The overall goal of this procedure is to measure responses of individual taste sensa on the labum of drosophila. This is accomplished by first building an electrophysiology rig and setting up all the necessary equipment. The second step is to prepare the fly for recording.
Next, an individual lum is stimulated with a taste compound while the neuronal response is simultaneously recorded. Ultimately, electrophysiological tip recording from individual label silla is used to show the responses of taste illa to different taste compounds. Visual demonstration of this method is critical as the fly preparation steps can be technically challenging and difficult to learn.
In this procedure, collect the newly ELOs flies from the well-maintained fly cultures. Then age them five to 10 days in fresh culture vials before recording, before the fly preparation, chill the microscope plate on ice for 15 to 30 minutes. Next, backfill the glass reference electrode with B and e solution.
And gently tap out any bubbles under the dissecting microscope. Use the forceps to break off a small section of the tip. Subsequently use capillary action to draw out any remaining bubbles with the tissue.
Then slide the B and E filled reference electrode over the wire of the electrode holder, and to be careful not to introduce any air bubbles after that, aspirate a fly into a P 200 pipette tip. Place it in an ice bucket and chill for 30 seconds to one minute. Next, remove the microscope plate from ice and position it under the microscope after wiping off the moisture.
Then gently tap the fly out of the pipette tip onto the microscope plate under low magnification. Remove the four legs with one pair of forceps while holding the Forex stable with the other pair of forceps. Position the fly on its ventral side with the dorsal side facing up.
Always be careful to avoid touching the label beum with the forceps during the preparation process to minimize mechanical damage while holding the fly in place with one pair of forceps, insert the reference electrode at the midline of the posterior dorsal thorax at approximately 45 degrees in the direction of the head. Then secure the reference electrode holder with modeling clay such that the fly is visible underneath the microscope at high magnification. Maneuver and angle the glass electrode at its neck and head, and slide the fly towards the reference electrode holder using two pairs of forceps.
Now, gently extend the probos with one pair of forceps and slide the fly further down the glass reference electrode until the tip of the electrode is inside the label beum and the probos is fully extended. In this step, secure the reference electrode holder to the micro manipulator mounted on the air table. Position one lobe of label labum in the microscope field of view under high magnification and in line with the humidified airstream.
After that, turn on the Humidified Airstream Computer Digital Acquisition System or DAS and Amplifier. Next, use a syringe and plastic tubing to draw a small amount of ultrapure water through the tube at least 10 times. To rinse the glass recording electrode, subsequently rinse the recording electrode with TAST at least five times.
Then fill the recording electrode approximately one third to halfway full with taste and remove it from the tubing. If there are air bubbles, tap to release them or simply refill the electrode. Now slide the electrode onto the silver wire of the head stage quickly and smoothly.
In order to avoid introducing air bubbles to stimulate the single send, first, use the micro manipulator to bring the recording electrode into alignment with the sens of interest. Next, press the foot pedal to trigger acquisition mode of the amplifier. Then advance the taste and filled recording electrode with the fine control knob of the micro manipulator carefully until it makes contact with the tip of the sens and start recording.
Remove the electrode after one to two seconds. Repeat the procedure again with other illa and wait at least one minute in between presentations to the same lum. If recording with a single taste and for a prolonged period of time, the taste and solution may dry out and the solution in the tip may become more concentrated.
This can be remedied by gently contacting the tip of the glass electrode with smooth paper to remove a small amount of liquid by capillary action to record the responses to another taste and rinse and load the recording electrode with new taste and repeat the procedure. Save data files periodically with identifying information such as date, genotype, and taste. This figure shows the response of an centum to a sugar sucrose.
The same centum does not respond to a bitter compound berberine, and this figure shows that an eye type sens, which contains a bitter responsive neuron, displays larger amplitude spikes in response to berberine and smaller amplitude spikes in response to sucrose. Shown here, a representative suboptimal electrophysiological results. This trace represents a complete lack of signal.
This is the 50 or 60 hertz noise from external electrical sources, and this is the stochastic noise. This trace represents that the Meno sensory neuron was firing alone while both bitter gustatory receptor neuron and the Meno sensory neuron were firing. After watching this video, you should have a good understanding of how to record neuronal responses from Oph Atory Silla on the labum.
