情報化学物質にトコジラミの嗅覚ニューロンの応答を検出するために単一感覚子録音を使用しました

The overall goal of the single sensillum recording is to investigate neuronal responses of the antennal olfactory sensillum to various odorants. This method can help to answer the key questions in the field of chemical ecology such as if the insect can detect the chemical odorants from their environment and how they process the chemical odorants. The main advantage of this technique is that it allows mirroring the single neuron activity of the function.

It provides more precise measurement of the sensitivity of neurons to the chemical odorants. The implication of this technique extended toward the development of attractants or repellants in the insect control. Because this technique can be used large amount of chemicals that are biologically important for the insect.

Begin this procedure by anesthetizing bed bugs on ice for two to three minutes. Then fix the insects antennae and body on a microscope cover slip with double sided tape and remove the legs with fine scissors. Use a small pin to gently adjust the antennae on the tape.

Next, rest the cover slip against a small ball of dental wax to facilitate manipulation and adjust it to a 90 degree angle for placing the recording electrode. Once secured, place the bed bug under a stereo microscope. Turn on the light source and adjust the intensity of illumination until the antennae is clearly presented.

Then focus the microscope on the second flagellum of the bed bug's antennae at a high magnification. In this step, connect the pre-amplifier with a signal acquisition controller, which is connected with the computer for signal recording and visualization. Next, open the software and click record from the menu bar.

Then choose wave to start recording the wave signals. Turn on the speaker connected to the pre-amplifier to present the tone of the neuronal responses from the antennal sensillum. Now insert the reference electrode into the abdomen of the stabilized bed bug.

After the reference electrode has been connected to the bed bug's abdomen, move the recording electrode towards the posterior end of the bed bug's antennae. When the recording electrode is in contact with the tip of the right antennae, locate the electrode at low magnification. Then adjust the recording electrode while gradually increasing the magnification until both the electrode and the antennal sensillum are in the same plane and clearly visible under the microscope.

After that, insert the recording electrode into the shaft of the sensillum using the micro-manipulator. Lower it deeper if the background noise is too high. Once clear action potentials are observed from the recorded sensillum, fill a micro pipette with botanical stimulus.

Use the micro pipette to deposit a 10 microliter aliquot of the stimulus onto a filter paper strip inside a glass pastier pipette. Accurately and directly recording electrodes to the sensillum is the key to the success of the experiment. Background noise can be partially reduced after inserting the electrodes a little deeper.

Next, connect the loaded pipette to the outlet of the stimulus flow tube and place the pipette tip into the small hole in the tube oriented towards the antennae. When all the connections are stabilized, depress the foot switch of the stimulus controller to deliver a stimulus into the continuous humidified air stream and start recording. The recording process will last for 10 seconds starting one second before the stimulation.

Count the action potentials offline for two 500 millisecond periods, one before, and one after the stimulation. To present another stimulus, label a new pipette with 001%eucalyptol to be tested. Apply 10 microliters of the stimulus before placing a small piece of filter paper onto it.

Then wait for two to five minutes until the stimulus is completely vaporized in the glass pipette. Attach the pipette onto the outlet of the stimulus flow tube. Subsequently, insert the pipette tip into the small hole of the tube oriented towards the antennae.

Depress the foot switch and start the 10 second recording. After that, disconnect the pipette and prepare another pipette with 01%eucalyptol. Test all the rest of the doses of eucalyptol on the antennal sensilla to observe the most dependent responses.

In this figure, the signal trace shows the typical neural response of an olfactory sensillum to the solvent which is used as the control in the single sensillum recording. Signal recording started one second before the 0.5 second of puff stimulus. The signal was recorded continuously for 10 seconds after initiating the stimulus puff.

This signal trace shows the extremely strong neural response of an olfactory sensillum to a botanical stimulus 10%positive betapynene. After the puff of stimulus is delivered to the olfactory sensillum, the olfactory sensory neurons inside this sensillum fire with high frequency into a long-lasting temporal dynamic. For in this procedure are the message that behavior by an insect can be performed in order to answer additional questions like what are the behavioral responses of insects to certain chemicals with strong neuron responses.

After watching this video, you should have a better understanding of how to record the insect olfactory neuron responses to the chemical stimulus.