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

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

1. Setup Assembly
<ol>
	<li>Manufacture the star-shaped arena (19.5 cm by 19.5 cm by 0.7 cm) out of polytetrafluoroethylene (PTFE) according to the provided drawing (Supplementary Materials, SupplementalSketch_StarShapedArena.pdf). The arena may be manufactured by a commercial or a custom facility.</li>
	<li>Acquire two glass plates (20.25 cm by 20.25 cm with thickness of 2 mm), and drill a hole (~0.7 cm in diameter) precisely in the center of one of the glass plates using a diamond-coated drill bit.</li>
	<li>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.

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			<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>
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			<td height="21" style="height:21px;width:335px;">Tubing and connectors</td>
			<td style="width:397px;"></td>
			<td style="width:244px;"></td>
			<td></td>
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		<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>
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		<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>
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			<td height="21" style="height:21px;">Tubing (ID:1/8)</td>
			<td>McMaster-Carr, IL, USA</td>
			<td>5108K43</td>
			<td>50Ft</td>
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		<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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			<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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			<td height="21" style="height:21px;">Push-to-connect tube fittings</td>
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			<td height="21" style="height:21px;">Barbed Tube Fittings (1/4MNPT-&#62;1/8BF)</td>
			<td>McMaster-Carr, IL, USA</td>
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			<td height="21" style="height:21px;">Barbed Tube Fittings (1/8MNPT-&#62;1/8BF)</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>
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			<td>2 pack (10)&#160;</td>
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			<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>
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			<td height="21" style="height:21px;">Hex head plug</td>
			<td>McMaster-Carr, IL, USA</td>
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			<td height="42" style="height:42px;width:335px;">Air pressure regulator, air filter and flowmeters</td>
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			<td>(Quantity needed)</td>
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			<td height="21" style="height:21px;">Labatory gas drying unit</td>
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			<td height="21" style="height:21px;">Multitube frames for 150-mm flowtubes</td>
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			<td height="21" style="height:21px;">Multitube frames for 150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
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			<td height="21" style="height:21px;">150-mm flowtubes</td>
			<td>Cole-Parmer, IL, USA</td>
			<td>R-03217-15</td>
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			<td>Cole-Parmer, IL, USA</td>
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			<td height="21" style="height:21px;">DAQ (NI USB-6009, National Instruments) and a&#160;</td>
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			<td style="width:244px;">NI USB-6009</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<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 ...

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 ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

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 ...

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

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 ...

Drosophila Adult Olfactory Shock Learning

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 ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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 ...

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

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 ...

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

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 ...

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

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 ...