Identifying novel genes associated with response to nicotine in a zebrafish model of drug dependence.

Abstract

Tobacco addiction is a leading preventable cause of death worldwide and places a heavy social and financial burden on society. There exists a substantial genetic variability in smoking behavior, the mechanisms of which are largely unknown. Despite significant advances in sequencing power, progress in the identification of genetic variants affecting smoking behavior based on human genome wide association studies has been slow. Thus this thesis investigates the utility of zebrafish as a model species in which to search for genetic variants affecting nicotine seeking. The work is based on the premise that as zebrafish are vertebrate with conserved neurochemical pathways and circuitry with humans, and the pathways involved in drug mediated reward and addiction are evolutionarily ancient, homologues of genes affecting zebrafish nicotine-seeking behavior will likely affect human smoking behavior. Thus results in zebrafish can be used to direct human genetic studies. The first result chapter addresses the hypothesis that zebrafish show conserved reward responses to common drugs of abuse. A conditioned place preference assay is used to assess zebrafish reward responses to stimulants, opioids, benzodiazepines and alcohol. The results indicate that, with the exception of benzodiazepines, reward responses are conserved, supporting the use of this model in a screen for genetic variants affecting nicotine preference. The second and third results chapters describe the findings of a pilot screen of ENU-mutagenized zebrafish provided by the Sanger Institute, Cambridge. I demonstrate that nicotine preference is heritable in fish as in Abstract 5 humans and identify 3 mutant lines that show increased or decreased nicotine place preference. Genotyping indicated that one of the families showing increased nicotine preference carries a predicted loss of function mutation in the slit3 gene. The involvement of this gene in nicotine preference was confirmed in a separate line. Further characterization of this line using qPCR showed slit3 mutants to have altered developmental expression of key nicotinic and dopaminergic genes. Having identified the slit3 gene as a locus affecting nicotine seeking in fish, I then tested the hypothesis that results in fish could be used to predict loci that affect human smoking behavior. Cohorts of patients were genotyped for 20 SNPs within the slit3 locus. Results of this analysis identified 1 novel SNP in the slit3 gene associated with smoking behavior in a cohort of individuals that were heavy smokers. This result was validated in cohorts of low and normal smoking prevalence. These data demonstrate the utility of behavioral assays in zebrafish to identify genes affecting human behavior and pave the way for the use of zebrafish to inform human studies exploring the genetic basis of drug seeking and behavioral disease.