Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae
Summary
Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).
Abstract
The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.
Protocol
Discussion
Olfactory cues are used by organisms to identify food sources, potential mates, and predators. Olfactory sensory neurons (OSNs) are the first relay center between external stimuli and higher centers of the brain where the information is further processed. In Drosophila melanogaster and Anopheles gambiae, OSNs are easily accessible and their electrical activity can be monitored while stimulated by odor puffs.
The single sensillum recording (SSR) technique explained in this vi…
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Paraffin oil | Odors | Fluka | 76235 | |
| High purity odors (>98%) | Odors | Sigma-Aldrich | Methyl acetate #296996 1-octen-3-ol #74950 |
|
| Filter paper strips | Odors | Fisherbrand | 05-714-1 | Chromatography paper |
| Connectors | Odors | Cole-Parmer | EW-06365-40 | 1/16×1/8″ |
| Glass vials | Odors | Agilent Technologies | 5182-0556 | |
| Air line plastic tubing | Odor Delivery | Python Products | 500PAL | |
| 1 serological pipette | Odor Delivery | Corning | 4101 | 10 mL |
| Plastic tubing | Odor Delivery | Cole-Parmer | EW-06418-0 | 0.050″x0.090″OD |
| Disposable borosilicate glass Pasteur pipettes | Odor Delivery | FisherBrand | 13-678-20A | 5-3/4 inches |
| Programmable stimulus controller | Odor Delivery | Syntech | CS-55 | |
| Anti-vibration table | Electrophysiology Equipment | TMC | 63533 | 36”Wx30”Dx29”H |
| Faraday cage | Electrophysiology Equipment | TMC | MI8133303 | |
| Inverted microscope | Electrophysiology Equipment | Nikon | E600FN ECLIPSE | Recording microscope |
| 10x and 100x objectives | Electrophysiology Equipment | Nikon | 10x Plan Fluor 100x L Plan | |
| Dissecting microscope | Electrophysiology Equipment | Nikon | EZ645 | electrode sharpening/insect prep microscope |
| Magnetic stands | Electrophysiology Equipment | Newport | MODEL 150 | |
| IDAC | Electrophysiology Equipment | Syntech | IDAC-4 | |
| Acquisition software | Electrophysiology Equipment | Syntech | Autospike | |
| 1 macromanipulator | Electrophysiology Equipment | NARISHIGE | MN-151 | Joystick manipulator Used for positioning reference electrode |
| 1 micromanipulator | Electrophysiology Equipment | EXFO | PCS-6000 | Used for positioning recording electrode |
| Crocodile clip | Electrophysiology Equipment | Pomona | AL-B-12-0 | |
| Electric cable | Electrophysiology Equipment | Pomona | B-36-0 | Test Cable Assembly |
| 2 electrode holders | Electrophysiology Equipment | Syntech | N/A | Electrode holders (set of 2) for tungsten wire electrode |
| AC probe | Electrophysiology Equipment | Syntech | N/A | Universal single ended probe (10xAC) |
| Tungsten electrodes | Electrophysiology Equipment | Microprobes | M210 | straight tungsten rods, 0.005“x3“ |
| Potassium hydroxide | Electrophysiology Equipment | Sigma-Aldrich | 221473 | |
| Syringe | Electrophysiology Equipment | BD | 301625 | 20 mL |
| Power supply | Electrophysiology Equipment | WILD HEERBRUGG 6V 40W | e.g MTR32 | |
| Vertical puller | Insect prep | Narishige | PB-7 | |
| Razor blade | Insect prep | VWR | 55411-050 | |
| Dental wax | Insect prep | Patterson | 091-1503 | |
| Microscope slide | Insect prep | FisherBrand | 12-550A | |
| Cover glass | Insect prep | FisherBrand | 12-541A | 18X18 #1.5 |
| Polypropylene mesh | Insect prep | Small Parts inc. | CMP-0500-B | |
| Glass electrode | Insect prep | Frederick Haer & Co. | 27-32-0-075 | Capillary tubing borosilicate 1.5mm OD x 1.12mm ID x 75 mm |
| Double-sided tape (3M) | Insect prep | 3M | MMM6652P3436 | Double-sided tape (3M) |
| Forceps | Insect prep | Fine Science Tools | 021×0053 | Dumont #5 Mirror Finish Forceps |
| Small plastic cup | Insect prep | VWR | 89009-662 | 7 x 5.7 (23/4 x 21/4) |
| Electric aspirator | Insect prep | Gempler’s | RHM200 |
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