High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

Summary

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

Abstract

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.

To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.

Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal´s dynamic behavioral reaction.

Introduction

The overarching goal of any neuroethological research is to establish a causal link between the activity states of single neurons or neuronal circuits and the behavior of an organism. To achieve this goal neuronal activity and behavior should be monitored under identical stimulus conditions and these stimulus conditions should ideally be similar to those the nervous system under scrutiny evolved to make sense of. Particularly when it comes to behavioral bioassays, these requirements have historically proven quite demanding in Drosophila melanogaster olfactory neuroethology.

Once released from the source, odor plumes rapidly break u....

Protocol

The construction and technical details of Flywalk have been described elsewhere²⁶ (in case of any problems of establishing this set up, further information can be obtained from MK). Here we focus on detailed instructions on the handling of the paradigm that will help to obtain reliable results.

1. Fly Handling

1. Rear flies in low to medium density cultures on food medium²⁷ under a 12 hr:12 hr light:dark regime at 23-25 °C and 70% relative humidity. To this end, allow 20-30 newly emerged adult flies to reproduce in a big food vial for 1 week, then discard adult flies and wait for offspring to emer....

Results

Because flies are allowed to distribute freely within their glass tubes between odor pulses and the odor pulse travels through the glass tubes at a constant speed flies encounter the odor at different times depending on their x-position at the time of stimulation. As a result, the onsets of the upwind trajectories evoked by a 500 msec pulse of an attractive 10^-3 dilution of ethyl acetate are delayed by about 1 sec for flies at the downwind end of their glass tubes compared to those of flies sitting closer to t.......

Discussion

Although the Flywalk system appears rather sophisticated at first glance, once set up and running it is easy to use and produces very robust results. To stress the consistency of the results produced with the bioassay it may be said that the representative results shown here were obtained almost 2 years after some of the results shown in a previous study²⁹ with a modified setup using a new tracking software and light source. Nevertheless, the attractant responses are - despite slightly higher response amplitud.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flywalk setup	Custom		details available upon request
stimulus device	Custom		details available upon request
LED cluster	Custom		details available upon request
HD Pro Webcam C920	Logitech, Lausanne, Switzerland
2 Computers
Flywalk Reloaded v1.0 software	Electricidade Em Pó (electricidadeempo.net)
Labview 11.0 software	National Instruments, Austin, TX
Standard fly food	Custom
Standard fly vials	Greiner bio-one GmbH, Frickenhausen, Germany
Standard fly vials	Greiner bio-one GmbH, Frickenhausen, Germany
aspirator	Custom
mineral oil	Sigma-Aldrich (www.sigmaaldrich.com)
odors	Sigma-Aldrich (www.sigmaaldrich.com)
200 µl PCR reaction tubes	Biozym Scientific GmbH, Oldendorf, Germany