We are using Drosophila melanogaster as a model system to understand the acute neural side effects of exposure to organic solvent inhalants. These inhalants, which are abused by a startling large number of adolescents, are commonly found in household cleaning solutions. We observed that a single solvent sedation causes changes in the expression of the slowpoke K+ channel gene and also induces solvent tolerance. We have demonstrated linkage between these phenomena. A mutation that eliminates slowpoke expression also prevents the acquisition of tolerance while transgenic slowpoke expression causes solvent resistance. Our interest is in the mechanics of how nerve cells activate the channel gene expression in response to solvent sedation. We postulate that gene induction induced by this drug experience might involve so-called """"""""epigenetic changes"""""""". We will catalog the epigenetic changes induced by sedation across the slowpoke promoter region and correlate these with behavioral tolerance using the chromatin immunoprecipitation assay. We have shown that sedation quickly produces an increase in CREB transcription factor binding at a conserved DNA element in the slowpoke promoter region followed by a spike of histone acetylation at this site. We will use tools unique to Drosophila to specifically block histone acetylation at this site to determine if it simultaneously blocks slowpoke induction and behavioral drug tolerance. Briefly, transposons carrying Gal4 UAS binding sites have been positioned within the slowpoke control region. A chimeric transcription factor made from a Gal4 DNA binding domain and a histone deacetylase will be used to suppress the sedation-induced acetylation spikes within the slowpoke control region. We will unambiguously test the cause and effect relationship between sedation, acetylation of these sites, gene induction and behavioral tolerance. In a related vein small chromosome deletions will be induced in the endogenous slowpoke promoter region using P element mediated mutagenesis (imprecise excision and HEI). These will be used to physically map the site of transcriptional control elements that regulate slowpoke epigenetic changes and gene expression and to determine if the promoter mutations eliminates the capacity for solvent tolerance. We will provide a unified description of how this gene senses and responds to the effects of abused solvent sedation. ? ? ?
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