To begin, place 25 millimeter glass cover slips into the wells of a six-well cell culture plate. Place a drop of polylysine in the center of each cover slip. After incubation is complete, wash each cover slip three times with distilled water.
Then wash the cover slips two times with a calcium-free saline solution. Next, install the covers in a recording chamber and fill it with calcium-free saline. Collect the wandering third instar larvae from the desired genetic cross of drosophila.
Transfer the larvae to a dish well, then wash the larvae three times with distilled water. Place the larvae in another glass dissection dish well containing 750 microliters of ice-cold, calcium-free saline solution. Now place a larvae under a stereo microscope.
With a pair of forceps, make a transverse cut across the back of the abdomen. Push the jaw with the forceps to turn the larvae inside out. Observe the ventral nerve cord next to the jaw.
Carefully remove the imaginal discs and remaining brain coddle tissues. Next, separate the ventral nerve cord with the central brain and optic lobes from the rest of the tissue. Transfer the ventral nerve cord to the recording chamber containing calcium-free saline solution.
To avoid interference, attach the remaining nerves to the bottom of the cover slip with forceps. To perform experiments on fat bodies, proceed to isolate the fat tissues from the turned-out larva. Place the isolated fat bodies expressing the fret sensors in the recording chamber.
To acquire images of tissues expressing sensors, turn on the illumination system of the microscope. Place the recording chamber containing ventral nerve cords or fat bodies on the stage of a fluorescent microscope. To view the GCaMP6f fluorescence, set the excitation wavelength to 488 nanometers.
For metabolites such as laconic, pyronic, ATP, or glucose sensors, set the excitation to 440 nanometers. Now set the image acquisition at regular intervals for GCaMP6f and metabolite sensor. Place the water immersion objective, ensuring it remains submerged.
Next, connect the recording chamber to a perfusion system. Use a low-flux peristaltic pump to extract the liquid from the chamber and maintain a constant flow of three milliliters per minute. Position the solution containing tubes 25 centimeters above the microscope stage.
Before any stimulation, obtain a stable fluorescence baseline by allowing the recording solution to flow for five to 10 minutes. Now, replace the flow solution with the stimulation solution containing glucose, pyruvate, or lactate for five minutes. Capture the images of the stimulated ventral nerve cord that was exposed to 80 micromolar picrotoxin to assess neuronal activity.
Laconic sensors in both glial cells and motor neurons respond to one millimolar lactate at a similar rate at the start of the pulse. However, the motor neurons reach a higher increase over the baseline during the five minute pulse. The glucose sensor's signal increased at a similar rate in the glial cells and neurons when exposed to five millimolar glucose.
During the glucose pulse, however, the signal in neurons increases more than in glial cells. Knocking out Chaski transporter reduced lactate transport in glial cells. Picrotoxin-induced increases in neuronal activity resulted in a transitory drop in ATP levels in the soma of motor neurons.
The laconic sensor was observed to be well-expressed in fat bodies. Increased fluorescence of the glucose sensors was seen when the fat bodies were exposed to increasing glucose concentration. Increased lactate and pyruvate concentrations resulted in a proportional increase in laconic and pyronic fluorescence.
