nicotine and alcohol reward behaviors, including the development of a new publicly available tool to further investigate nAChRs in specific cell populations.