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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 up into thin filaments with turbulent diffusion caused by air movement being the main determinant of odor distribution¹. As a result, an insect navigating towards an odor source experiences intermittent stimulation with odor packages interspersed with variable intervals of clean air. Both walking and flying insects - including Drosophila- have been demonstrated to exploit this intermittent stimulation regime for navigation by surging upwind upon plume encounter and predominantly moving cross-wind in the absence of odors^2-5. Whereas stimulation procedures in physiological experiments largely mimic those an insect may experience in its natural environment by either providing single puffs of odors interspersed with extended periods of clean air or dynamic stimulation sequences^6-11, many behavioral bioassays used in Drosophila neuroethology such as trap assay, open-field arenas or T-maze rely on odor-gradients^12-15. However, because odor gradients by definition are variable in concentration depending on the distance from the odor source, a particular behavior cannot be attributed to a precise odor concentration using these paradigms. In addition, the slope of an odor gradient critically depends on the physicochemical properties of the odorant. A gradient of a highly volatile compound will be shallower than that created by a less volatile compound and therefore also harder to track for an organism relying on measuring concentration differences in space as the only means of navigation^16-20, which may lead to a misinterpretation of olfactory preferences particularly in choice assays. This effect is also highly detrimental when investigating behavior towards odor mixtures because it leads to different blend component ratios at every point in space and therefore again precludes a clear correlation between physiology and behavior.

While vinegar flies tend to aggregate on fermenting fruit, they are solitary in their navigation towards food sources and oviposition sites. Nevertheless, rather than testing individual animals many behavioral paradigms used in Drosophila neuroethology examine the odor-guided behavior of cohorts of flies and attraction is scored as the fraction of flies choosing the odor over a control stimulus. These cohort experiments have contributed greatly to the understanding of fly neuroethology and many of the observations made by using them could be confirmed in single-fly experiments. However, it has been observed that flies can influence each other´s decision²¹ and in extreme cases the evaluation of an odor can switch from indifference to avoidance depending on population density²². Additionally, results from these kinds of experiments often provide only the endpoint of a sequence of behavioral decisions rather than observing what the fly is doing while it is doing it, which would be desirable when attempting to correlate behavior with neuronal activity. These rather low-resolution cohort experiments are contrasted by high-resolution single-fly methods such as tethered flight arenas and treadmills which allow for a direct observation of behavioral responses at the time the stimulus is presented^20,23,24. Nevertheless, cohort experiments are still popular, because they are very efficient and provide robust results even at comparably low sample sizes because inter-individual and inter-trial variability are partially averaged out due to the observation of populations over extended periods of time. While tethered flight and treadmill probably provide the gold standard concerning stimulus presentation and temporal resolution, the arenas used are designed for single animals and it is therefore time-consuming to obtain sample sizes necessary for a statistical analysis. Several other approaches have recently been developed that allow an efficient acquisition of high-resolution behavioral data in combination with a well-defined stimulus regime. These include unsupervised 3D-tracking of multiple vinegar flies in a windtunnel in combination with an accurate 3D-model of the odor plume⁵, tracking of multiple individual flies in choice chambers supplied with airstreams from both sides²⁵ and the Flywalk paradigm²⁶.

In Flywalk, 15 individual flies are situated in small glass tubes and continuously monitored by an overhead camera under red-light conditions. Odors are added to a continuous airstream of 20 cm/sec and travel through the glass tubes at a constant speed. The airstream is humidified by passing it through 250 ml bottles containing distilled water (humidifiers) before entering the odor delivery system. The flies´ positions are recorded within a square region of interest (ROI) encompassing most of the length of the odor tubes (but excluding the outer edges of the tubes (approximately 5 mm at each side) where the flies cannot move further up- or downwind) around the time of odor presentation (Figure 1A, B). Fly identities are kept constant by the tracking system throughout the experiment on the basis of their Y-positions (i.e. their glass tube limits). Odor stimulation is achieved using a multi-component stimulus device which allows the presentation of up to 8 single odors and all possible mixtures thereof^26,29 (Figure 1B). The course of an experiment is controlled by a computer regulating the odor delivery system and collecting temperature and humidity information (computer 1, Figure 1C). This computer also controls a datalogger (start/stop recording) on a second computer which continuously tracks fly positions at 20 frames per sec (computer 2). Fly positions, odor valve status (i.e. time-point of valve opening), odor ID, temperature and humidity around odor stimulation cycles are logged on computer 2. This way information on odor and fly positions are synchronized and exported as .csv-files which can be further processed and analyzed using custom-written analysis routines. Because the whole system is computer-controlled, no human intervention is necessary during an experimental session.

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Protokół

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 emerge.
2. Collect 30-40 newly emerged (age <24 hr) adult flies and age them on in new a vial containing food medium²⁷ for 3-5 days.
3. Twenty-four hr before the start of the behavioral experiment: Transfer all 30-40 previously collected 3-5 d old (see 2.2) flies to a new vial containing a moist rubber foam plug or a moist tissue paper using an aspirator.
   NOTE: Do not anaesthetize flies using CO₂.

2. Preparation of the Flywalk Setup 

1. Use 250 ml bottles as humidifiers. Fill humidifiers with 100 ml of distilled water.
2. Prepare odor vials.
   1. Prepare 500 µl 10^-3 dilutions of the pure odors ethyl acetate, ethyl butyrate, isopentyl acetate and 2,3-butanedione in the solvent mineral oil.
   2. Attach two ball check valves per odor vial. Note that check valves allow for uni-directional airflow only. Therefore, connect check valves in such a way that air can enter the vial on one side and leave it on the other side.
   3. Remove the lid of a 200 µl PCR reaction tube. Pipette 100 µl of every odor dilution into a separate reaction tube and place the tubes in separate odor vials. Also prepare one odor vial containing only the solvent mineral oil.
   4. Tightly seal the odor vials by closing them using stainless steel plugs and rubber gaskets.
   5. Connect the 5 odor vials (4 containing odors and 1 containing mineral oil) to the odor delivery system. Make sure to connect them in the right flow direction. A wrong connection will not only compromise the planned experiment, but it may also contaminate the delivery system. 
3. Check for leaks by sealing the outlet of the mixing chamber of the stimulus device. Make sure, that all airflows before the stimulus device now progressively drop to zero. If not, check for leaks which can now be identified by the hissing sound of air leaving the system.
4. Carefully transfer 15 individual flies to 15 individual glass tubes using an aspirator and close glass tubes on both sides using the corresponding adapters. 
   Note: Because the system must be hermetically sealed for successful experiments, ensure that adapters fit glass tubes tightly and note that glass tubes may break during this step. Take care to avoid injuries by wearing protective gloves and goggles.
5. Connect the glass tubes to the Flywalk setup and, from here on, wait for at least 15 min before starting the experiment to allow flies to habituate to the new environment.
6. After attaching glass tubes: check the readout of the downstream digital flow meters on computer 1 if the 16 airflows after the glass tubes add up to the airflow entering the system. Also check on computer 1, if humidity is between 60% and 80%.
7. Design stimulus protocol controlling the sequence and timing of odor stimuli presented to the flies. To obtain e.g. the described data, present 4 odors and the control (mineral oil) singly and all possible ternary and the quarternary mixtures of the odors simultaneously for 40 times each. Set pulse duration to 500 msec at an interstimulus interval of 90 sec and randomize stimulus sequence.
8. Switch on the light source (LED-cluster; λ = 630). Make sure to provide enough light for efficient tracking without increasing the temperature inside the glass tubes.
9. Set up a region of interest of the tracking system by dragging a frame across the area to be monitored in such a way that all 15 glass tubes are included and approximately 5 mm of the edges of the tubes are excluded.
10. Set up 14 parallel separation lines between individual tubes in the tracking system by changing their Y-positions in the corresponding script to keep individual flies identifiable throughout the experiment. Make sure to position them in such a way that there is always one glass tube between two such separator lines, because only one fly will be tracked between any set of two lines.
11. Make sure to set camera parameters in such a way that flies are reliably tracked throughout the glass tubes. If flies are lost at the edges of the region of interest, increase brightness or gain of the tracking software. Avoid mechanical vibrations of the tracking system. Track using commercial software according to manufacturer's protocol.
12. Start experiment by starting the stimulus protocol. Record flies´ XY-coordinates at 20 fps (frames per sec) and log in combination with the odor valve status in text files.

3. Data Analysis

NOTE: The following steps in the data analysis are automatized using custom-written routines programmed in R. Because these steps are crucial to obtain meaningful results the analysis will nevertheless be presented in a step-by-step manner. The raw data for the analysis are .csv-files containing synchronized information on odor valve status, pulse number in the experiment and 15 fly x-positions in cm on a common time axis for one odor stimulation cycle. Custom code for data analysis can be provided upon request.

1. Open .csv-file, find time-point of valve opening signified by a change in the column representing the valve status.
2. Calculate the linear function of odor position of the form
   f(t) = s*t + i
   where t is time in the stimulation cycle, s is the wind speed (here 20 cm/sec) and the intercept i can be calculated using the time-point the odor enters the tubes at position 0 (valve opening plus delay).
3. Find the time point at which odor and fly x-position intersect for every fly and set this time-point to 0. Note: This way fly positions are aligned to each individual´s encounter with the odor.
4. Exclude flies sitting at the very edges of the region of interest.
5. Calculate speed from X-positions by dividing displacement along the x-axis by the time interval (100 msec) and repeat procedure for every stimulation cycle.
6. To obtain speed time-courses as shown in Figure 2E calculate mean speed time-course for every fly and odor and from those the mean time-course for a given odor.
7. To obtain net displacement as shown in Figure 3C calculate the net displacement within 4 sec after the odor pulse for every tracking event and afterwards the mean net displacement per fly and odor.

4. Cleaning Procedure

1. Clean Glass Tubes
   1. Remove flies and adapters from glass tubes and soak glass tubes in detergent.
   2. Rinse glass tubes under running distilled water and dry them using pressurized air.
   3. Heat glass tubes at 200 °C for 8 hr.
2. Clean Odor Delivery System
   1. Remove all odor vials and tubing from the central mixing chamber.
   2. Remove tubing adapters from the mixing chamber.
   3. Clean mixing chamber by rinsing it with laboratory cleaning solution and solvents (e.g. ethanol, acetone). Perform these steps under the laboratory hood.
   4. Dry mixing chamber using pressurized air and heat it at 200 °C for 8 hr.
3. Clean Odor Vials and Check Valves
   1. Remove steel plug (discard rubber gasket) and check valves from odor vials and soak all components in laboratory cleaning solution.
   2. Sonicate components in an ultrasound bath and rinse them with distilled water.
   3. Clean all components except check valves with ethanol and acetone. Perform these steps under the laboratory hood.
   4. Dry components using pressurized air and heat them at 200 °C for 8 hr.
   5. Clean check valves on the inside by flushing them with ethanol and acetone using a syringe (consider flow direction). Perform these steps under the laboratory hood wearing laboratory goggles. Because acetone attacks rubber parts, immediately dry check valves by flushing them with pressurized air.
   6. Remove residual odors by pulsing air through check valves for several days. Use an incubator at 60 °C and a 1 sec air on/1 sec air off regime for this cleaning step.

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Wyniki

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...

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Dyskusje

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...

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Materiały

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