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Encyclopedia of Experiments

Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila


Fruit flies have a complex olfactory system, can discriminate hundreds of odorants, and use their sense of smell to direct behavioral decisions. This video describes the four-way olfactometer assay, a method to study olfactory behavioral responses in flies. The featured protocol clip shows how to handle test flies and what are critical steps while performing the assay.


This protocol is an excerpt from Lin et al., Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer, J. Vis. Exp. (2016).

1. Behavioral Responses to Attractive and Repellent Odorants

  1. Switch on the temperature controller and set it to 25 °C.
  2. Connect the odorant chambers (control and test odorants) by inserting the tubing to the outlet of odorant chamber and to the push-to-connect fitting on the behavior box.
  3. Check the flow rate in each quadrant by using the airflow meter to make sure that the control and odorant airstreams are equal to 100 ml/min.
  4. Clean the PTFE fly arena and the glass plates with 70% ethanol 2-3 times and allow them to fully air dry (~3-4 min).
  5. Affix the glass plates to the arena with clamps.
  6. Transfer flies without CO2 anesthesia into the arena through the hole in one of the glass plates. After the transfer, place a circular mesh on the hole to prevent flies from escaping.
    NOTE: CO2 anesthesia has been shown to affect Drosophila behavior and should not be used within 24 hr of a behavioral experiment.
  7. Place the arena with flies into the light-tight chamber, connect the four control air streams by connecting the tubing attached to the push-to-connect fitting on the behavior box to the arena corners, close the door of the chamber and wait 10-15 min to let the flies acclimatize to the new environment. If possible, switch the lights off in the room where the experiments are performed, to avoid possible minimal light leak that may bias the experimental outcome.
  8. Run a 5-10 min control experiment, in which flies are exposed to 4 control air streams.
  9. Analyze the data immediately (see Data analysis section below) to make sure that the flies are distributed uniformly in the arena, and the Attraction Index is close to 0. This step is essential, as it verifies that there are no uncontrolled sources of preference or avoidance within the arena (e.g. light leaking from the outside, uneven temperature distribution, uneven arena, odor contamination, etc.). If the flies are distributed unequally or their locomotor activity is low, discard the flies, clean the arena again (Step 1.4) and use a new batch of flies to repeat the experiment.
  10. Connect the test odorant chamber to the setup by switching on the 3-way valves or re-plugging the connector tubes.
  11. Run test experiment for 5-10 min and analyze the data as discussed in section 2 below (Figure 1). Recordings longer than 20 min can result in data files that may be difficult to computationally process. If longer experimental recordings are desired, rapidly stop and re-start the tracking program. This results in a ~10 sec gap between experimental recordings.
  12. Discard flies.
  13. Clean arena and glass plates with 70% ethanol (Step 1.4) and replace connector tubes within the light-tight enclosure. To expedite experiments, a new clean arena can be used, and the dirty arena cleaned while performing experimental runs.
  14. Run another experiment with a new batch of flies, if required. If several experiments are run on the same day, take extreme care to ensure that no odorant is left in the system from a previous test run. This is normally not a problem with low concentrations of odorants or with CO2, but for highly concentrated stimuli up to a 24 hr gap between experimental runs may be needed. In addition, all tubing after the flow-tubes can be replaced if odorant contamination is suspected during control experiments. Always leave the dry air on between the experiments to continuously flush the system.

2. Data Analysis

NOTE: The suggested fly tracking acquisition software (detailed in Materials), tracks flies in real time during acquisition, and saves the time stamp and coordinates of all detected flies in *.dat format. We have developed a custom-made Matlab routine to convert the data into a Matlab format, and to analyze the data. Code examples are provided in Supplementary Materials, but details of implementation will depend on the software used for data acquisition.

  1. Load the raw data. Create a spatial mask that follows the contours of the arena and apply the mask to the raw data to remove all data points that fall outside of the arena as they represent noise (Figure 2A, Supplementary Code MaskSpatialFiltering.m, Score.m, DrawCircularMask.m).
  2. Remove all data points that move at a speed below 0.163 cm/s for longer than 3s, as this data is likely to be noise or generated by non-moving flies (Figure 2B, Supplementary Code TemporalFiltering.m).
  3. Visualize remaining data points by plotting them out all at once or as single trajectories (Figure 1, Supplementary Code SingleTrajectoryViewer.m).
    NOTE: The location of odor boundaries in the four-field likely depends on a number of factors, such as the characteristics of each odorant and the airflow rates being used. For example, highly volatile odorants will likely fill the odor quadrant more fully than less volatile odorants. Thus, it is likely that each odorant may exhibit slightly different odor boundaries. The use of a photoionization detector to measure odor boundaries can be problematic as it uses a vacuum to sample air from a particular spot, and so disrupts the odorant concentration at that spot. Nonetheless, odor boundaries can be quickly estimated based on fly behavioral data. For example, an odor boundary based on cumulated fly tracks in response to different odors can be clearly observed in Figure 1C and 1D.
  4. Calculate an attraction index to determine whether control experiments generate no preference response, and also to access the response to an odorant (or optogenetic) stimulus. To calculate an Attraction Index (AI), use the last 5 min of a control or test recording. To obtain a measure of attraction that falls between +1 (absolute attraction) and -1 (absolute repulsion), the following formula is used to calculate the AI:
    Equation 1
    where Ntest is the number of data points in the test quadrant, Ncontrol is the average number of data points in the three control quadrants. This measure is intuitive as no preference would be indicated by near-zero values. However, it does not correctly indicate the proportion of the total number of flies that are located in the odorant quadrant. To obtain this measure, a Percentage Index (PI) may be used:
    Equation 2
    where Ntest is the number of data points in the test quadrant, and Ntotal is the total number of data points in all four quadrants. This formula provides a measure that falls between 0 and 1, with 0.25 corresponding to no behavioral preference (Figure 1E and 2C, Supplementary Code AttractionIndex.m).
  5. Run 5-10 repeats of each experimental condition, using a new group of flies for each repeat. Compare the attraction indexes between conditions or against controls by using the Kolmogorov-Smirnov non-parametric test (Figure 1F, kstest2 function in Matlab).

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Representative Results

Figure 1
Figure 1: Example Data Generated using a Four-field Olfactory Assay. (A) Schematic of the four-field arena. (B) Neutral responses are observed when all four quadrants contain only dry air perfusion. (C) Attraction responses to a 6.25% dilution of apple cider vinegar perfused from the left upper quadrant. (D) Repulsion behaviors triggered by 10% ethyl propionate. In Figure 2B-2D, a single trajectory from the acquired data is plotted. A color gradient is used to signify the time course of recording, with blue and red colors being the start and end of recordings, respectively. (E) Comparison of the Attraction Index (AI) and the Percentage Index (PI). (F) Average AI's of 3- 6 experiments with no odor (Control), Apple Cider Vinegar (ACV) and 10% Ethyl Propionate (EP). Error bars indicate SEM. Statistical difference was evaluated by the Kolmogorov-Smirnov test. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Example Data Generated by the Data Analysis Steps. (A) Spatial filtering of the data, performed by MaskSpatialFiltering.m to remove data points that fall outside of the arena. Red circles show initial positions of the circles that are used to define the borders of the arena. Black circles are the final positions, acquired by fitting the circle outlines to the data (grey shaded area inside the four-field). Red dots and black arrowheads indicate data points that will be removed from the dataset after this filtering step. (B) Temporal filtering of data, performed by TemporalFiltering.m. This filtering step removes data points that move very slowly or not at all, as they are likely to be generated by non-moving flies or by dirt/reflections from the arena. A red dot surrounded by a dashed red box indicates positions of ~6,000 data points with identical coordinates that will be removed by this filtering step. (C) Attraction Index (AI) and Percentage Index (PI), calculated in 10-sec bins over the last 5 min of an experiment by AttractionIndex.m. Temporal profiles of these indexes contain information about the dynamics of behavioral responses and may be used for detailed analysis of behaviors. Please click here to view a larger version of this figure.

Supplementary Code File. Please click here to view this file (Right click to download).

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Name Company Catalog Number Comments
Air delivery system (Quantity needed)
Tubing and connectors
Thermoplastic NPT(F) Manifolds Cole-Parmer, IL, USA R-31522-31 1
Hex reducing nipple (1/4MNPT->1/8MNPT) McMaster-Carr, IL, USA 5232T314 1
Tubing (ID:1/8) McMaster-Carr, IL, USA 5108K43 50 Ft
Tubing (ID:1/16) McMaster-Carr, IL, USA 52355K41 100 Ft
Barbed tube fittings McMaster-Carr, IL, USA 5117K71 1 pack
Push-to-connect tube fittings McMaster-Carr, IL, USA 5779K102 4
Barbed Tube Fittings (1/4MNPT->1/8BF) McMaster-Carr, IL, USA 5463K439 1 pack (10)
Barbed Tube Fittings (1/8MNPT->1/8BF) McMaster-Carr, IL, USA 5463K438 2 pack (10)
Barbed Tube Fittings (1/8MNPT->1/16BF) McMaster-Carr, IL, USA 5463K4 2 pack (10)
Barbed Tube Fittings (1/4MNPT->1/4BF) McMaster-Carr, IL, USA 5670K84 1
Hex head plug McMaster-Carr, IL, USA 48335K152 1
Air pressure regulator, air filter and flowmeters (Quantity needed)
Labatory gas drying unit W A HAMMOND DRIERITE CO LTD, OH, USA Model: L68-NP-303; stock #26840 1
Multitube frames for 150 mm flowtubes Cole-Parmer, IL, USA R03215-30 1
Multitube frames for 150 mm flowtubes Cole-Parmer, IL, USA R03215-76 1
150 mm flowtubes Cole-Parmer, IL, USA R-03217-15 9
Valve Cartridge Cole-Parmer, IL, USA R-03218-72 9
Precision Air regulator McMaster-Carr, IL, USA 6162K13 1
Soleniod valves Automate Scientific, Berkeley, CA 02-10i 4
Solenoid valve controller ValveLink 8.2, Automate Scientific, Berkeley, CA 18-Jan 1
Electronic flow meter Honeywell AWM3100V 1
DAQ (NI USB-6009, National Instruments) and a National Instruments NI USB-6009 1
Power supply Extech Instruments 382200 1
Odor chambers
Polypropylene Wide Mouth jar 2 oz; 60 ml Nalgene 562118-0002 At least 5 are required per experiment, but a separate chamber is required for each dillution of each odorant. Available at Container Store, part #635114)
Glass odor chamber, 0.25 oz Sunburst Bottle LB4B At least 5 are required per experiment
"In" valve for odor chamber Smart Products, Inc., CA, USA 214224PB-0011S000-4074 1 of these parts is used per odor chamber but they need to be replaced frequently
"Out" valve for odor chamber Smart Products, Inc., CA, USA 224214PB-0011S000-4074 1 of these parts is used per odor chamber but they need to be replaced frequently
O ring RT Dygert International, MN, USA AS568-029 Buna-N O-R 1 pack (100)
Fly arena, camera and behavior boxes (Quantity needed)
Behavior and camera box material Interstate plastics, CA, USA ABS black extruded (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php) 1803 sq inch
Teflon for fly arena and odor chamber inserts, 3/8" thick, 12" x 12" McMaster-Carr, IL, USA 8545K27 1
Glass plates, 1/8" Thick, 9" x 9" McMaster-Carr, IL, USA 8476K191 2
Dual action thermoelectric controller WAtronix Inc, CA, USA DA12V-K-0 1
IR LED array Advanced Illumination, Rochester, VT, USA AL4554-88024, PS24-TL 2 LED arrays and one power supply
Air conditioner Unit Melcor Store MAA280T-12 1
Imaging system (Quantity needed)
Cosmicar/Pentax C21211TH (12.5 mm F/1.4) C-mount Lens B AND H PHOTO AND ELECTRONICS CORP, NY, USA PEC21211 KP 1
B+W 40,5 093 IR filter B AND H PHOTO AND ELECTRONICS CORP, NY, USA 65-072442 1
TiFFEN 40.5 mm Circular polarizer Amazon 1
IR Videocamera Industrial Vision Source, FL, USA Sony XC-EI50 (SY-XC-E150) 1
USB video converter The Imagingsource, NC, USA DFG/USB2-It 1
iFlySpy2 (fly tracking software) Julian Brown, Stanford, Calfornia: julianrbrown@gmail.com iFlySpy2 1
IC Capture software The Imagingsource, NC, USA (http://www.theimagingsource.com/)
Miscellaneous (Quantity needed)
Dremel rotary tool Dremel, Racine, WI, USA Dremel 8000-03 1
Diamond-coated drill bits for glass cutting Available from various suppliers; MSC industrial Supply Co, Melville, NY 90606328 1


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