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Representative Results






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

Published: August 20th, 2016



1The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, 2MRC Clinical Sciences Center, Imperial College London

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.

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 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 neurob....

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1. Setup Assembly

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

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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.......

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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.......

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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 Lundström 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).


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Name Company Catalog Number Comments
Air delivery system  (Quantity needed)
Tubing and connectors
Thermoplastic NPT(F) Manifolds Cole-Parmer, IL, USA R-31522-31 1
Hex reducing  nipple (1/4MNPT->1/8MNPT) McMaster-Carr, IL, USA 5232T314 1
Tubing (ID:1/8) McMaster-Carr, IL, USA 5108K43 50Ft
Tubing (ID:1/16) McMaster-Carr, IL, USA 52355K41 100Ft
Barbed tube fittings McMaster-Carr, IL, USA 5117K71 1pack
Push-to-connect tube fittings McMaster-Carr, IL, USA 5779K102 4
Barbed Tube Fittings (1/4MNPT->1/8BF) McMaster-Carr, IL, USA 5463K439 1 pack (10)
Barbed Tube Fittings (1/8MNPT->1/8BF) McMaster-Carr, IL, USA 5463K438 2 pack (10) 
Barbed Tube Fittings (1/8MNPT->1/16BF) McMaster-Carr, IL, USA 5463K4 2 pack (10) 
Barbed Tube Fittings (1/4MNPT->1/4BF) McMaster-Carr, IL, USA   5670K84 1
Hex head plug McMaster-Carr, IL, USA 48335K152 1
Air pressure regulator, air filter and flowmeters (Quantity needed)
Labatory gas drying unit W A HAMMOND DRIERITE CO LTD, OH, USA Model: L68-NP-303; stock #26840 1
Multitube frames for 150-mm flowtubes Cole-Parmer, IL, USA R03215-30 1
Multitube frames for 150-mm flowtubes Cole-Parmer, IL, USA R03215-76 1
150-mm flowtubes Cole-Parmer, IL, USA R-03217-15 9
Valve Cartridge Cole-Parmer, IL, USA R-03218-72 9
Precision Air regulator McMaster-Carr, IL, USA 6162K13 1
Soleniod valves Automate Scientific, Berkeley, CA 02-10i 4
Solenoid valve controller ValveLink 8.2, Automate Scientific, Berkeley, CA 01-18 1
Electronic flow meter Honeywell AWM3100V 1
DAQ (NI USB-6009, National Instruments) and a  National Instruments NI USB-6009 1
Power supply Extech Instruments 382200 1
Odor chambers
Polypropylene Wide Mouth jar 2oz; 60ml Nalgene 562118-0002 At least 5 are required per experiment, but a separate chamber is required for each dillution of each odorant. Available at Container Store, part #635114)
Glass odor chamber, 0.25 oz Sunburst Bottle LB4B At least 5 are required per experiment 
"In" valve for odor chamber Smart Products, Inc., CA, USA 214224PB-0011S000-4074 1 of these parts is used per odor chamber but they need to be replaced frequently
"Out" valve for odor chamber Smart Products, Inc., CA, USA 224214PB-0011S000-4074 1 of these parts is used per odor chamber but they need to be replaced frequently
O ring RT Dygert International, MN, USA AS568-029 Buna-N O-R 1 pack (100)
Fly arena, camera and behavior boxes (Quantity needed)
Behavior and camera box material Interstate plastics, CA, USA ABS black extruded ( 1803 sq inch
Teflon for fly arena and odor chamber inserts, 3/8" thick, 12"x12" McMaster-Carr, IL, USA 8545K27  1
Glass plates, 1/8" Thick, 9"x 9" McMaster-Carr, IL, USA 8476K191  2
Dual action thermoelectric controller WAtronix Inc, CA, USA DA12V-K-0 1
IR LED array Advanced Illumination, Rochester, VT, USA AL4554-88024, PS24-TL 2 LED arrays and one power supply
Air conditioner Unit Melcor Store  MAA280T-12 1
Imaging system (Quantity needed)
Cosmicar/Pentax C21211TH (12.5mm F/1.4) C-mount Lens B AND H PHOTO AND ELECTRONICS CORP, NY, USA PEC21211 KP 1
B+W 40,5 093 IR filter B AND H PHOTO AND ELECTRONICS CORP, NY, USA 65-072442 1
TiFFEN 40.5mm Circular polarizer Amazon 1
IR Videocamera Industrial Vision Source, FL, USA Sony XC-EI50 (SY-XC-E150) 1
USB video converter The Imagingsource, NC, USA DFG/USB2-It 1
iFlySpy2 (fly tracking software) Julian Brown, Stanford, Calfornia: iFlySpy2 1
IC Capture 2.2 software The Imagingsource, NC, USA (
Miscellaneous (Quantity needed)
Dremel rotary tool Dremel, Racine, WI, USA Dremel 8000-03  1
Diamond-coated drill bits for glass cutting Available from various suppliers; MSC industrial Supply Co, Melville, NY 90606328 1

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