This technique is significant because it can measure glucose uptake in specific cells in live Drosophila tissues. The FRET-based sensor provides a direct measure of changes in intracellular glucose levels. This technique allows researchers to evaluate changes in glucose levels in real time, within living motor neurons from ALS models versus controls.
A challenging aspect is imaging the same set of neurons after changing the buffer. Leaving the sample on the microscope helps ensure that the VMC moves as little as possible. 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 length-wise 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 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 buffer. 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 six motor neurons in focus located along the ventral nerve cord midline. Then acquire images every 10 seconds for 10 minutes. These images represent the baseline.
For glucose stimulation, remove the trehalose sucrose containing HL3 buffer using a Pasteur pipette and add five 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 CZI files or any file type supported by the imaging software with a file name including date, genetic background, experimental condition and channels used. Reuse the parameters to image all the genotypes. For image processing open the Fiji-ImageJ imaging software, then open the CZI files, choose view stack with hyper stack and set color mode to gray scale and select split channels into separate windows.Next.
Process the images using drift correction plug-ins, turbo reg and stack reg. Each channel is corrected separately by selecting stack reg under plug-ins and then choosing transformation to translation. Manually choose the regions of interest or ROIs by tracing all motor neurons that remain in focus over the entire time series.
Then use the ROI manager to save each motor neuron outline. Use the multi measure function to measure the mean gray value in each ROI at each time point. Then copy the ROIs traced from the first channel to the second channel for the pre stimulation image.
For data analysis, calculate the FRET by CFP ratio using the mean gray values for the FRET and CFP channels. Changes in the FRET by CFP ratios reflect alterations in the intracellular glucose concentration. Then plot FRET by CFP ratios for each ROI and time point versus time.
A genetically encoded FRET-based glucose sensor was used to measure glucose uptake in motor neurons. When glucose is not bound to the sensor, CFP excitation causes CFP emission only. And when glucose binds, the YFP signal can be detected due to FRET occurring between CFP and YFP.
Shown here are images of FRET and CFP signals in glucose sensor controls in TDP-43 mutant neurons under baseline and stimulation conditions. A representative motor neuron is outlined in each image as an example of ROI. Upon glucose stimulation, control motor neurons expressing glucose sensors, exhibit a small increase in glucose uptake while neurons expressing TDP-43 showed a significantly higher glucose uptake.
Further FRET by CFP ratios of TDP-43 mutant motor neurons were normalized to glucose sensor controls at each time point. Under baseline conditions, there was no difference between the genotypes. However, upon glucose stimulation, a rapid and significant increase in glucose uptake was observed in TDP-43 mutants compared to the control.
In a bar graph representation, glucose stimulation in neurons expressing the glucose sensor demonstrated a small but significant increase in FRET by CFP ratio compared to the baseline. In contrast neurons expressing TDP-43 showed a significant increase in ratio upon stimulation compared to the baseline. The net difference in FRET by CFP ratios between motor neurons expressing TDP-43 and glucose sensor is around 10.9%Time management is critical for this procedure so that the neurons do not die over the course of imaging.
It's important to image immediately following dissections to avoid this. This technique highlighted the importance of glucose metabolism in neurons and has led our group to further investigate the role of glycolysis in motor neuron regeneration.
