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

Summary

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Abstract

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

Introduction

Alcohol use disorder (AUD) is an enormous health problem worldwide (reviewed in ¹). Although the mechanisms driving the development of AUD are complex, these disorders have a major genetic component (e.g., ²). The large heritability of AUD and the conserved behavioral responses to ethanol across many species (reviewed in ^3,4) have generated strong interest in using genetic model organisms to investigate the involvement of specific genes in ethanol-related behaviors toward better understanding the molecular basis of AUD. The fruit fly, Drosophila melanogaster, has emerged as a leading model organism for exploring ....

Protocol

1. Day Before Assay

1. Collect flies into fresh food vials in groups of 11 (single sex) under brief (1-5 min) CO₂.
2. Allow flies to recover O/N in food vials in an environmentally controlled space (typically 25 °C, 60% relative humidity, 12 hr light/dark cycle).
3. Prepare ethanol solution(s) by diluting pure (100%) ethanol in purified (≥18 MΩ) water to final concentration(s) appropriate for the planned experiment. Allow solution(s) to return to RT O/N.
   Note: Dilution of ethanol is exothermic.

2. Day of Assay

1. For each vial of flies to be tested, prepare (i) a clean....

Results

The raw data from this ethanol sedation assay are the numbers of flies that are sedated as a function of ethanol vapor exposure time. Raw data are converted to the percent active flies as a function of time (primary data, Figures 1A, B, D-F). Sensitivity to ethanol sedation from the primary data can be quantitated as Sedation Time 50 (ST50), the time required for 50% of flies to become sedated or aea under the curve (AUC), via interpolation from curve fi.......

Discussion

Straightforward assays that reproducibly quantitate meaningful phenotypes are of great value for the analysis of behavior. The work described here addresses several practical aspects of an assay for measuring ethanol sedation sensitivity and rapid tolerance in Drosophila. Although not a focus of this work, behavioral analyses are facilitated by maintaining the environment and genetic background constant for test subjects within a study. Furthermore, comparisons should typically be made between groups of flies re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
food vials	VWR	89092-772	narrow
Flugs	Genesee/flystuff.com	49-102	narrow
silicone stopper	Fisher Scientific	09-704-1l	#4
ethanol	Pharmaco-Aaper	111000200	200 proof