Richard A. Radcliffe - US grants

DESCRIPTION (provided by applicant): Behavioral sensitivity to alcohol can be modified as a function of both environmental and genetic factors. The underlying basis of this effect is the ability of neurons to adapt in response to environmental perturbations including, but not limited to alcohol exposure, and the specific nature of this response is dependent on genetic makeup. These processes may be an important feature in the etiology of alcoholism. Evidence indicates that drugs acting on dopamine (DA) neurons are able to alter the behavioral response to alcohol in rodents. Thus, 24 hrs after a single treatment with the DA antagonist haloperidol or the indirect DA agonist methamphetamine, rats become more or less sensitive to alcohol, respectively, and "rapid" tolerance develops one day after a single dose of alcohol. These acute drug responses will be exploited to investigate the global gene expression changes occurring in DA pathways that contribute to altered alcohol sensitivity. Specific Aim 1 will fully characterize the behavioral response to alcohol (loss of righting reflex) 24 hrs after acute treatment with haloperidol, alcohol, or methamphetamine in replicate inbred High and Low Alcohol Sensitive rat strains. Gene expression analyses will be performed in Specific Aim 2. Rats will be sacrificed at 8 hrs following administration of a single optimal dose of each drug and the striatum and ventral midbrain will be dissected. RNA will be extracted from these structures and gene expression will be determined with the use of the Affymetrix GeneChip system. This will provide a total of 16 experimental conditions comprised of different drug/genotype combinations. It is postulated that each of these conditions will show different, but overlapping gene expression profiles. The genes that are important in the altered alcohol response will be identified by clustering and discriminative analysis procedures in a comparison of the expression profiles and the behavioral responses among the 16 conditions. Specific Aim 3 will validate important gene expression changes with the use of ribonuclease protection assays and/or real-time quantitative PCR. The results of the proposed studies will offer insight into the neuroadaptive processes that contribute to alcohol abuse and will also provide more information from which to further investigate alcohol related neuroadaptation.

DESCRIPTION (provided by applicant): Behavioral sensitivity to alcohol can be modified as a function of both environmental and genetic factors. The underlying basis of this effect is the ability of neurons to adapt in response to environmental perturbations including, but not limited to alcohol exposure, and the specific nature of this response is dependent on genetic makeup. These processes may be an important feature in the etiology of alcoholism. Evidence indicates that drugs acting on dopamine (DA) neurons are able to alter the behavioral response to alcohol in rodents. Thus, 24 hrs after a single treatment with the DA antagonist haloperidol or the indirect DA agonist methamphetamine, rats become more or less sensitive to alcohol, respectively, and "rapid" tolerance develops one day after a single dose of alcohol. These acute drug responses will be exploited to investigate the global gene expression changes occurring in DA pathways that contribute to altered alcohol sensitivity. Specific Aim 1 will fully characterize the behavioral response to alcohol (loss of righting reflex) 24 hrs after acute treatment with haloperidol, alcohol, or methamphetamine in replicate inbred High and Low Alcohol Sensitive rat strains. Gene expression analyses will be performed in Specific Aim 2. Rats will be sacrificed at 8 hrs following administration of a single optimal dose of each drug and the striatum and ventral midbrain will be dissected. RNA will be extracted from these structures and gene expression will be determined with the use of the Affymetrix GeneChip system. This will provide a total of 16 experimental conditions comprised of different drug/genotype combinations. It is postulated that each of these conditions will show different, but overlapping gene expression profiles. The genes that are important in the altered alcohol response will be identified by clustering and discriminative analysis procedures in a comparison of the expression profiles and the behavioral responses among the 16 conditions. Specific Aim 3 will validate important gene expression changes with the use of ribonuclease protection assays and/or real-time quantitative PCR. The results of the proposed studies will offer insight into the neuroadaptive processes that contribute to alcohol abuse and will also provide more information from which to further investigate alcohol related neuroadaptation.

DESCRIPTION (provided by applicant): Acute functional tolerance (AFT) to ethanol is a well known phenomenon that is thought to influence individual differences in risk liability for alcoholism, but the underlying molecular substrates of AFT remain a mystery. In this application, we are proposing to test the hypothesis that the zebrafish (Danio rerio) will exhibit AFT to the behavioral disruptions produced by ethanol, with the main goal of the proposal to characterize and develop the zebrafish as a model organism in the study of the molecular basis of AFT. Zebrafish provide numerous advantages for genetic studies including short generation time, large numbers of offspring, low cost and space demands, a growing body of information on the genetics of zebrafish, and the ease with which genetic manipulations can be accomplished. Another important advantage of zebrafish in AFT studies is that a steady-state blood and brain ethanol concentration can easily be maintained since the ethanol is administered via equilibration with the holding tank solution; in essence, an extremely simple ethanol clamp. AFT for certain behaviors has been demonstrated in goldfish, but it has not been examined in zebrafish. The Specific Aims of this project are to screen several established zebrafish behaviors for the acquisition of AFT, determine if there is naturally occurring genetic variation for AFT by testing several inbred zebrafish strains, and to develop methods for automated, high-throughput mutagenesis screens for AFT. Future studies will implement the high-throughput screens on large numbers of mutagenized zebrafish with the goal of mapping the molecular basis of AFT in zebrafish.

DESCRIPTION (provided by applicant): An important genetic risk factor for the development of alcoholism is differential sensitivity to an acute dose of alcohol. Acute alcohol responses are a function of the combined effects of initial sensitivity and acute functional tolerance (AFT), bot of which are influenced by genetic factors. Using inbred mouse strains, we have been using a paradigm known as rapid tolerance - tolerance that develops within 24 hrs following a single exposure to alcohol - as a tool to investigate the genetics of acute alcohol responses. We have found that the Inbred Long and Short Sleep mouse strains (ILS and ISS) differ considerably in their ability to develop rapid tolerance using the loss of righting reflex test (LORR) as the measure of sensitivity. This strain- dependent difference appears to be mediated at least partly by differential effects on AFT. We hypothesize that genetic variance in rapid tolerance occurs as a result of genotype-dependent differences in baseline gene expression, in alcohol-mediated effects on gene expression, and in differences in gene sequence and structure. Thus, we propose to exploit the rapid tolerance model to examine the molecular and genetic basis of acute responses using Next Generation high-throughput deep sequencing technologies. The genetics of rapid tolerance, initial sensitivity, and AFT will be investigated using the LXS recombinant inbred (RI) mouse strain panel which was derived from the ILS and ISS. Expression profiling will be conducted using quantitative RNA sequencing (RNA-seq) with which it is possible to investigate effects on alternative splicing as well as on transcript abundance. The following six Specific Aims are being proposed: 1) determine relationships between initial sensitivity, AFT, and rapid tolerance for the LORR response in the LXS RIs; 2) map quantitative trait loci (QTLs) for the responses determined in Aim 1; 3) sequence the full genomes of the ILS and ISS; 4) conduct expression profiling in the brains of the LXS RIs; 5) map expression QTLs (eQTLs) for genes identified in Aim 4 and for genes that occur within the behavioral QTL intervals determined in Aim 2; and 6) confirm expression results for genes identified in Aims 4 and 5. We propose that the results of these experiments will offer insight into the nature of genetic variance for acute alcohol sensitivity. This in turn will contribute to a deeper understanding of genetic risk for human alcoholism. PUBLIC HEALTH RELEVANCE: The initiation and maintenance of alcoholism is influenced by both environmental and genetic factors. This project aims to identify genes that influence variation in acute alcohol sensitivity, a trait that is thought to contribute to genetic risk for alcoholism. Such knowledge is essential for a complete understanding of the molecular basis of alcoholism and for the development of new or improved strategies for its treatment.

DESCRIPTION (provided by applicant): An important genetic risk factor for the development of alcoholism is differential sensitivity to an acute dose of alcohol. Using segregating populations derived from selectively bred rat lines, we have mapped quantitative trait loci (QTLs) on rat chromosomes 1 and 2 for an acute response to alcohol, the duration of the loss of righting reflex (LORR). We have subsequently generated congenic and recombinant congenic lines that have confirmed the QTLs while simultaneously reducing their genomic size. We hypothesize that there are one or more genes within the QTL intervals that contribute to genetic variance for LORR through polymorphism- related effects on gene expression and/or on the structure of the genes'products. A second important hypothesis is that these same genes will influence genetic variance in alcohol reward-related behaviors. To address these hypotheses, the project will carry out four Specific Aims: 1) Fine-map the chromosome 1 and 2 QTLs using the recombinant congenic strategy;2) Identify candidate genes in the fine-mapped areas of the chromosome 1 and 2 QTLs;3) Identify enriched functional pathways and gene networks that are affected by the QTLs;and 4) Determine if the chromosome 1 and 2 QTLs are pleiotropic with alcohol reward-related behaviors. Contemporary genetic and genomic methods and analytical approaches, including high-throughput DNA and RNA sequencing, will be used to accomplish the goals of the Aims. We propose that the results of these experiments will offer insight into the nature of genetic variance for acute alcohol sensitivity. This in turn will contribute to a deeper understanding of genetic risk for human alcoholism. PUBLIC HEALTH RELEVANCE: The initiation and maintenance of alcoholism is influenced by both environmental and genetic factors. This project aims to identify genes that influence variation in acute alcohol sensitivity, a trait that is thought to contribute to genetic risk for alcoholism. Such knowledge is essential for a complete understanding of the molecular basis of alcoholism and for the development of new or improved strategies for its treatment.

? DESCRIPTION (provided by applicant): Genetics clearly contributes to individual risk for nicotine dependence in humans and variations in nicotine sensitivity in experimental animals. In mice, several behavioral and physiological responses to nicotine have been demonstrated to be influenced by genetics. However, none of the specific genes that contribute to the genetic influence on nicotine sensitivity in mice have been identified. These genes remain an untapped source of information that almost certainly will improve our understanding of what drives individual differences in nicotine sensitivity. For example, the recent discovery that variants in human CHRNA5 are associated with risk for nicotine dependence in humans led to follow-up studies in rodents which not only helped to identify a specific neuronal pathway critical for controlling the level of nicotine consumption, but also demonstrated that this gene impacts individual differences in nicotine self-administration not by increased sensitivity to the reinforcng effects of nicotine at low doses but rather a lack of the loss of reinforcement at aversive doses. This is just one of many examples where the identification of a gene that contributes to individual variability in a phenotypic measure can lead to significant insights into the underlying biology of the measure. We previously have mapped chromosomal regions that harbor genes or genes that contribute to individual differences in oral nicotine intake in mice and it is the goal f this project to follow up this initial finding to identify the genes that contribute to variation i nicotine intake. For this, we will again map chromosomal regions that impact nicotine intake but this time in a panel of mice that will allow us to define with much greater precision the regions i the genome that harbor genes that impact nicotine intake. We will follow this up with selective breeding to produce lines of mice that differ in nicotine intake. The selection process should produce mouse lines that are enriched for alleles that are involved in increasing or decreasing nicotine intake. Finally, we will perform whole genome sequencing on the selected lines. We will use these sequencing data to establish whether the chromosomal regions identified through mapping are enriched through the selection process and to identify all variants within the region. We also will analyze the sequence data for other potential chromosomal regions that have been enriched through selection for nicotine consumption. This multi-tiered approach should allow us to substantially narrow the search for variants that influence nicotine intake and potentially lead to the identification of causal variants (functional variants that contribute to individual variabiity in nicotine consumption).

PROJECT SUMMARY/ABSTRACT Less than 1 in 10 individuals who attempt to quit smoking remain abstinent for 1 year. This poor quit rate is driven, in part, by the fact that currently available drugs used to aid in smoking cessation are only moderately effective, at best. Thus, there is a great need to develop novel drugs that are more effective for smoking cessation. It is the goal of this project to use a novel genetic strategy to identify new biological targets for the potential development of novel smoking cessation drugs. The genetic strategy is based upon identifying modifier genes that alter nicotine responses in mice that have a null mutation in Chrna5, the gene that codes for the nicotinic receptor ?5 subunit. In effect, modifier genes are genes that contribute to physiological and/or molecular processes that are important for the behavior of interest but that generally go undetected in the absence of a perturbation in the gene that they modify. Because variants in Chrna5 alter risk for nicotine dependence in humans and studies in rodents clearly demonstrate that Chrna5 is critical for many nicotine- related behaviors, we believe that identifying genes that modify the effect of Chrna5 deletion on nicotine behaviors will uncover new genes relevant to nicotine dependence that may serve as novel targets for novel smoking cessation pharmacotherapies. Importantly, the behaviors that we plan to screen for modifiers are not only dependent upon Chrna5, but also dependent uponthe medial habenula-IPN pathway, a neural pathway that is thought to play a critical role in nicotine dependence. To identify genetic modifiers of the effect of Chrna5 deletion on nicotine behaviors, we propose 3 aims. In specific aim 1, we will breed the Chrna5 null mutation onto each of the B6-ChrA/J chromosome substitution strains (CSS) and identify chromosomes that harbor modifier genes for the effect of Chrna5 deletion on three nicotine behaviors, oral nicotine intake, somatic signs of nicotine withdrawal, and nicotine conditioned place preference. For specific aim 2, we will fine map those chromosomes that harbor modifier genes using sequential congenic strains. Typically, 3 generations of congenic strains starting from a CSS strain provides mapping resolution equivalent to that of any high resolution mapping population. Finally, in specific aim 3, we will use RNA-seq to identify genes whose expression is altered by the identified modifier genes. Importantly, we will use a state of the art genetic strategy that will allow us to examine gene expression in a neural cell population that is highly relevant to the behaviors: Chrna5 expressing cells of the interpeduncular nucleus. By combining the results of this aim with modifier loci identified through aims 1 and 2, we expect to narrow the list of potential candidate modifier genes and identify pathways specifically impacted by the modifier genes. In short, we believe that this strategy will lead to the identification of previously unknown genes and/or genetic pathways that contribute to the physiological and/or molecular processes important for the response to nicotine. These genes and/or pathways may serve as novel targets for the development of new pharmacotherapies to aid in smoking cessation.