Transcriptional response to amphetamine in Drosophila dopamine transporter (DAT) loss-of-function mutant

We used Drosophila genetic and behavioral models to examine AMPH-induced transcriptional changes in DAT-dependent manner, as those would be the most relevant to the stimulatory effects of the drug in the brain. We previously showed that flies respond to AMPH by increasing their locomotor activity and decreasing their sleep in a dopamine-dependent manner. Flies that carry a loss-of-function mutation in the gene encoding the Drosophila DAT homolog (dDATfmn, henceforth referred to as DAT mutants) exhibit heightened activity levels at baseline, consistent with increased levels of extracellular dopamine caused by the impairment of reuptake. In this study we compared gene expression changes in response to AMPH in brains of isogenic w1118 strain (WT) and DAT mutants. We found genes involved in the control of mRNA translation to be significantly upregulated in response to AMPH in a DAT-dependent manner. WT and DAT flies were divided into treatment and control groups resulting in four experimental groups: WT-AMPH, WT-Vehicle for the WT and DAT-AMPH, DAT-Vehicle for the DAT mutant. Each group was housed in polycarbonate tubes containing food consisting of 1% agar and 3% sucrose delivered in water (vehicle) or AMPH solution (10 mM) for 48 hours. Flies were continuously monitored for movement by a single infrared beam in the center of the tube using Trikinetics Drosophila Activity Monitors (TriKinetics, Waltham, MA). Four replicates from each of the four groups were used for subsequent whole brain RNA-Seq gene expression analysis. To ensure sufficient read coverage, we performed two, and in one case, three sequencing runs, except for the samples M1_3_WT0 and M4_17_DAT10, where a single run was sufficient.