This method can help answer key questions in the fields of insect toxicology and insect physiology by measuring the electrogenesis of the Drosophila melanogaster central nervous system. This allows one to test a wide range of scientific hypotheses and aid in the discovery of new insecticide modes of action. The main advantage of this technique is that it provides a simple, reproducible, and relatively high-throughput system with minimal financial input to study the nervous system of Drosophila.
To start, open the acquisition/analysis software. Click Setup in the main toolbar, and select Channel Settings, which will open a dialog box. Reduce the number of total channels to three.
Channel one will record the raw signal from the amplifier with a range of 100 millivolts. Channel two will be used to count the number of events from channel one that are above a given threshold. For this, click on the Calculation tab to open a drop-down menu for channel two.
Select Cyclic Measurements, which will open a second dialog box. Select channel one as the source. Then, on the drop-down menu measurement, select Unit Spike at Events.
Finally, in the detection settings area, from the drop-down menu, select Simple Threshold. The threshold level is input on the detection adjustment window to be set above background noise. To convert the electrical activity into a rate plot expressed in hertz, select Arithmetic on the third channel.
On the new window, input 1000 times smoothsec, parentheses channel two comma two. Then, select at the units drop-down menu the frequency unit hertz. Close the dialog boxes to return to the main screen.
The y-axis of channel three should be expressed in hertz and the x-axis in time. Dissect the larval Drosophila CNS by first identifying and extracting a wandering third-instar Drosophila melanogaster from the culture vial and placing it into 200 microliters of saline. Then, grasp the mouth hooks with a pair of fine forceps, and grab the abdomen of the maggot with a second pair of forceps, with light pressure, to avoid tearing the thin cuticular layer.
Gently pull the mouth hooks and abdomen in different directions to separate the caudal end of the maggot from the head region and expose the viscera of the maggot. The CNS will be intertwined with the trachea and digestive tract. Tease the CNS out of the digestive tract and trachea with forceps.
The dissection of the Drosophila central nervous system from the maggot is a critical step for success. The dissected ganglia must be intact, without any significant damage to the descending motor nerves. A larger number of motor nerves attached to the ganglia will increase baseline firing frequencies.
If necessary, disrupt the blood-brain barrier by manually transecting the CNS posterior to the cerebral lobes with Vannas spring scissors. The transection should be done based on the physiochemical properties of the chemical being used. The red line is the suggested transection point and the transected CNS with intact descending peripheral nerve trunks shown here.
To begin, first pull the glass pipette electrode from borosilicate glass capillaries to a resistance of five 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 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 three to five 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. Shown here is a representative nerve discharge trace before and after exposure to DMSO.
The arrow indicates the time of application. A limited response to DMSO is seen. Increasing doses of propoxur amplified the spike discharge frequency of the transected Drosophila CNS in a concentration-dependent manner, whereas the neurodepressant GABA reduced the spike discharge frequency in a concentration-dependent manner.
While attempting this procedure, it's important to remember that it is an ex vivo experiment, and therefore the conditions of the saline, such as pH and temperature, is critical for prolonged activity of the central nervous system preparation. Similarly, the lack of extraneous electrical activity, an effective Faraday cage, and a reduction of 60-hertz noise are critical for success of this assay. The data collected through this assay can help identify the specific mode of action of insecticides.
Subsequent validation of these data through other methods such as voltage-clamp electrophysiology, biochemical analyses, and additional pharmacological assays can be performed.
