Fireveis olfactometeranalyse: En metode for å vurdere luktfrie svar i Drosophila
Published: April 30, 2023
Abstract
Kilde: Lin, C.C., et al. Olfactory Behaviors Analysert av Computer Tracking Of Drosophila i et fire-kvadrant Olfactometer. J. Vis. Utt. (2016).
Fruktfluer har et komplekst olfaktorisk system, kan diskriminere hundrevis av luktstoffer og bruke luktesansen til å lede atferdsbeslutninger. Denne videoen beskriver den fireveis olfactometeranalysen, en metode for å studere olfaktoriske atferdsresponser hos fluer. Det utvalgte protokollklippet viser hvordan du håndterer testfluer og hva som er kritiske trinn mens du utfører analysen.
Protocol
Denne protokollen er et utdrag fra Lin et al., Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer, J. Vis. Exp. (2016). 1. Atferdsrespons på attraktive og avstøtende luktstoffer Slå på temperaturregulatoren og sett den på 25 °C. Koble luktkamrene (kontroll og test luktstoffer) ved å sette slangen inn i utløpet av luktkammeret og til push-to-connect-beslaget på atferdsboksen. Kontroller…
Representative Results
Figur 1: Eksempeldata generert ved hjelp av en olfaktorisk analyse med fire felt. (A) Skjematisk for firefeltsarenaen. (B) Nøytrale reaksjoner observeres når alle fire kvadrantene bare inneholder tørrluftperfusjon. (C) Tiltrekning svar på en 6,25% fortynning av eple cider eddik perfused fra venstre øvre kvadrant. (D) Avstøtningsvirkemåter utløst a…
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 |
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).