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 border="0" cellpadding="0" cellspacing="0" width="1935">
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			<td height="21" style="height:21px;width:335px;">Air delivery system&#160;</td>
			<td style="width:397px;"></td>
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			<td style="width:960px;">(Quantity needed)</td>
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			<td height="21" style="height:21px;">Thermoplastic NPT(F) Manifolds</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R-31522-31</td>
			<td>1</td>
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			<td height="21" style="height:21px;">Hex reducing&#160; nipple (1/4MNPT-&#62;1/8MNPT)</td>
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			<td>5232T314</td>
			<td>1</td>
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			<td>5108K43</td>
			<td>50Ft</td>
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			<td>52355K41</td>
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			<td>1 pack (10)</td>
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			<td>5463K438</td>
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			<td>Model: L68-NP-303; stock #26840</td>
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			<td height="21" style="height:21px;">Multitube frames for 150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R03215-76</td>
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			<td height="21" style="height:21px;">150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
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			<td>9</td>
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			<td>Cole-Parmer, IL, USA</td>
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			<td>9</td>
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			<td height="21" style="height:21px;width:335px;">Precision Air regulator</td>
			<td>McMaster-Carr, IL, USA</td>
			<td style="width:244px;">6162K13</td>
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			<td>Automate Scientific, Berkeley, CA</td>
			<td style="width:244px;">02-10i</td>
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			<td style="width:244px;">382200</td>
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			<td height="21" style="height:21px;width:335px;">Odor chambers</td>
			<td style="width:397px;"></td>
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			<td height="21" style="height:21px;width:335px;">Polypropylene Wide Mouth jar 2oz; 60ml</td>
			<td style="width:397px;">Nalgene</td>
			<td style="width:244px;">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>
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			<td height="21" style="height:21px;width:335px;">Glass odor chamber, 0.25 oz</td>
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			<td style="width:244px;">LB4B</td>
			<td>At least 5 are required per experiment&#160;</td>
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			<td height="21" style="height:21px;width:335px;">&#34;In&#34; valve for odor chamber</td>
			<td style="width:397px;">Smart Products, Inc., CA, USA</td>
			<td style="width:244px;">214224PB-0011S000-4074</td>
			<td>1 of these parts is used per odor chamber but they need to be replaced frequently</td>
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			<td style="width:397px;">Smart Products, Inc., CA, USA</td>
			<td style="width:244px;">224214PB-0011S000-4074</td>
			<td>1 of these parts is used per odor chamber but they need to be replaced frequently</td>
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			<td style="width:397px;">RT Dygert International, MN, USA</td>
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			<td>1 pack (100)</td>
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			<td height="21" style="height:21px;width:335px;">Fly arena, camera and behavior boxes</td>
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			<td style="width:397px;">Interstate plastics, CA, USA</td>
			<td style="width:244px;">ABS black extruded (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php)</td>
			<td>1803 sq inch</td>
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			<td height="42" style="height:42px;width:335px;">Teflon for fly arena and odor chamber inserts, 3/8&#34; thick, 12&#34;x12&#34;</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>8545K27&#160;</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">Glass plates, 1/8&#34; Thick, 9&#34;x 9&#34;</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>8476K191&#160;</td>
			<td>2</td>
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			<td height="21" style="height:21px;width:335px;">Dual action thermoelectric controller</td>
			<td>WAtronix Inc, CA, USA</td>
			<td style="width:244px;">DA12V-K-0</td>
			<td>1</td>
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			<td>Advanced Illumination, Rochester, VT, USA</td>
			<td style="width:244px;">AL4554-88024, PS24-TL</td>
			<td>2 LED arrays and one power supply</td>
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			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td style="width:244px;">PEC21211 KP</td>
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			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
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			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td style="width:244px;">SODC700</td>
			<td>1</td>
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			<td height="28" style="height:28px;">B+W 40,5 093 IR filter</td>
			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td>65-072442</td>
			<td>1</td>
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			<td style="width:397px;">Industrial Vision Source, FL, USA</td>
			<td>Sony XC-EI50 (SY-XC-E150)</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">USB video converter</td>
			<td>The Imagingsource, NC, USA</td>
			<td style="width:244px;">DFG/USB2-It</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">iFlySpy2 (fly tracking software)</td>
			<td style="width:397px;">Julian Brown, Stanford, Calfornia: julianrbrown@gmail.com</td>
			<td style="width:244px;">iFlySpy2</td>
			<td>1</td>
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			<td height="63" style="height:63px;width:335px;">IC Capture 2.2 software</td>
			<td style="width:397px;">The Imagingsource, NC, USA (http://www.theimagingsource.com/en_US/products/software/iccapture/)</td>
			<td style="width:244px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:335px;">Miscellaneous</td>
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			<td>(Quantity needed)</td>
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			<td height="21" style="height:21px;width:335px;">Dremel rotary tool</td>
			<td style="width:397px;">Dremel, Racine, WI, USA</td>
			<td style="width:244px;">Dremel 8000-03&#160;</td>
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			<td height="42" style="height:42px;width:335px;">Diamond-coated drill bits for glass cutting</td>
			<td style="width:397px;">Available from various suppliers; MSC industrial Supply Co, Melville, NY</td>
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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 ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

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

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

Assessment of Social Interaction Behaviors

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila Adult Olfactory Shock Learning

A Computer-assisted Multi-electrode Patch-clamp System

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters