Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Our lab investigates how animals detect and respond to odors, taste, and some pheromone. We've made significant strides in understanding the molecular basis of chemoreception. We've made several advances in the field, including the discovery of the first insect odor receptors, the discovery of the first insect taste receptors.

And the elucidation of basic principles of the logic of odor and taste coding. This technique allows the measurement of test neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of responses that occur after a testing stimulus.

It also allows the measurement of testing neuron activity to the river of volatile compounds. The classical technique for test electrophysiology was unable to measure test responses to hydrophobic compounds, such as long-chain pheromones that have very loose mobility in water. However, with the help of this technique, we can overcome this limitation by separating the recording electrode from the glass stimulus capillary.

Our research explores the functioning of chemosensory systems in animals, focusing on their role in feeding and mating choices. We're currently examining non-coating RNAs and micro peptides and fruit flies'olfactory systems, and how mosquitoes and tsetse flies use these systems to find human hosts. Begin by placing 10 to 15 newly emerged drosophila flies into fresh standard culture vials.

Maintain these vials at 25 degrees Celsius and 60%relative humidity in a 12 hour light and dark cycle. To prepare a glass capillary for holding the stimulus, select a bora silicate glass capillary. And use a pipette puller instrument to pull the glass capillary.

Then take tungsten rods for both reference and recording electrodes. Dip these electrodes repeatedly for several seconds in either a 10%potassium nitrate solution, or a potassium hydroxide solution to sharpen them to a one micrometer tip diameter. To draw a single fly from the vial into an aspirator, carefully withdraw the aspirator and place a finger over the end of the aspirator to trap the fly.

Now, keep the end of the aspirator in the 200 microliter pipette tip and use the end to push the fly forward headfirst towards the narrow end of the pipette tip. Using a razor blade, trim both ends of the pipette tip anterior and posterior to the fly. With the help of clay or a small piece of cotton, further push the fly forward until half of its head protrudes from the end of the trimmed pipette tip.

Employ forceps to gently expose the labellum at the front of the head. Under a stereo microscope position the labellum laterally on a cover slip, ensuring one lobe with its 31 taste sensilla is exposed. To begin, place the slide containing the drosophila labellum on the microscope stage under a low magnification.

Using a manual micro manipulator, insert the reference electrode into the eye opposite to the eye with the recording electrode. Now use a motorized micro manipulator to focus the tip of the glass stimulus capillary at the end of the field of view under both low magnification and high magnification objectives. Then employ a second motorized micromanipulator under low magnification and bring the recording electrode close to the labellum.

Under high magnification, insert the recording electrode into the base of a taste sensillum. Continue the insertion until the sound of neuronal firing activity is heard from the audio output of the IDAC system. Once a stable signal is detected, press the start recording button to initiate the recording of the signal using the software associated with the IDAC system.

Using the motorized manipulator, adjust the tip of the stimulus glass capillary so that it covers the tip of the taste sensillum. To conclude the stimulus, retract the glass stimulus capillary from the sensillum using the motorized manipulator. Sensillum spikes are categorized into two classes based on amplitude.

Larger spikes are attributed to the bitter sensitive neuron, while smaller spikes are from the sugar responsive neuron. The bitter sensitive neuron in the S-5 sensillum responded to DEET odor, indicating sensory activation without physical contact. And on response was observed in the I-1 sensillum when contacting a bitter stimulus, followed by a stronger off response upon contact termination.