Andrew Davies - US grants

[unreadable] DESCRIPTION (provided by applicant): Abuse of alcohol creates enormous social and financial problems. An improved understanding of the molecular basis of alcohol intoxication is likely to improve our treatments for alcohol poisoning and withdrawal, and further our understanding of alcohol tolerance and addiction, the development of which is likely to be rooted in the acute effects of alcohol. The overall goal of this project is to identify molecular targets of alcohol that mediate the behavioral effects of the drug using the model organism, Caenorhabditis elegans. The level of conservation of the nervous system components in C. elegans and humans is such that the targets of alcohol found in C. elegans are likely to be present in the human nervous system. Given the link between acute alcohol response and predisposition to alcoholism, the human homologs of the genes identified in this study are likely to be valid candidates for genes that predispose an individual to alcoholism. The following specific aims describe the means by which these ethanol targets will be identified. 1. Isolate mutations affecting alcohol targets or regulators of those targets using forward genetics. The premise of this aim is that a mutation that eliminates a target of alcohol will display increased resistance. A behavioral screen of the progeny of randomly-mutagenized slo-1 mutant animals will be employed that is selective for mutants that display increased levels of resistance to the effect of alcohol on locomotion. Using a slo-1 mutation will ensure that the screen will identify targets of alcohol other than SLO-1. 2. Carry out behavioral and pharmacological characterization of the mutants and a detailed examination of their alcohol response across a variety of behaviors to provide insight into the nature of the mutant defect in the animals, as well as providing a means of prioritizing which of the mutants to study. 3. Positional map and clone the prioritized genes using a combination of genetic mapping and a candidate gene approach that will utilize RNA interference to test the effect of loss of function of candidates on alcohol sensitivity. 4. Determine the expression pattern of the resistance genes using green fluorescent protein. This aim along with Aim 2 will assist in defining a function for the genes (if not already known) and will provide a better understanding of the mechanism by which mutations in these genes result in ethanol resistance. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY There are two, partially overlapping goals of this research: (1) comprehensively define the array of G protein- coupled neuropeptide receptors that act to modulate and mediate acute actions of ethanol in vivo. (2) investigate the impact of specific behavioral states on acute sensitivity to ethanol. We will characterize the roles of all neuropeptide receptors in C. elegans in basal locomotion behaviors and ethanol-induced behavioral effects. This comprehensive assessment will identify receptors that positively and negatively regulate the neuronal circuit that controls locomotion and those receptors that act to promote or negatively regulate ethanol actions. We will assess both the level of initial sensitivity to ethanol and the time-dependent development of acute functional tolerance to the drug. In addition, for those receptors that act to modify responses to ethanol, we will classify any interactions with the neuropeptidase nep-2, which is orthologous to the mammalian neprilysin protein. The mammalian neprilysin protein is involved in the regulation of levels of multiple important signaling peptides, including enkephalins, tachykinin, substance P and others. A mutation in the nep-2 gene produces an ethanol-resistant behavioral phenotype. We hypothesize that a peptide target of NEP-2, which is likely to be elevated in a nep-2 mutant background, acts to counteract acute effects of ethanol via increased signaling through a neuropeptide receptor. The proposed experiments will identify that receptor and test the hypothesis that the receptor acts in a defined neuronal circuit that controls behavioral state decisions. Our preliminary data has identified several mutants that affect both ethanol responses and a behavioral state decision that affects exploratory behavior. The circuit that regulates that decision is well defined, and includes the sites of action of neuropeptides and serotonin. We will define networks of genes that act to regulate that behavioral decision and ethanol responses, and test specific neurons in the controlling circuit for their role in regulating ethanol responses. The relationship between an emotional (or affective) state in humans and the problematic use of drugs of abuse is of significant interest. The successful outcome of this proposed research will provide a better understanding of how specific behavioral states, controlled by a known regulatory circuit, can impact the acute responses to an abused drug. There is a significant correlation between the level of an individual?s initial response to alcohol and their likelihood to develop an alcohol use disorder (AUD). Genetic variation in any of the human orthologs of the C. elegans genes identified in this study has the potential to alter an individual?s level of response to ethanol, and therefore could impact that individual?s predisposition to develop an AUD later in life.