Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Summary

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Abstract

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)^1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors^4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Introduction

The ability to adapt and respond to the external environment is critical for the survival of all animals. An animal needs to avoid dangers, seek out food and find mates, and learn from previous experiences. Sensory systems function to receive a variety of stimuli, such as visual, chemical and mechanosensory, and send these signals to the central nervous system to be interpreted and decoded. The brain then directs appropriate motor behaviors based on the perceived environment, such as foraging for food or escaping from a predator. Understanding how sensory systems detect the external world, and how the brain decodes and directs decisions, is a major challenge in neurobiology.

Drosophila melanogaster is a powerful model system for investigating how neural circuits guide behaviors. Besides being simple and inexpensive to maintain, Drosophila exhibit many diverse and complex stereotyped behaviors, yet do so with a compact nervous system of about 100,000 neurons. Powerful genetic techniques exist for manipulating the Drosophila genome, and thousands of transgenic lines have been generated that selectively and reproducibly label the same subsets of neurons^10-13. These transgenic lines can be used to selectively manipulate the activity of the labeled neurons (activate or inhibit), and these manipulations can be used to investigate how neural functions guide behaviors.

Multiple behavioral assays have been developed for studying various Drosophila behaviors. Drosophila, like many animals, use their sense of smell for guiding many behavioral choices, such as finding food, finding mates, and avoiding dangers. Olfaction is therefore a good sensory system for investigating how external stimuli are detected and interpreted by an animal's nervous system to guide appropriate choices. As such, a number of assays have been developed for investigating larval and adult olfactory behaviors. Traditionally, olfactory behaviors in Drosophila were assayed by a two-choice T-maze paradigm, which can be used for assaying innate and learned olfactory behaviors³. In this assay, about 50 flies are given a choice between two tubes: one tube contains the odor in question and the other contains a control odorant (usually the odor solvent). The flies are given a set period of time to make a choice, and then the number of flies that are in the different chambers are counted. Although the T-maze is a simple assay for many experiments, there are several limitations. For example, olfactory behaviors are measured at only one time point, and different choices made before this time point are discarded. Similarly, the individual behaviors of the flies within the population are neglected. In addition, the T-maze requires manual counting of flies, which might introduce errors. Finally, since there are only two measured choices, this reduces the statistical power often required to detect subtle behavioral changes. An alternative to a two-choice T-maze is a four-quadrant (four-field) olfactometer^14-18. In this assay, animals explore an arena in which each of the four corners of the arena is filled with a potential source of odorized air. The arena has a puckered star shape to maximize the formation of four experimentally defined odor quadrants. If odor is supplied in one of the corners then it is contained only in that one quadrant. The behaviors of the animals can be tracked as they enter and leave the odor quadrant, and easily compared to their behavior in the three control quadrants. The four-quadrant olfactometer assay thus records spatial and temporal behavioral response to the odor stimuli over a large experimental arena.

The four-quadrant olfactometer was first developed by Pettersson et al.¹⁵ and Vet et al.¹⁷ to investigate the olfactory behavioral responses of individual parasitic Hymenoptera. Faucher et al.¹⁸ and Semmelhack and Wang¹⁶ adapted the setup to monitor the olfactory responses of individual Drosophila. The four-quadrant olfactometer is equally sensitive to attractive and repulsive responses, allowing for a wide range of test odorants and conditions. Custom-written fly tracking software, developed by Alex Katsov¹⁹ and currently maintained by Julian Brown (detailed in Materials), introduced additional advantages to more recent implementations of the four-quadrant olfactometer^14,20-23. It is now possible to assay up to 100 flies simultaneously at high spatial (27.5 pixels/cm) and temporal (30 frames per sec) resolution, which allows extracting various parameters, such as position, speed and acceleration of flies at any time point. This enables investigations into the dynamics of the flies' behavioral responses to odors²⁰. It should be noted, however, that the identity of individual flies within the population during the entire tracking period is not maintained. Instead, each fly track is recorded for as long as two fly tracks do not intersect. At which point, new tracks are assigned after the flies diverge. By incorporating other video-capturing software (detailed in Materials Table), the same configuration allows flexible tracking periods and could be used to track flies for up to 24 hr by taking images at a lower frame rate. This option was used to study egg-laying behaviors of flies and compare their body positions with ovipositional preferences¹⁴. The four-field olfactometer may also be used to study responses to multimodal (e.g. olfactory and visual) stimuli, or to combine optogenetic⁹ or thermogenetic²¹ stimulation with presentations of sensory stimuli. Furthermore, the high temporal resolution allows the extraction of trajectories for each individual fly in the ensemble data set. Therefore, the method allows investigation into olfactory-guided population behaviors and also individual social interactions. The data generated by this assay are robust and highly reproducible, allowing for the use of the four-field olfactometer for behavioral screens.

We describe here the setup assembly for a four-quadrant olfactometer. We further demonstrate its use in assaying olfactory attraction in response to apple cider vinegar and repulsion in response to highly concentrated ethyl propionate. Finally, we describe and provide example code for the analysis of the recorded fly tracking data.

Protocol

1. Setup Assembly

1. Manufacture the star-shaped arena (19.5 cm by 19.5 cm by 0.7 cm) out of polytetrafluoroethylene (PTFE) according to the provided drawing (Supplementary Materials, SupplementalSketch_StarShapedArena.pdf). The arena may be manufactured by a commercial or a custom facility.
2. Acquire two glass plates (20.25 cm by 20.25 cm with thickness of 2 mm), and drill a hole (~0.7 cm in diameter) precisely in the center of one of the glass plates using a diamond-coated drill bit.
3. Manufacture a light-tight behavior box for the behavioral arena. Also manufacture a light-tight camera box for the infra-red CCD video camera according to the provided drawings (Supplementary Materials, SupplementalSketch_LightTightBox.pdf). The boxes may be manufactured by a commercial or a custom facility.
4. Mount the air conditioner unit on the posterior wall and the LED arrays on the side walls of the behavior box. Place the temperature probe in the behavior box through a side hole for real time temperature feedback and adjustment (see Figures 1 and 2 for details).
5. Attach the IR filter and circular polarizer to the camera, and mount the assembly into the camera box. The behavior box and camera box are separated by a glass window for better temperature control of the behavior box (see Figures 1 and 2 for details).
6. Connect the Infrared CCD camera to a camera adaptor. Connect the camera adaptor to a USB converter. Connect the USB converter to a USB port on the computer for data acquisition.
7. Install the driver for the video converter on the computer according to the manufacturer's instructions. Optionally, install image processing software provided by the manufacturer of the USB video converter to access a wider range of camera settings and acquisition parameters.
8. Connect the air conditioner unit (through "output" on the back of temperature controller) and the temperature probe (through "thermocouple" on the back of temperature controller) to the temperature controller. Place the probe into the behavior box.
   Note: The temperature control system in our layout is capable of maintaining the box temperature between 18 °C and 30 °C. Higher or lower ambient temperatures could be useful for thermogenetic (dTrpA1, TrpM8 or shibire^ts) experiments to manipulate neuronal activity or inhibit synaptic transmission. For most experiments, the temperature is maintained at 25 °C.
9. Assemble the odor delivery system in the following steps (please see Figure 1B for detailed schematics and connection fittings):
   1. Use the air pressure regulator to control the air input from the central air system. Connect a carbon air filter (filled with charcoal) to the pressure regulator to purify the air from the central air system.
   2. Assemble the flow control system consisting of multiple channels regulated by high-resolution flowmeter tubes.
   3. Connect the output from the carbon air filter to the flowmeter tubes via a manifold as shown in Figure 1B and 2F. Direct the output of the flowmeter tubes through electronically controlled 3-way solenoid valves to regulate if clean air leaving the flowmeters is expelled into the room or entered into custom-made odor chambers²⁴.
   4. Install the solenoid valve controller according to the manufacturer's manual.
10. Install the electronic air flow meter by connecting it to a data acquisition device (DAQ) and a power supply according to the manufacturer's manual. Install the DAQ interface software to verify equal flow rates in each quadrant of the arena before every experiment.

2. Olfactory Stimuli Preparation

1. Prepare 5 odorant chambers²⁴ that consist of a plastic outer container, glass inner container, a custom-made PTFE lid insert, original container lid with central part removed, and two one-way valves.
   Note: An O-ring around the PTFE lid could be used to prevent air leak from the odor chamber during odor perfusion. See Figure 1 for schematic and Figure 2 for photos of the chambers.
2. Use four odorant chambers for solvent controls, and one chamber for a test odorant. Fill the glass containers with 1,000 µl of solvent or odorant dilution (test odorants + appropriate solvents, mix thoroughly before experiments), place the glass container inside the corresponding plastic chamber (do not spill out the liquid into the plastic chamber) and tighten the lid. Make sure to always use a clean chamber for the test odorant and solvent controls.
   Note: Olfactory attraction may be triggered by 1/16 dilution of apple cider vinegar (5% acidity) in water. In contrast, olfactory repulsion behavior may be studied by using a 10% dilution of ethyl propionate in mineral oil. Control stimuli in these cases are odor chambers with pure mineral oil.

3. Fly Preparation

1. Rear flies on standard cornmeal medium. Place 30 male and 30 female parental flies in a standard bottle, and let them lay eggs for 5 days at 25 °C or room temperature.
2. For each experiment, collect newly eclosed (<1 day old) 25 male and 25 female flies under brief CO₂ anesthesia.
3. Keep flies in a vial with standard fly medium for 2-4 days.
4. 40-42 hr before the experiment, transfer the flies without CO₂ anesthesia to a vial with ~10 ml 1% agarose gel. This will keep the flies humidified without food, which helps to increase their locomotor activity.
   Note: More than 90% of the flies should survive the starvation. Some genotypes are less healthy and may not make it through a 40 hr starvation. In those cases, shorter periods such as 24-28 hr are acceptable but should be kept the same for all experimental conditions and repeats.

4. 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 behavior²⁵ 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 4.4) 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 5 below (also see Reference 14 and Figure 3). 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 4.4), 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

5. 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 4A, 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 4B, Supplementary Code TemporalFiltering.m).
3. Visualize remaining data points by plotting them out all at once or as single trajectories (Figure 3, 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 Figures 3C and 3D.
4. Calculate an attraction index to determine whether control experiments generate no preference response, and also to access the response to an odorant (or optogenetic⁹) 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:
   
   where N_test is the number of data points in the test quadrant, N_control 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:
   
   where N_test is the number of data points in the test quadrant, and N_total 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 3E and 4C, 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 3F, kstest2 function in Matlab).

Results

The four-quadrant olfactometer assay records and analyzes the walking activities of many flies over a large behavioral space. Odorants can be introduced into the air-streams that enter one, two, three, or all four quadrants. In the absence of odors, the flies will freely move between all four quadrants. This behavior is crucial to observe as it indicates that un-intentional biases have not been introduced into the assay. These biases can include light, temperature fluctuations, difference...

Discussion

The four-field olfactometer described here is a versatile behavioral system for studying the olfactory responses of large populations of wild-type and mutant Drosophila flies. Each experiment takes ~1 hr (including setup, experimental runs, and cleaning), and 4-6 experiments can be routinely performed each day. A typical assay using 40-50 flies for 5 minutes generates approximately 450,000 tracked data points for analysis. The described configuration may also be used, with minor modifications, to monitor movemen...

Materials

List of materials used in this article

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	01-18	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
CCXC-12P05N Interconnect Cable	B AND H PHOTO AND ELECTRONICS CORP, NY, USA	SOCCXC12P05N	1
DC-700 Camera Adapter	B AND H PHOTO AND ELECTRONICS CORP, NY, USA	SODC700	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 2.2 software	The Imagingsource, NC, USA (http://www.theimagingsource.com/en_US/products/software/iccapture/)
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