The FlyBar: Administering Alcohol to Flies

Summary

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

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.

Introduction

Drosophila melanogaster demonstrate biphasic behavioral responses to alcohol¹ that are analogous to human responses to this drug^2,3. Upon initial exposure to low concentrations of alcohol, flies exhibit increased locomotor activity, replaced by a lack of motor coordination, the loss of postural control and righting reflexes (Loss of Righting Reflex: LoRR), and sedation (complete lack of motor activity in response to mechanical stimulation) as exposure to alcohol progresses^4-9. The endogenous circadian clock is a strong modulator of alcohol sensitivity and toxicity as observed in mice^10,11, rats¹², and hum....

Protocol

1. Assembly of the FlyBar

Rationale and overview: The system is designed to administer controlled percentages of alcohol vapor to flies. Note: Figure 1 provides a schematic overview of the FlyBar set-up as described below in three stages (assembly of the air flow, set-up of the alcohol and water bottles, and assembly of the observation vials). In short, a steady airflow is split into two fractions that are bubbled through alcohol and water, respectively, mixed, and administered to 4 observation vials.

1. Assembly of the Airflow
   1. Connect a short piece of flexible silicone tubing to either building air o....

Results

Circadian Modulation of Alcohol Sensitivity using the 50% LoRR as a Marker.

A representative example showing circadian modulation in alcohol sensitivity during the day is presented in Figure 2.  LoRR was measured at six time points during the 2^nd day of DD in Canton-S and the 50% LoRR was determined for each time point. Analysis showed a significant effect of time of day (ANOVA: F_5,45=7.39, p<0.001, N=6-10 per time point). The Fisher LSD test .......

Discussion

The costs of alcohol abuse and alcoholism for society is tremendous, both in terms of human²⁹ and economic costs^30,31. Drosophila as a model offers a fast and versatile system to quickly examine the behavioral responses of a large number of individuals and as such has been extensively used for both alcohol^5,7,32-34 and circadian research^35-37.

Here, we described a straightforward protocol for the controlled administration of alcohol vapor to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alcohol 190 proof	Various
Aerator	Local pet store		We use Whisper 60
Silicone tubing 1/8”	VWR	408060-0030
120° Y-connector	VWR	82017-256
Quick disconnects	VWR	46600-048
Plastic tube clamps	Bell-art products	132250000	Either this or next
Miniature air regulator	McMaster-Carr	8727K11	Either this or previous
Miniature air regulator mounting bracket	McMaster-Carr	9891K66
Gilmont size 12 flow meter	VWR	29895-242
Tool clips	McMaster-Carr	1722A43	To hold flow meters
Vial	VWR	89092-722
Rubber stopper with two holes	VWR	59585-186	Fits in vials
5 mm Pyrex glass tubes	Trikinetics	PGT5x65	Fits best in previous stopper
Teflon tape	Hardware store		To achieve snug fit in stoppers if necessary
Rubber stopper with two holes	VWR	59582-122	Fits our bottles
Disposable glass pipets	VWR	53283-768	Cut to length and bend by heating
Very fine nylon netting	VWR	Various
15 watt bulbs	Hardware store		Overhead red light
Photographic red safe light filters			Overhead red light
Mini flashlights with red filters	Mag-light