Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The brain is a tissue with a high-energy consumption that mostly uses glucose as fuel. On the other hand, experimental data suggests that other metabolites like ketone bodies and monocarboxylates might be useful as energy sources. However, there is a still debate on which brain cells are the producers and primary consumers.

Lactate has been demonstrated to be a relevant metabolite in vertebrates to drive brain activity in vivo. Our groups and others have shown in Drosophila the importance of monocarboxylate molecules to fuel the high-energy requirements in neurons, as well as the need of glial-derived lactate and ketone bodies in memory formation. We have described monocarboxylate transporters in the drosophila brain and determined that the transfer of lactate from glial cells to neurons is necessary to maintain the neural activity during high-demand periods.

The main advantage is to have the possibility to determine the intracellular dynamic of glucose and its metabolites, such as lactate, pyruvate and ATP. In glial cells and neurons during basal and high neural activity. Additionally, to measure the transfer of these molecules in other living tissues, such as the fat bodies.

The use of Drosophila as in-vivo model for the study of brain energy metabolisms using a simple setup. This will allow the modeling of metabolic diseases or to better understand the energy management in the normal brain or during the development of neurodegenerative diseases. To begin, place a 60 millimeter Petri dish covered with 1%agar and PBS in an egg-laying chamber.

Put a drop of liquid yeast in the center of the plate to encourage egg laying. Next, introduce 300 Drosophila flies into the chamber. Keep the flies at 25 degrees Celsius for three days.

Replace the Petri dish in freshly dissolved yeast twice daily. On the fourth day, let the flies lay eggs for four hours. After replacing the dish, allow the flies to lay eggs for three more hours on the fresh plate.

After three hours, place the plaque with the eggs at 25 degrees Celsius for 24 hours. From this plaque, collect 50 to 100 newly hatched larvae and place them in a plastic vial containing standard feed. To begin, place 25 millimeter glass cover slips into the wells of a six-well cell culture plate.

Place a drop of Poly-L-lysine 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 stereomicroscope.

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 larvae. 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 sensors 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 to 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.