Conserved Role of unc-79 in Ethanol Responses in Lightweight Mutant Mice

Abstract

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants

“…QTL approaches have been successful in mapping and cloning ENU-induced mutations (12-14, 54, 55). In several cases cloning has been facilitated by lethality exhibited by the homozygous mutant, turning a quantitative behavioral trait into a qualitative viability trait that can be used for genetic mapping (12,13). We believe a combination of low-resolution mapping using QTL analysis with the recent advances in next-generation sequencing technology will provide a feasible route to cloning of these mutants (11).…”

“…Increasing the number of animals tested is often prohibitive with mice owing to cost, and instead the threshold for detection of mutants is raised to as high as 3 SDs to minimize falsepositive results. In addition, locomotor output assays are often used as endophenotypes for complex emotionality, and these outputs can be affected by systems independent of the behavioral pathway being investigated, leading to identification of animals with pleiotropic phenotypes (12,13). Moreover, mutations in genes known to regulate behaviors such as learning and memory, psychostimulant response, and open field behavior usually only produce a shift in the mean of 1 to 2 SDs (11,(13)(14)(15).…”

“…In addition, locomotor output assays are often used as endophenotypes for complex emotionality, and these outputs can be affected by systems independent of the behavioral pathway being investigated, leading to identification of animals with pleiotropic phenotypes (12,13). Moreover, mutations in genes known to regulate behaviors such as learning and memory, psychostimulant response, and open field behavior usually only produce a shift in the mean of 1 to 2 SDs (11,(13)(14)(15). Thus, when genetic screens are conducted to search for "strong" (greater than 3 SDs) mutants, the investigator is attempting to discover mutants that are more extreme than what would normally be found if a key gene, known to mediate the behavior, was deleted.…”

A Novel Cholinergic Action of Alcohol and the Development of Tolerance to That Effect inCaenorhabditis elegans

“…Mutations in multiple genes have previously been shown to generate altered phenotypes compared to wild-type animals upon treatment with ethanol (Davies et al 2003(Davies et al , 2004Kayser et al 2003;Kapfhamer et al 2008;Graham et al 2009;Speca et al 2010;Bettinger et al 2012;Bhandari et al 2012;Jee et al 2013;Raabe et al 2014). We examined two of these genes that have robust mutant ethanol response phenotypes, slo-1 and npr-1.…”

“…C. elegans are sensitive to acute ethanol exposure, demonstrating an increased speed of locomotion at low ethanol concentrations (Graham et al 2009) and decreased amplitude of body bends and decreased rates of crawling, swimming, egg laying, and pharyngeal pumping at higher concentrations of ethanol (Davies et al 2003;Mitchell et al 2007;Speca et al 2010). Ethanol also produces disinhibition of certain crawling behaviors that would normally be inhibited in a liquid environment (Topper et al 2014).…”