Assessing Glucose Uptake in the Motor Neurons of Drosophila Larvae with Upregulated Glucose Metabolism

Place control and mutant Drosophila larvae into separate dishes containing a glucose-free buffer.

Dissect the larvae to remove internal organs, exposing the central nervous system or CNS.

The CNS includes a ventral nerve cord harboring motor neurons. In the mutant larvae, these neurons exhibit upregulated glucose metabolism.

Motor neurons in both groups express intracellular glucose sensors containing donor and acceptor fluorophores.

Position the larvae under a confocal microscope. Upon excitation, the donor fluorophore emits fluorescence.

Locate the motor neurons using this fluorescence.

Replace the buffer with a glucose-supplemented solution.

Glucose enters the neurons, binding to the sensors and triggering a conformational change that brings the two fluorophores into proximity.

Upon excitation, the acceptor absorbs the donor-emitted energy, termed fluorescence resonance energy transfer or FRET, and emits fluorescence.

The FRET signal correlates with glucose uptake. A higher acceptor signal intensity in the mutant larvae compared to the control indicates upregulated glucose metabolism.

Before beginning the dissections, turn on the imaging microscope and lasers. Then, collect a wandering third instar larva. Rinse it with double-distilled water and place it in a drop of HL3 buffer on a previously prepared elastomer-lined dish.

Next, under a dissecting microscope, use a pair of forceps to pin the anterior and posterior ends of the larva, dorsal side up, by carefully stretching the larva lengthwise with insect pins. Using a pair of angled iris scissors, make an incision just above the posterior pin. Then make a vertical cut from the incision towards the anterior end of the larva.

After adding a few drops of HL3 buffer, if needed, remove the trachea and the rest of the floating organs without disturbing the central nervous system. Then, pin the flaps, stretching the body wall, to expose the central nervous system while keeping the neuromuscular system intact.

For image acquisition, use an upright confocal microscope with a 40 times water immersion lens. Then, select the 405-nanometer laser to excite the CFP. Next, optimize the acquisition parameters, such as scan speed, average, objective, zoom pinhole size, and spatial resolution. Adjust the gain such that the signal is in the optimal dynamic range, and use the same parameters for all genotypes.

To image motor neurons within the ventral nerve cord, place the silicone dish with the dissected sample under the lens, and lower the lens so that it comes in contact with the trehalose sucrose HL3. Ensure that the lens is completely submerged in the buffer and add more buffer if required.

Next, using the CFP and FRET channels, manually select an optical selection consisting of at least 6 motor neurons in focus, located along the ventral nerve cord midline. Then, acquire images every 10 seconds or 10 minutes. These images represent the baseline.

For glucose stimulation, remove the trehalose sucrose containing HL3 buffer using a Pasteur pipette, and add 5 millimolar glucose supplemented HL3 buffer without moving the ventral nerve cord. Then, acquire images every 10 seconds for another 10 minutes.

These images represent the stimulation phase. Save the images as dot czi files, or any file type supported by the imaging software with a file name including date, genetic background, experimental condition, and channels used. Reused the parameters to image all the genotypes.