Ensaio do fatoômetro de quatro vias: um método para avaliar respostas odorant-cued em Drosophila

Published: April 30, 2023

Abstract

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

Moscas frutíferas têm um sistema olfativo complexo, podem discriminar centenas de odores, e usar seu olfato para direcionar decisões comportamentais. Este vídeo descreve o ensaio de fatoômetro de quatro vias, um método para estudar respostas comportamentais olfativas em moscas. O clipe de protocolo em destaque mostra como lidar com moscas de teste e quais são etapas críticas durante a execução do ensaio.

Protocol

Este protocolo é um trecho de Lin et al., Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer, J. Vis. Exp. (2016). 1. Respostas comportamentais a odores atraentes e repelentes Ligue o controlador de temperatura e coloque-o a 25 °C. Conecte as câmaras odorantes (odores de controle e teste) inserindo a tubulação na saída da câmara odoante e ao encaixe push-to-connect na caixa de comportamento. …

Representative Results

Figura 1: Exemplos de dados gerados usando um ensaio olfativo de quatro campos. (A) Esquema da arena de quatro campos. (B) As respostas neutras são observadas quando todos os quatro quadrantes contêm apenas perfusão de ar seco. (C) Respostas de atração a uma diluição de 6,25% de vinagre de cidra de maçã perfumado do quadrante superior esquerdo. (D</strong…

Materials

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 50 Ft
Tubing (ID:1/16) McMaster-Carr, IL, USA 52355K41 100 Ft
Barbed tube fittings McMaster-Carr, IL, USA 5117K71 1 pack
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 18-Jan 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 2 oz; 60 ml 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 (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php) 1803 sq inch
Teflon for fly arena and odor chamber inserts, 3/8" thick, 12" x 12" 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 (12.5 mm F/1.4) C-mount Lens B AND H PHOTO AND ELECTRONICS CORP, NY, USA PEC21211 KP 1
CCXC-12P05N Interconnect Cable B AND H PHOTO AND ELECTRONICS CORP, NY, USA SOCCXC12P05N 1
DC-700 Camera Adapter B AND H PHOTO AND ELECTRONICS CORP, NY, USA SODC700 1
B+W 40,5 093 IR filter B AND H PHOTO AND ELECTRONICS CORP, NY, USA 65-072442 1
TiFFEN 40.5 mm 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: julianrbrown@gmail.com iFlySpy2 1
IC Capture software The Imagingsource, NC, USA (http://www.theimagingsource.com/)
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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Citazione di questo articolo
Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila. J. Vis. Exp. (Pending Publication), e20140, doi: (2023).

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