Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol. Alcoholism is a complex condition of compulsive alcohol use that results in devastating physical and social consequences. The development of this affliction is believed to arise in part by homeostatic adaptations in the brain that lead to the development of alcohol tolerance and dependence. These adaptations are strongly influenced by a great number of genetic and environmental conditions. Identifying the relevant factors that define alcohol tolerance and dependence has been a major objective of neurobiology research for many decades. Here we use a novel genomic approach that exploits the analysis of epigenetic modifications and the power of Drosophila genetics to identify a network of genes with a potential role in the neuroadaptations that lead to alcohol tolerance. Gene-expression profiling and subsequent gene ontology analysis revealed that the group of genes identified here is highly enriched with genes involved in the activity-dependent modulation of synaptic transmission. Because of the strong conservation of regulatory gene networks between Drosophila and mammals, we believe that the network identified here will serve as a powerful guide for the identification of the regulatory events that lead to human alcohol tolerance.

Significance: Quantifying the preference for particular foods is difficult in Drosophila because of the animal’s small meal size. It is common for fly feeding assays to induce or require starvation conditions. We have developed BARCODE, a method of tracking consumption preference by tagging foods with different oligonucleotides and later performing qPCR from the fly body to determine its feeding history. Because a fraction of the ingested oligomer persists for about a week, this assay provides a long-term record of the feeding pattern. We use this assay to study sexually dimorphic differences in ethanol consumption preference in Drosophila . BARCODE is also suitable for multiplex feeding studies in which the preference between more than two foods is simultaneously compared.

Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.