Molecular and genetic mechanisms of ethanol tolerance in the fruit fly

Krishnan, Harish Ravikumar, 1975-
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Tolerance to the sedative effects of ethanol is an acute physiological change that can lead to more chronic phenotypes such as dependence and addiction. Ethanol tolerance in the adult nervous system of the fruit fly, Drosophila melanogaster, is dependent on the calcium-activated potassium channel (BK) gene, slowpoke. This gene is highly conserved between flies and humans and critically involved in neuronal communication in the brain, where it modulates action potential duration and regulates the firing rate of neurons. Adult Drosophila acquire rapid functional tolerance to ethanol after a single sedation due in part to a transcriptional up-regulation of the slowpoke message. Genes can be regulated at multiple levels, for instance, at the level of transcription, level of mRNA splicing, mRNA editing, post-translational modifications to the protein and protein localization. Using a heat inducible transgene expressing slowpoke in a null mutant background, I show that flies are able to acquire tolerance in the absence of transgenic induction. This suggests that slowpoke mediated rapid tolerance to ethanol involves a transcription-independent mechanism. Regulation of gene expression involves post-translational modifications to histones which cause chromatin decondensation and recruit transcription factors at the promoter to initiate transcription. Using the chromatin immunoprecipitation assay followed by real-time quantitative PCR, I show that the slowpoke promoter has a distinct spatio-temporal pattern of histone acetylation following a single sedative dose of ethanol. Increased acetylation at conserved control elements in the promoter is responsible for the transcriptional up-regulation of the gene following ethanol sedation. It is well known that anesthetics and abused drugs preferentially modulate specific molecular pathways in defined regions of the brain. In this study I have identified brain structures where slowpoke induction may play a role to elicit tolerance. Using the binary GAL4/UAS system to express slowpoke in different parts of the Drosophila nervous system I show that slowpoke induction in the mushroom bodies of the adult fly brain, a region critical for olfactory learning and memory, is important for ethanol tolerance. A survey of brain structures mediating drug tolerance in the genetically tractable Drosophila could potentially reveal evolutionarily conserved molecular pathways regulating tolerance. Using temperature sensitive mutants to block neuronal transmission I show that tolerance is a cell autonomous property of the nervous system that is independent of synaptic communication and not an emergent property of the whole brain. I also show that to elicit tolerance the presence of drug is necessary and reduced neuronal signaling does not phenocopy or mimic tolerance. I have identified Drosophila shibire as a critical gene mediating rapid tolerance to ethanol in flies. This gene is the Drosophila homolog of dynamin and regulates vesicle recycling at the synapse and neuronal communication in the brain. Two independent alleles of shibire, shits1 and shits2 are incapable of acquiring tolerance to ethanol. Increased activity of SLOWPOKE channels is predicted to enhance the firing frequency of neurons. This may augment the recovery of flies from sedation leading to the observed behavioral tolerance. DYNAMIN also functions at the synapse to mediate rapid neurotransmission and critically modulate neuronal excitability. This suggests that rapid tolerance to ethanol sedation may be a neuroadaptive state regulated by homeostatic changes in neuronal excitability that lead to faster recovery from sedation.