This article describes a method for recording neuronal activity from the transected central nervous system (CNS) of Drosophila larvae. The technique involves monitoring action potentials to assess the effects of various test agents on neuronal firing frequency.
Place a transected central nervous system, or CNS, of a Drosophila larva inside a recording chamber filled with saline solution.
The transected CNS includes the ventral ganglia and descending peripheral nerves.
Place a suction electrode at the posterior end of the transected CNS. Connect the chamber to a grounding wire to reduce background noise.
Apply negative pressure to draw a peripheral nerve into the electrode and initiate extracellular recording of the nerve activity.
Monitor the spontaneous generation of action potentials by neurons over time, termed the baseline firing frequency, and allow it to stabilize.
Add a solvent as a control. Record the activity to ensure the firing frequency remains consistent with the baseline.
Introduce the target test agent dissolved in the solvent and record the firing frequency.
An increase in frequency exhibits the agent's excitatory effect, whereas a decrease in frequency indicates an inhibitory effect.
To begin, first, pull the glass pipette electrode from borosilicate glass capillaries to a resistance of 5 to 15 megaohms. Insert the transected CNS into a wax chamber containing 200 microliters of saline. Clamp an uncoated insect pin with an alligator clip soldered to the ground wire, and insert the pin into the saline to complete the circuit. Using the micromanipulators, orient the electrode to the caudal end of the transected CNS.
Eliminate background noise by adjusting the threshold level in the acquisition analysis software prior to connecting the peripheral nerve trunks. Apply slight negative pressure on the syringe to draw peripheral nerves into the suction electrode. Start the recording on the data acquisition software, and allow the baseline firing rate to equilibrate for five minutes prior to collecting baseline firing rate data.
After five minutes, add 200 microliters of saline and vehicle to bring the total volume of the chamber to 400 microliters to begin recording control firing rates. Discard the preparation and recording if the pattern of firing of control treatment is not similar to the example shown here.
When baseline has been established after 3 to 5 minutes of recording, withdraw 200 microliters of saline and add 200 microliters of the experimental agent solubilized in saline. Label this time point of drug application in the acquisition analysis software by including a comment that includes the drug and the final concentration.
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