Angela Renee Ozburn, B.S., Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Opposing ethanol self-administration behaviors are observed when comparing two strains of mice: C57BL/6J x NZB/B1NJ F1 hybrids (B6xNZB) avoid ethanol (avoiders) after experience with high concentrations of ethanol and C57BL/6J x FVB/NJ F1 hybrids (BGxFVB) do not (revelers). The overarching hypothesis of this application is that these hybrids differ in sensitivity to the aversive, but not rewarding or anxiolytic, properties of ethanol and differential levels of FosB and deltaFosB in specific cell types and brain regions reflects experience dependent differences in voluntary ethanol self-administration. Hybrid sensitivity to aversive, rewarding, and anxiolytic properties of ethanol will be determined by employing the following tests; conditioned taste aversion, conditioned place preference, modified two bottle choice, and elevated plus maze. To define neurocircuits engaged by ethanol avoidance and revelry, FosB and deltaFosB immunoreactivity will be measured in ethanol experienced hybrids. Neuronal cell types of FosB and deltaFosB expressing neurons will also be determined. Effects of strain, dose, and quantity of FosB/deltaFosB positive nuclei (per brain region and cell type) will be determined. These experiments will contribute to the understanding of alcohol use and abuse. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Repeated exposure to drugs of abuse leads to long lasting changes in reward- and stress-related neuronal circuitry. Important components of this circuitry include the caudate-putamen, nucleus accumbens, central nucleus of the amygdala, and the ventral tegmental area (VTA). Several studies have suggested a role for molecular components of the circadian clock as key players in drug reward and drug-associated responses. McClung et al. (2005) identified a key role for the circadian locomotor output cycles kaput (CLOCK) gene in the regulation of drug reward. Mice bearing a dominant negative mutation in the CLOCK gene (CLOCK 19 mice) exhibit increased cocaine sensitivity and preference. Furthermore, these mice exhibit increased locomotor activity, reduced anxiety-like and depression-like behavior, increased intracranial self-stimulation (ICSS) at a lower threshold, and increased dopaminergic cell activity in the VTA (McClung et al., 2005;Roybal et al., 2007). The goal of the proposed research training plan is to identify the role of CLOCK in specific brain regions as a mediator of ethanol-related behavior. First, we will determine how chronic binge ethanol consumption regulates expression levels of CLOCK in reward- and stress-related regions of the brain using in situ hybridization. Second, we will determine how a dominant negative mutation of CLOCK (CLOCK 19 mice) affects measures of operant oral ethanol self-administration. Finally, we will determine if CLOCK function in the VTA is important in modulating ethanol intake and relapse-like drinking behavior using RNAi. Further examination of the role of circadian genes in alcohol-related behaviors will have exciting translational significance for treatment, as many recovering addicts exhibit circadian disruptions that persist in recovery and contribute to relapse. I have a strong interest to perform alcohol research at the molecular and behavioral levels. The goal of this training plan is to provide me with detailed expertise in the use of cutting edge techniques and approaches, conversion of data into high-visibility publications, and prepare me for a transition to independence. I am motivated, diligent, and dedicated to advancing the treatment of alcoholism. This plan, combined with the excellent group of researchers at UTSW will allow me to obtain my goals outlined in this proposal, as well as prepare me for a successful independent research career in the alcohol and drug abuse field. The funding of this NRSA application is instrumental in my success. PUBLIC HEALTH RELEVANCE: The proposed work will provide insights into the role of circadian genes in alcohol consumption and withdrawal from chronic binge-drinking, which may contribute to increased drug craving and relapse. Application of this knowledge may provide insights into new treatment strategies during drug withdrawal thereby decreasing the relapse rate and improving treatment outcomes.

Project Summary Alcohol has wide-reaching effects on the nervous system. The mechanism of action for alcohol is complex, where alcohol interacts specifically and non-specifically with many targets (e.g. receptors, lipids, extra-cellular matrix, protein function, etc.). However, the fundamental mechanisms that underlie the effects of alcohol on different behaviors are poorly understood. Thus, more research is required to identify the key molecules essential for different alcohol behaviors (sensitivity to sedation, withdrawal, tolerance, and drinking) that may be targeted to yield new treatments. We focus here on the role of the BK channel, a calcium and voltage-gated potassium channel, in behavioral responses to alcohol. The highly conserved BK potassium channel is a direct target of ethanol that might be modified to reduce alcohol behaviors with minimal side effects. Unbiased genetic screens revealed that the BK channel represented by far the most important ethanol target for intoxication in Caenorhabditis elegans. Acute ethanol directly activates the BK channel to depress general neuronal activity and behaviors in worm. The BK channel was subsequently implicated as important in various behavioral responses to alcohol in flies, rodents and humans. New research also suggests that the channel may be targeted in a way to minimize side effects. A novel BK channel mutation has been identified (T352I) that prevents effects of intoxication and alcohol withdrawal in a C. elegans model. This mutation alters a single residue that is conserved in worm, mouse and human BK channels. Patch-clamp recordings confirmed that the human BK T352I channel was insensitive to activation by ethanol, but otherwise had normal conductance, K+ selectivity, and only subtle differences in voltage dependence. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes critical for behavioral responses. These results suggest that knocking in the T352I mutation in rodent models may alter ethanol-dependent behaviors without causing gross behavioral impairments, which would advance our understanding of the role of the BK channel in different ethanol-mediated behaviors. For this proposal, we will determine whether the BK channel represents a major target of ethanol to modify behaviors in mammals. This will be done by performing quantitative analysis of alcohol-related behaviors in our new mouse engineered with the BK T352I mutation via CRISPR/Cas9; this mouse was generated with our collaborators Drs. John Pierce, Gregg Homanics, and William Shawlot. We will test whether the T352I mutation reduces sensitivity to ethanol sedation, withdrawal, tolerance, and drinking. We hypothesize that the reduced sensitivity seen in C. elegans carrying the T352I BK mutation will be recapitulated in more complex but analogous behaviors in mutant mice. These studies have the potential to determine whether the BK channel represents a major contributor across different alcohol behaviors in mammals. They will also help elucidate the molecular role of the BK channel in alcohol withdrawal and its potential as a treatment avenue during withdrawal.