Steven L. McIntire, MD. Ph.D. - US grants

DESCRIPTION (provided by applicant): Ethanol is one of the most widely abused substances in the world, yet the molecular mechanisms of action of ethanol is poorly understood. Ethanol has been shown to disrupt ion channel functions in vitro through interactions with hydrophobic domains of channel subunit proteins. It is still unclear, however, how these effects of ethanol relate to the behavioral changes observed in humans and other organisms. Unlike other drugs of abuse, ethanol is likely to act through multiple molecular targets rather than upon a single major neurotransmitter receptor or transporter. We have initiated studies using C. elegans as a model system to understand the neurobiological effects of ethanol. We have shown that C. elegans exhibits similar behavioral responses to ethanol as mammalian systems including acute intoxication, adaptive neural responses or acute tolerance and state-dependency. We propose to isolate mutants that are hypersensitive to the intoxicating effects of ethanol on different behaviors. We have devised screens that provide for the isolation of mutants hypersensitive to the effects of ethanol on locomotory and egg laying behaviors. We will also identify mutants defective in acute tolerance to ethanol and mutants exhibiting enhanced tolerance. A final screen provides for an enrichment in mutants that are insensitive to the state-dependent effects of ethanol. Secondary behavioral characterizations and pharmacological assays will be carried out to determine whether the mutations affect neural activity and whether the phenotypes are likely to be ethanol specific. We will map the mutations to specific chromosomal regions and molecularly characterize many of the mutants. The genes identified are likely to encode relevant targets of ethanol, either as direct targets or as members of a pathway affected by ethanol. The studies should also lead to a greater understanding of the molecular mechanisms mediating the effects of ethanol invertebrate systems. The determination of the mechanisms of action of ethanol should provide the basis for a directed approach to the development of therapeutics to treat alcoholism and alcoholic neurological disorders.

DESCRIPTION (provided by applicant): Ethanol is one of the most widely abused substances in the world, yet the molecular mechanisms of action of ethanol are poorly understood. Ethanol has been shown to disrupt ion channel functions in vitro through interactions with hydrophobic domains of channel subunit proteins and an understanding of the mechanisms that are likely to underlie some of the acute effects of ethanol has begun to emerge. It is still unclear, however, how many of the more complex behavioral responses to ethanol, such as tolerance and dependence, are mediated at a molecular level. We have pursued studies in C. elegans, as a model system, to understand the different molecular mechanisms of action of ethanol. Through genetic studies, we have identified proteins that are targets of ethanol or required for normal ethanol responses of C. elegans. In many cases, homologous proteins are also thought to be required for behavioral responses to ethanol in mammalian systems. We have demonstrated and characterized a form of acute tolerance in C. elegans that can be observed during sustained but brief ethanol exposures. After more prolonged exposure to ethanol and development of tolerance, wild type animals exhibit an intriguing change in their behavior that consists of an "ethanol-seeking" response, which we have quantified in a preference-type assay. This change is strengthened with longer ethanol exposures of several days. We have demonstrated that this response is partially dependent on dopaminergic and serotonergic function, as mutants with known defects in dopamine or serotonin synthesis exhibit substantially reduced ethanol-seeking. In addition, we have identified two novel mutants that are defective in the development of ethanol-seeking behavior. These ethanol-seeking defective mutants (esd-1 and esd-2) are also defective in the development of tolerance to ethanol. A subset of mutants defective in ethanol tolerance are found to be defective in ethanol seeking behavior, suggesting that the molecular mechanisms underlying the development of tolerance and ethanol-seeking behavior partially overlap. We now propose to complete the mapping of esd-1 and esd-2 and to clone and characterize the corresponding genes to gain insight into the molecular mechanisms that underlie this change in the desirability of ethanol over time. We will also pursue further detailed characterizations of the ethanol seeking response in wild type animals, and we will test additional mutants for defects in ethanol-seeking, as observed with esd-1 and esd-2. The determination of novel molecular mechanisms that underlie ethanol-seeking in C. elegans may enhance our understanding of changes in ethanol preference in vertebrate systems. PUBLIC HEALTH RELEVANCE: This project seeks to identify and characterize at a molecular level the mechanisms that underlie ethanol- seeking behavior in C. elegans or changes in the desirability of ethanol. Such mechanisms may be conserved. A greater understanding of the molecular pathways mediating such behavioral changes could lead to the development of medications to treat individuals who are alcohol dependent.