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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

The FlyBar: Administering Alcohol to Flies

Published: May 18th, 2014

DOI:

10.3791/50442

1Department of Biological Science, Florida State University, 2Department of Biology and Biochemistry, Biology of Behavior Institute, University of Houston

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.

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.

Drosophila melanogaster demonstrate biphasic behavioral responses to alcohol1 that are analogous to human responses to this drug2,3. 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 progresses4-9. The endogenous circadian clock is a strong modulator of alcohol sensitivity and toxicity as observed in mice10,11, rats12, and hum....

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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 .......

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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 2nd 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: F5,45=7.39, p<0.001, N=6-10 per time point). The Fisher LSD.......

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The costs of alcohol abuse and alcoholism for society is tremendous, both in terms of human29 and economic costs30,31. 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 alcohol5,7,32-34 and circadian research35-37.

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

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Funding for this research was provided by a Program in Neuroscience Award from the Florida State University College of Medicine and support from the Department of Biological Science at FSU.  Additional Funding was provided by a Grant-in-Aid from the Alcohol Beverage Manufacturer’s Research Fund.

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Name Company Catalog Number Comments
Alcohol 190 proof Various
Name of Material Company Catalog Number Comments
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

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