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>Manufact...

<ol>
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R., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+sparse+olfactory+inputs+are+sufficient+to+mediate+innate+aversion+in+Drosophila.">Extremely sparse olfactory inputs are sufficient to mediate innate aversion in Drosophila.</a> PLoS One. 10 (4), e0125986 (2015).</li><li>Ronderos, D. S., Lin, C. C., Potter, C. J., Smith, D. 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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">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Air delivery system&#160;</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td style="width:960px;">(Quantity needed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tubing and connectors</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hex reducing&#160; nipple (1/4MNPT-&#62;1/8MNPT)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5232T314</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing (ID:1/8)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5108K43</td>
			<td>50Ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing (ID:1/16)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>52355K41</td>
			<td>100Ft</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Barbed tube fittings</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5117K71</td>
			<td>1pack</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Push-to-connect tube fittings</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5779K102</td>
			<td>4</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Barbed Tube Fittings (1/4MNPT-&#62;1/8BF)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5463K439</td>
			<td>1 pack (10)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Barbed Tube Fittings (1/8MNPT-&#62;1/8BF)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5463K438</td>
			<td>2 pack (10)&#160;</td>
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			<td height="21" style="height:21px;">Barbed Tube Fittings (1/8MNPT-&#62;1/16BF)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5463K4</td>
			<td>2 pack (10)&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Barbed Tube Fittings (1/4MNPT-&#62;1/4BF)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>&#160; 5670K84</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hex head plug</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>48335K152</td>
			<td>1</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Air pressure regulator, air filter and flowmeters</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td>(Quantity needed)</td>
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			<td height="21" style="height:21px;">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>
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			<td height="21" style="height:21px;">Multitube frames for 150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R03215-30</td>
			<td>1</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Multitube frames for 150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R03215-76</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R-03217-15</td>
			<td>9</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Valve Cartridge</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R-03218-72</td>
			<td>9</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Precision Air regulator</td>
			<td>McMaster-Carr, IL, USA</td>
			<td style="width:244px;">6162K13</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">Soleniod valves</td>
			<td>Automate Scientific, Berkeley, CA</td>
			<td style="width:244px;">02-10i</td>
			<td>4</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Solenoid valve controller</td>
			<td>ValveLink 8.2, Automate Scientific, Berkeley, CA</td>
			<td style="width:244px;">01-18</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">Electronic flow meter</td>
			<td>Honeywell</td>
			<td style="width:244px;">AWM3100V</td>
			<td>1</td>
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		<tr height="21">
			<td height="21" style="height:21px;">DAQ (NI USB-6009, National Instruments) and a&#160;</td>
			<td>National Instruments</td>
			<td style="width:244px;">NI USB-6009</td>
			<td>1</td>
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			<td height="21" style="height:21px;width:335px;">Power supply</td>
			<td>Extech Instruments</td>
			<td style="width:244px;">382200</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Odor chambers</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass odor chamber, 0.25 oz</td>
			<td style="width:397px;">Sunburst Bottle</td>
			<td style="width:244px;">LB4B</td>
			<td>At least 5 are required per experiment&#160;</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">&#34;Out&#34; valve for odor chamber</td>
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">O ring</td>
			<td style="width:397px;">RT Dygert International, MN, USA</td>
			<td style="width:244px;">AS568-029 Buna-N O-R</td>
			<td>1 pack (100)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fly arena, camera and behavior boxes</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td>(Quantity needed)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:335px;">Behavior and camera box material</td>
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">IR LED array</td>
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Air conditioner Unit</td>
			<td style="width:397px;">Melcor Store</td>
			<td style="width:244px;">&#160;MAA280T-12</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Imaging system</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td>(Quantity needed)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cosmicar/Pentax C21211TH (12.5mm F/1.4) C-mount Lens</td>
			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td style="width:244px;">PEC21211 KP</td>
			<td>1</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CCXC-12P05N Interconnect Cable</td>
			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td style="width:244px;">SOCCXC12P05N</td>
			<td>1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">DC-700 Camera Adapter</td>
			<td style="width:397px;">B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td>
			<td style="width:244px;">SODC700</td>
			<td>1</td>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TiFFEN 40.5mm Circular polarizer</td>
			<td style="width:397px;">Amazon</td>
			<td style="width:244px;"></td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">IR Videocamera</td>
			<td style="width:397px;">Industrial Vision Source, FL, USA</td>
			<td>Sony XC-EI50 (SY-XC-E150)</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="63">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Miscellaneous</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td>(Quantity needed)</td>
		</tr>
		<tr height="21">
			<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>
			<td>1</td>
		</tr>
		<tr height="42">
			<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>
			<td style="width:244px;">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 ...

The overall goal of this setup is to assay olfactory behaviors of flies in a four quadrant olfactometer using a provided automated fly tracking software script. This method can help address key questions regarding how olfactory neural systems detect the external world, and how the brain decodes and directs behavioral decisions. The main advantage of this technique is that it's versatile and robust, and allows repeat testing of olfactory responses by population of flies in a large experimental arena.For this protocol, prepare five odorant chambers that consist of a plastic outer container, glass inner container, and a custom-made PTFE lid insert. The insert is an original container lid with the central part removed, and two attached one way valves. Use four of the odorant chambers for solvent controls, and one chamber for a test odorant.Make sure each chamber is clean, and then load an odorant container with one milliliter of solvent or odorant dilution. Then, check the position of the container in the plastic chamber, and ensure that no liquid has been spilt. Secure the lid using the screwtop and an O-ring on the PTFE insert to prevent leaks.Rear the flies on standard cornmeal medium. In a standard bottle, place 30 males and 30 females, and allow eggs to be laid for five days at room temperature. From such bottles, collect newly eclosed flies.For each experimental cohort, collect 25 male and 25 female flies under brief carbon dioxide anesthesia and let the cohort age on standard fly medium for two to four days. 40 to 42 hours before the experiment, transfer the cohort to a vial with about 10 milliliters of one percent agarose gel for humidity without food. Starvation helps to increase the whole motion, and more than 90%of the flies should survive the starvation process.If more than 10%die, which can happen in a weak genotype, try using a one day starvation for all the flies in the experiment. At least five minutes in advance, switch on the temperature controller and set it to 25 degrees Celsius. Then, connect the odorant chambers to the arena via a plastic tube.Check the airflow rate in each quadrant of the arena using an electronic flow meter. Make sure that the control and odorant air streams are both running at 100 milliliters per minute. Flies are very sensitive to airflow, and it is critical that the airflow for each quadrant is verified before each experiment.Now, clean the arena and the glass plates using 70%ethanol. Wipe all the parts down two to three times, and allow them to fully air dry before proceeding. Next, clamp the glass plates to the arena.Then, transfer the flies into the arena without anesthesia. Using gravity, let them enter through the hole in one of the glass plates, and then cover the hole with a circular mesh. Now, place the fly loaded arena into the light-tight chamber.Then, connect the four control air streams to each arena corner. Close the door of the chamber and let the flies acclimatize to the new environment for 10 to 15 minutes. After the acclimation period, run a five to 10 minute control experiment in which flies are exposed to four control air streams.It is essential to now analyze the data immediately. If the flies are not evenly distributed in the arena, the arena must be reset. It is critical to test the behavior of control flies to just clean air.This will quickly verify that all background conditions are normal. Leaking light, temperature imbalance, tilted arena, or an odor contamination can all cause problems. If necessary, discard the flies and clean the arena again.If an odorant contamination is suspected, replace all the tubing. Continue repeating the control test with new cohorts until the animals show no preference for any of the four quadrants. Then, connect the test odorant chamber to the setup using a three way valve, or reconnect the tubing and run a test experiment for five to 10 minutes.For longer experimental recordings, rapidly stop and restart the tracking program every 20 minutes, or the data files will be too large. Between tests, discard the flies, clean the arena and glass plates with 70%ethanol, and replace the connector tubes. Keep the dry airflow going to continually flush the system.For efficiency, it is good to have two arenas so cleaning can be done during runs. If several experiments are wrong on the same day, take extreme care to ensure that no odor is left in the system from a previous run. This is normally not a problem with low concentrations of odorant or with CO2, but for highly concentrated stimuli, up to a 24 hour gap between experiment runs may be needed.For the data analysis, use the provided custom-made Matlab routine to convert the data into a Matlab format. Load the raw data file made in real time by the tracking software, and create a spacial mask that follows the contours of the arena. Apply the mask to the raw data to remove all the data points that fall outside of the arena, as they represent noise.Then, remove all the data points that move at less than 0.163 centimeters per second for longer than three seconds. This data is likely to be noise, or non-moving flies. Now, visualize the remaining data points by plotting all of them through the recording periods.The data can also be viewed as single trajectories. Odor boundaries can be quickly estimated based on the fly behavioral data. Then, calculate the attraction index, and make statistical tests as described in the text protocol.25 male and 25 female flies were tested in the behavioral apparatus. In response to dry air, flies explored the entire arena with no preference for any quadrant. The line shows the tracked trajectory of one fly.When apple cider vinegar was introduced into the top-left quadrant air stream, most of the flies were attracted to that odor quadrant. When a repellant odorant, 10%ethyl propionate, was introduced in the top-left quadrant, that quadrant was avoided. The attractive index of that quadrant scored 0.68.An attractive index is the most widely-used index in olfactory response measurement. A value above or below zero indicates attraction or repulsion. The script compensated for slight variations in arena position between trials.Data points that might have been noise or non-moving flies were removed. While the test can be run for any duration, it is interesting to view changes in the cohort's movements across 10 second bins to appreciate odor habituation and other phenomena. After watching this video, you should have a good understanding of how to effectively run and analyze olfactory behaviors in a four-field assay.Once mastered, each olfactory experiment can be completed in about an hour, and we routinely run four to six experiments during one day.

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

Research

JoVE Journal

Neuroscience

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 ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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 ...

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.