Contrasting Influences ofDrosophila white/mini-whiteon Ethanol Sensitivity in Two Different Behavioral Assays

Abstract

Background The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white, a derivative of the endogenous gene white. Whether the mini-white transgenic marker or the endogenous white gene influence behavioral responses to acute ethanol exposure in flies has not been systematically investigated. Methods We manipulated mini-white and white expression via (i) transposons marked with mini-white, (ii) RNAi against mini-white and white and (iii) a null allele of white. We assessed ethanol sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of ethanol) and an assay based on ethanol-induced sedation. Results In eRING assays, ethanol-induced impairment of climbing correlated inversely with expression of the mini-white marker from a series of transposon insertions. Additionally, flies harboring a null allele of white or flies with RNAi-mediated knockdown of mini-white were significantly more sensitive to ethanol in eRING assays than controls expressing endogenous white or the mini-white marker. In contrast, ethanol sensitivity and rapid tolerance measured in the ethanol sedation assay were not affected by decreased expression of mini-white or endogenous white in flies. Conclusions Ethanol sensitivity measured in the eRING assay is noticeably influenced by white and mini-white, making eRING problematic for studies on ethanol-related behavior in Drosophila using transgenes marked with mini-white. In contrast, the ethanol sedation assay described here is a suitable behavioral paradigm for studies on ethanol sedation and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-white.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in <em>Drosophila</em>

“…In a previous article from our group on the genetics of ethanol sensitivity and rapid tolerance in Drosophila , a behavioral assay based on ethanol vapor-induced sedation of flies was used 18 . Testing in this assay was initiated by transferring live adult flies without anesthesia to empty food vials, trapping the flies in the vials with a cellulose acetate plug, adding ethanol to the top ( i.e.…”

“…Testing in this assay was initiated by transferring live adult flies without anesthesia to empty food vials, trapping the flies in the vials with a cellulose acetate plug, adding ethanol to the top ( i.e. non-fly side) of the cellulose acetate plug, and sealing the vial containing flies, cellulose acetate plug and ethanol with a silicone stopper (see schematic in Figure S3 , reference 18 ). Multiple vials representing different groups of flies were assessed in parallel, increasing throughput of this assay.…”

“…In a standard experiment, flies in vials were tapped gently at 6 min intervals and, after a 30 sec recovery, the number of sedated flies in each vial was counted and converted to percent active flies. Flies absorbed ethanol vapor from the cellulose acetate plug in a time-dependent fashion, causing progressive increases in internal ethanol 18 and sedation (c.f. reference 18 and Figure 1A and 1B in this report).…”

Genetics and genomics of alcohol responses in Drosophila

“…The Clic gene was identified as the highest-ranked alcohol-responsive gene in mouse and human transcriptome studies. Mutations in the C. elegans orthologues produced ethanol resistance (Bhandari et al , 2012) as did genetic knockdown of Clic in flies (Chan et al , 2014). Clic encodes a mysterious protein that functions as an anion-specific ion channel (Littler et al , 2008).…”

Genomewide Association Study of Alcohol Dependence Identifies Risk Loci Altering Ethanol‐Response Behaviors in Model Organisms

“…There are also differences in ethanol-response measures available for different MOs (e.g. AFT has not been demonstrated in flies despite direct efforts to elicit this response (Chan et al, 2014)). Within species, we observe consilience across studies for effects of 1) Klf-3 and binding partner Ctbp1 on AFT and 2) genes influencing intracellular calcium levels on initial sensitivity in worms, and 2) Col6a3 on HIC in mice.…”