The overall goal of this procedure is to introduce a detailed and highly effective protocol to make single sensillum recordings from insect palp Sensilla Basiconica. Since mastered can help answer key questions in the dissection chemoreception field. By allowing of specific odorant receptor in palp sensilla.
The main advantage of this is that use a recorder Sensilla Basiconia of locust of the palps. To prepare the tungsten electrodes first attach a new tungsten wire into a micro manipulator and observe it under 40 times magnification. Now sharpen the wire by delivering 10 volts to it while using a syringe to apply 10%sodium nitrite.
Then reduce the power to five volts and over one minute repeatedly dip four millimeters of the sharpened wire into sodium nitrite solution. When in use the tip must be fine enough to penetrate the cuticle of a locust palp olfactory sensillum. Next prepare the stimulus solutions.
Dilute each of the chemical stimulus substances in mineral oil. Make 10%dilutions of 1-Nonanol and Nonanoic acid in mineral oil. Then make a series of tenfold serial dilutions of E-2-Hexenal and Hexanal in mineral oil.
Next prepare Pasteur tubes loaded with stimuli. Insert filter paper strips into the tubes and add 10 microliters of the diluted stimulus solutions to the filter paper strips. Then plug the tubes with one milliliter pipette tips.
From the recordings make a locust maxillary palp holder from a glass slide, a piece of plastic, a cover slip and double sided tape and rubber tape. Attach the plastic piece to a corner of the slide using double sided tape and then use another piece of tape to attach the cover slip to the plastic piece. Then attach a small piece of red rubber tape onto the cover glass to make it a non-slip surface.
Next using the tape attach a tungsten wire 1.5 millimeters parallel to the inside edge of the platform and fix both ends of the wire onto the platform. This completes the holder. Rear the locust on fresh wheat seedlings and at 60%humidity 28 to 30 degrees Celsius and with an 18 hour light cycle.
To collect the antenna select one day to three day old fifth instar Locust nymphs. Remove the antenna using fine scissors so they cannot interfere with the recording. To secure the locust use a 1.5 millimeter tube cut in 1/2 vertically.
Leave the ventral region and the head of the locust exposed. Then fix the locust and tube to the glass slide with double sided adhesive tape. Next pull the right maxillary palp out and position it onto the platform.
Put the tungsten wire at the fourth segment of the palp. Now surround the tungsten wire with blue Taft putty about two millimeters from the maxillary palp. It is critical to the to most properly set up the platform with a centimeter locust maxillary palp holder and to use the tungsten wire to comprise the four segment or the palp.
Now view the preparation at 100%times magnification and position the palp perpendicularly to the recording electrode. Next using a micromanipulator insert a tungsten reference electrode into the locust eye. Then move the recording electrode close to the maxillary palp.
Next position the odor delivery device about one centimeter from the maxillary palp. Now open the recording software and set the recording parameters. For the recording scale use 500 microvolts.
For the high cut off of the filter use 300 hertz. Set the low cutoff to 200 hertz and set the pre-trigger to 10 seconds. Now switch the microscope to a high magnification and insert the recording electrode into the base of a basiconic sensillum on the maxillary palp.
Then delicately adjust the recording electrode to obtain good spontaneous spikes. It is very important that the recording electrode penetrates into the base of the sensillum and that once into the sensillum are prepared. Now open the stimulus controller to deliver a continuous airstream at 20 millimeters per second.
Next set the stimulation time to one second and record signals for 10 seconds starting 10 seconds before the onset of the stimulus pulse. Now collect the data. Two sensilla subtypes on the locust maxillary palp were distinguishable based on their response dynamics to the chemical stimulants 1-Nonanol and Nonanoic acid.
The odorants Hexanal and E-2-Hexanal naturally occurring in food sources can evoke a locust palp opening response. In response E-2-Hexanal pb1 neurons have longer action potentials. Comparing the mean changes in spiking during the five seconds before stimulation to the five seconds after stimulation shows that the response to 1-Nonanol is significantly higher than Nonanoic acid in the pb1 neurons.
And the reverse is true for pb2 neurons. The response by these neurons to E-2-Hexenal and hexanal is also dose dependent. The two neuron types have a different dose response pattern to E-2-Hexenal, but the response to hexanal is mostly the same.
After watching this video you should have a good understanding of how to perform the highly effective single sensilla recording on locust palp Sensilla Basonica. Once mastered the technique can be done if it is performed properly. When attempting this procedure it's important the tube of the palp from when the recording electrode is inserted into the base of sensillum.
After this development paved the way for research in the field of insect physiology to the and cellular mechanisms of incellular olfaction in the mouth part.
