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The overall goal of this technique is to present odorants of low volatility effectively to olfactory receptor neurons and drosophila for electrophysiological recording. Those neurons are housed in the trichoid sensilla and respond to long chain cuticular pheromones. This method can help answer key questions in the field of insect pheromone communication by providing an improved odor delivery technique.

The main advantage of this technique is that it allows odorants which were previously inaccessible due to their low volatility to be effectively presented during in vivo recording. Specifically, pheromones are presented from close distances that simulate the range between courting males and target females. The preparation of the odor delivery cartridge component for AT4 recordings begins with removing 0.9 centimeters from the narrow end of a 200-microliter pipette tip.

Then, from a second pipette tip, remove 1.7 centimeters from the narrow end, and 1.5 centimeters from the wide end to make a 1.8 centimeter length. Next, use forceps to place a 1/8 inch diameter disk of filter paper at the tip of the 1.8 centimeter section. Leave a small opening through which air can pass.

Then secure the pipette sections together with the smaller section angled downward to aim at the preparation. Now use a P10 to apply the odorant at the desired dilution to the filter paper. Then, to evaporate the solvent, let the loaded cartridges sit in a vacuum desiccator for an hour.

First, assemble a fly preparation slide. On a glass slide, attach a glass cover slip with modeling clay, to create an angle of about three degrees between the slide and cover slip. Then attach double-sided tape at two places, the inner edge of the cover slip and the slide immediately below the cover slip.

Always use fresh tape. Now collect the fly of interest in an aspirator tube. Then fit a 200-microliter pipette tip over the end of the tube, and simultaneously flick the tube while blowing, to push the fly into the end of the pipette tip.

Then use a razor blade to cut the tip just below the body of the fly. Now carefully load molding clay into the wide end of the tip to force the fly's head into the smaller opening. Once both antennae are exposed, stop packing in clay.

Now use forceps to position the prep onto the slide. Position the clipeus to face horizontally with the lateral side of the antenna against the taped cover slip surface. Then place the holding rod on the arista to secure the antenna to the tape.

Next, place the prep onto the stage of the rig. Confirm that the trichoids are visible along the distal lateral edge of the third segment of the antenna. The sensilla should be clearly silhouetted against the background.

Keep the prep under a constant humidified air flow delivered via separate tube at a distance of about two centimeters. To take recordings, insert the reference electrode into the clipeus using a swift and smooth motion. Place it just below the cuticle where it will be in contact with the hemolymph.

Next, slowly lower the recording electrode until it enters the same plane of view as the target sensillum. Then insert the recording electrode into the sensillar base. Before proceeding, ensure that there is a high signal-to-noise ratio, with identifiable spikes from AT4-A and AT4-C neurons.

The AT4-A neurons should fire no faster than 20 hertz. Now connect the cartridge to the odorant delivery tube. Begin with the solvent control and progress towards the highest odorant concentration.

In this case, a palmitoleic acid cartridge is attached. Next, use the micromanipulator to maneuver the cartridge towards the prep while aiming it to the head. Position the cartridge tip pointing directly at the antenna from a distance of about four millimeters.

The cartridge must also be about one millimeter above the slide. Precise positioning of the cartridge with respect to the prep is the single most critical step of this procedure. Consistent neuronal responses are contingent on the reproducible position of the cartridge across trials.

The cartridge might be closely bordered by the electrodes and the slide. It should ideally be at least four millimeters from either electrode. For stimulation, set the odorant flow controller to 500 millisecond bursts, and use a 500 milliliter per minute flow rate.

For each stimulus burst, record the electrical activity for 10 seconds. After an odorant application, carefully retract the cartridge before replacing it with a cartridge containing the next highest concentration. After taking all the recordings for the day, thoroughly rinse the recording electrode with distilled water.

Using the described methods on seven-day-old male Berlin wild-type flies, the relative efficacy of the trans and cis palmitoleic acid was compared. Trans-palmitoleic was a stronger stimulant of Or47b olfactory receptor neurons. From each fly, only a single neuron was recorded.

The distance between the opening of the odor cartridge to the head of the fly had a significant influence on neuronal responses. At four millimeters there were many more spikes fired in response to palmitoleic acid. At a distance of 11 millimeters, there were hardly any observable responses by the O47b neurons.

Close-range presentation of palmitoleic acid was quite important for studying its effects as an odorant. We hope that after watching this video you will have a clear understanding of how to present palmitoleic acid or other low-volatility odorants into your drosophila olfactory receptor neurons during in vivo single sensillum recording. Once this technique is mastered, recording an entire dosage curve from a single fly can be achieved in around 20 minutes.

After its development, this technique paved the way for researchers in the field of insect olfaction to explore the neurophysiology and signal transduction mechanisms for pheromone detection.