Name: olfactory behaviors assayed by computer tracking of drosophila in a four-quadrant olfactometer
Uploaded: 2016-08-20T15:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer at JoVE.com

We thank Terry Shelley for manufacturing the fly arena and the light-tight enclosure, Liz Marr for help with fly stock maintenance, and Xiaojing Gao and Junjie Luo for help with the Matlab code used for data analysis. We thank Johan Lundstr&#246;m at the Monell Chemical Senses Center for demonstrating his odor delivery setup. This work was supported by grants from the Whitehall Foundation (CJP) and NIH NIDCD (R01DC013070, CJP).

1. Setup Assembly
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Install the driver for the video converter on the computer according to the manufacturer&#39;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.</li>
	<li>Connect the air conditioner unit (through &#34;output&#34; on the back of temperature controller) and the temperature probe (through &#34;thermocouple&#34; 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 &#176;C and 30 &#176;C. Higher or lower ambient temperatures could be useful for thermogenetic (dTrpA1, TrpM8 or shibirets) experiments to manipulate neuronal activity or inhibit synaptic transmission. For most experiments, the temperature is maintained at 25 &#176;C.</li>
	<li>Assemble the odor delivery system in the following steps (please see Figure 1B for detailed schematics and connection fittings):
	<ol>
		<li>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.</li>
		<li>Assemble the flow control system consisting of multiple channels regulated by high-resolution flowmeter tubes.</li>
		<li>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.</li>
		<li>Install the solenoid valve controller according to the manufacturer&#39;s manual.</li>
	</ol>
	</li>
	<li>Install the electronic air flow meter by connecting it to a data acquisition device (DAQ) and a power supply according to the manufacturer&#39;s manual. Install the DAQ interface software to verify equal flow rates in each quadrant of the arena before every experiment.</li>
</ol>
2. Olfactory Stimuli Preparation
<ol>
	<li>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.</li>
	<li>Use four odorant chambers for solvent controls, and one chamber for a test odorant. Fill the glass containers with 1,000 &#181;l 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.</li>
</ol>
3. Fly Preparation
<ol>
	<li>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 &#176;C or room temperature.</li>
	<li>For each experiment, collect newly eclosed (&#60;1 day old) 25 male and 25 female flies under brief CO2 anesthesia.</li>
	<li>Keep flies in a vial with standard fly medium for 2-4 days.</li>
	<li>40-42 hr before the experiment, transfer the flies without CO2 anesthesia&#160;to a vial with ~10 ml&#160;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.</li>
</ol>
4. Behavioral Responses to Attractive and Repellent Odorants
<ol>
	<li>Switch on the temperature controller and set it to 25 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Affix the glass plates to the arena with clamps.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Run a 5-10 min control experiment, in which flies are exposed to 4 control air streams.</li>
	<li>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.</li>
	<li>Connect the test odorant chamber to the setup by switching on the 3-way valves or re-plugging the connector tubes.</li>
	<li>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&#160;sec gap between experimental recordings.</li>
	<li>Discard flies.</li>
	<li>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.</li>
	<li>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</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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: 
	<img alt="Equation 1" src="/files/ftp_upload/54346/54346eq1.jpg" /> 
	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: 
	<img alt="Equation 2" src="/files/ftp_upload/54346/54346eq2.jpg" /> 
	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 3E and 4C, Supplementary Code AttractionIndex.m).</li>
	<li>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).</li>
</ol>

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The authors declare that they have no competing financial interests.

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...

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 neurons10-13. 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&#39;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) olfactometer14-18. 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 olfactometer14,20-23. 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&#39; 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.

<table><tbody><tr><td>&lt;strong&gt;Air delivery system&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Tubing and connectors&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermoplastic NPT(F) Manifolds</td><td>Cole-Parmer, IL, USA</td><td>R-31522-31</td><td>1</td></tr><tr><td>Hex reducing&amp;nbsp; nipple (1/4MNPT-&gt;1/8MNPT)</td><td>McMaster-Carr, IL, USA</td><td>5232T314</td><td>1</td></tr><tr><td>Tubing (ID:1/8)</td><td>McMaster-Carr, IL, USA</td><td>5108K43</td><td>50 Ft</td></tr><tr><td>Tubing (ID:1/16)</td><td>McMaster-Carr, IL, USA</td><td>52355K41</td><td>100 Ft</td></tr><tr><td>Barbed tube fittings</td><td>McMaster-Carr, IL, USA</td><td>5117K71</td><td>1 pack</td></tr><tr><td>Push-to-connect tube fittings</td><td>McMaster-Carr, IL, USA</td><td>5779K102</td><td>4</td></tr><tr><td>Barbed Tube Fittings (1/4MNPT-&gt;1/8BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K439</td><td>1 pack (10)</td></tr><tr><td>Barbed Tube Fittings (1/8MNPT-&gt;1/8BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K438</td><td>2 pack (10)&amp;nbsp;</td></tr><tr><td>Barbed Tube Fittings (1/8MNPT-&gt;1/16BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K4</td><td>2 pack (10)&amp;nbsp;</td></tr><tr><td>Barbed Tube Fittings (1/4MNPT-&gt;1/4BF)</td><td>McMaster-Carr, IL, USA</td><td>&amp;nbsp; 5670K84</td><td>1</td></tr><tr><td>Hex head plug</td><td>McMaster-Carr, IL, USA</td><td>48335K152</td><td>1</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Air pressure regulator, air filter and flowmeters&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Labatory gas drying unit</td><td>W A HAMMOND DRIERITE CO LTD, OH, USA</td><td>Model: L68-NP-303; stock #26840</td><td>1</td></tr><tr><td>Multitube frames for 150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R03215-30</td><td>1</td></tr><tr><td>Multitube frames for 150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R03215-76</td><td>1</td></tr><tr><td>150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R-03217-15</td><td>9</td></tr><tr><td>Valve Cartridge</td><td>Cole-Parmer, IL, USA</td><td>R-03218-72</td><td>9</td></tr><tr><td>Precision Air regulator</td><td>McMaster-Carr, IL, USA</td><td>6162K13</td><td>1</td></tr><tr><td>Soleniod valves</td><td>Automate Scientific, Berkeley, CA</td><td>02-10i</td><td>4</td></tr><tr><td>Solenoid valve controller</td><td>ValveLink 8.2, Automate Scientific, Berkeley, CA</td><td>01-18</td><td>1</td></tr><tr><td>Electronic flow meter</td><td>Honeywell</td><td>AWM3100V</td><td>1</td></tr><tr><td>DAQ (NI USB-6009, National Instruments) and a&amp;nbsp;</td><td>National Instruments</td><td>NI USB-6009</td><td>1</td></tr><tr><td>Power supply</td><td>Extech Instruments</td><td>382200</td><td>1</td></tr><tr><td>Odor chambers</td><td></td><td></td><td></td></tr><tr><td>Polypropylene Wide Mouth jar 2 oz; 60 ml</td><td>Nalgene</td><td>562118-0002</td><td>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)</td></tr><tr><td>Glass odor chamber, 0.25 oz</td><td>Sunburst Bottle</td><td>LB4B</td><td>At least 5 are required per experiment&amp;nbsp;</td></tr><tr><td>&quot;In&quot; valve for odor chamber</td><td>Smart Products, Inc., CA, USA</td><td>214224PB-0011S000-4074</td><td>1 of these parts is used per odor chamber but they need to be replaced frequently</td></tr><tr><td>&quot;Out&quot; valve for odor chamber</td><td>Smart Products, Inc., CA, USA</td><td>224214PB-0011S000-4074</td><td>1 of these parts is used per odor chamber but they need to be replaced frequently</td></tr><tr><td>O ring</td><td>RT Dygert International, MN, USA</td><td>AS568-029 Buna-N O-R</td><td>1 pack (100)</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Fly arena, camera and behavior boxes&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Behavior and camera box material</td><td>Interstate plastics, CA, USA</td><td>ABS black extruded (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php)</td><td>1803 sq inch</td></tr><tr><td>Teflon for fly arena and odor chamber inserts, 3/8&quot; thick, 12&quot; x 12&quot;</td><td>McMaster-Carr, IL, USA</td><td>8545K27&amp;nbsp;</td><td>1</td></tr><tr><td>Glass plates, 1/8&quot; Thick, 9&quot; x 9&quot;</td><td>McMaster-Carr, IL, USA</td><td>8476K191&amp;nbsp;</td><td>2</td></tr><tr><td>Dual action thermoelectric controller</td><td>WAtronix Inc, CA, USA</td><td>DA12V-K-0</td><td>1</td></tr><tr><td>IR LED array</td><td>Advanced Illumination, Rochester, VT, USA</td><td>AL4554-88024, PS24-TL</td><td>2 LED arrays and one power supply</td></tr><tr><td>Air conditioner Unit</td><td>Melcor Store</td><td>&amp;nbsp;MAA280T-12</td><td>1</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Imaging system&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Cosmicar/Pentax C21211TH (12.5 mm F/1.4) C-mount Lens</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>PEC21211 KP</td><td>1</td></tr><tr><td>CCXC-12P05N Interconnect Cable</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>SOCCXC12P05N</td><td>1</td></tr><tr><td>DC-700 Camera Adapter</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>SODC700</td><td>1</td></tr><tr><td>B+W 40,5 093 IR filter</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>65-072442</td><td>1</td></tr><tr><td>TiFFEN 40.5 mm Circular polarizer</td><td>Amazon</td><td></td><td>1</td></tr><tr><td>IR Videocamera</td><td>Industrial Vision Source, FL, USA</td><td>Sony XC-EI50 (SY-XC-E150)</td><td>1</td></tr><tr><td>USB video converter</td><td>The Imagingsource, NC, USA</td><td>DFG/USB2-It</td><td>1</td></tr><tr><td>iFlySpy2 (fly tracking software)</td><td>Julian Brown, Stanford, Calfornia: julianrbrown@gmail.com</td><td>iFlySpy2</td><td>1</td></tr><tr><td>IC Capture 2.2 software</td><td>The Imagingsource, NC, USA (http://www.theimagingsource.com/en_US/products/software/iccapture/)</td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Miscellaneous&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Dremel rotary tool</td><td>Dremel, Racine, WI, USA</td><td>Dremel 8000-03&amp;nbsp;</td><td>1</td></tr><tr><td>Diamond-coated drill bits for glass cutting</td><td>Available from various suppliers; MSC industrial Supply Co, Melville, NY</td><td>90606328</td><td>1</td></tr></tbody></table>

olfactory behaviors assayed by computer tracking of drosophila in a four-quadrant olfactometer

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 behaviors4-11. 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.

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...

Watch this Scientific Journal Video about Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer at JoVE.com

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

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.

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

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

olfactory-behaviors-assayed-computer-tracking-drosophila-four

Research

JoVE Journal

Neuroscience

15.6K Views.  Johns Hopkins University School of Medicine. 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.

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

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

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

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&#180;s dynamic behavioral reaction.

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

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

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

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

Source: Lin, C.C., et al. <a href="https://www.jove.com/video/54346/" target="_blank">Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer</a>. J. Vis. Exp. (2016).
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.

Czterokierunkowy test olfaktometryczny: metoda oceny reakcji zapachowych u Drosophila

Source: Lin, C.C., et al. Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer. J. Vis. Exp. (2016).
Fruit flies ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

Drosophila Y-maze Assay: A Method to Assess Olfactory Responses in Flies

Source: Simonnet, M. M., et al. <a href="https://www.jove.com/video/51241/" target="_blank">Testing Drosophila Olfaction with a Y-maze Assay</a>. J. Vis. Exp. (2014).
This video describes a method to test olfactory exploratory behavior in the fruit fly, called the Y-maze. The featured protocol clip demonstrates how to set up and conduct the assay.

Drosophila Test labiryntu Y: metoda oceny reakcji węchowych u much

Source: Simonnet, M. M., et al. Testing Drosophila Olfaction with a Y-maze Assay. J. Vis. Exp. (2014).
This video describes a method to test olfactory ...

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

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