The FlyBar: Administering Alcohol to Flies

Abstract

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes. Video LinkThe video component of this article can be found at

Aging and circadian dysfunction increase alcohol sensitivity and exacerbate mortality in Drosophila melanogaster

“…To better understand how the circadian regulation of alcohol sensitivity changes with age, we examined a second alcohol behavior, alcohol-induced sedation, across age groups. Sedation is the induction of a sleep state characterized by immobility (De Nobrega and Lyons, 2016, van der Linde et al, 2014). We observed a robust rhythm in alcohol-induced sedation in young (3 d old) flies (Fig.…”

“…Alcohol delivery occurred using an alcohol vapor system in the dark as previously described (De Nobrega and Lyons, 2016, van der Linde et al, 2014, van der Linde and Lyons, 2011). Briefly, four tubes of flies (approximately 30 flies per tube) were exposed to a mixture of alcohol vapor and humidified air at a predetermined ratio of 95% alcohol and de-ionized water (Koptec, Declon Labs, Inc. King of Prussia, PA).…”

“…Sedation was performed as previously (De Nobrega and Lyons, 2016, van der Linde et al, 2014). Flies were exposed to 40% alcohol vapor on the second day of DD, except for 3 d flies that were exposed to 50% alcohol vapor to avoid tolerance development within the time period to sedation.…”

Circadian Modulation of Alcohol-Induced Sedation and Recovery in Male and Female Drosophila

“…Exposure to alcohol vapor was performed as described previously (van der Linde and Lyons, 2011; van der Linde et al, 2014). Four tubes of Drosophila (3–5 days old; ~30 flies per tube) were exposed to ethanol vapor by mixing proportional fractions of an airstream bubbled through deionized water with an airstream bubbled through 95% ethanol (Koptec) to achieve the pre-determined percentage of ethanol vapor.…”

“…The loss-of-righting reflex assay was performed as previously described in Drosophila (van der Linde and Lyons, 2011; van der Linde et al, 2014). Prior to the start of alcohol exposure, flies were habituated to dark room conditions for one hour (25° Celsius, 60 – 70 % humidity).…”