This method can help answer key questions about Drosophila brain metabolism and how over-proliferation of glial cells affects metabolic reprogramming, whether diet or behavior alters metabolism. The main advantage of this technique is that fly brains can be metabolically analyzed as one whole intact tissue with reproducible results obtained from measuring just one brain at a time. Though this method can provide insight into the metabolism of the fly brain, it can also be applied to other tissues and model systems such as imaginal discs and C.elegans, respectively.
To prepare the micro-tissue restraints, from the storage container select one micro-tissue restraint per brain that is being measured, plus at least three for use as restraint-only controls. Using a mesh basket and a six-well plate, rinse the restraints with seventy percent ethanol and wash them with de-ionized water three times, each time for two minutes. Perform additional water washes if the restraints still give off an alcohol odor.
Wash the micro-tissue restraints in assay media and leave them in the solution until they are ready for use. To dissect the Drosophila melanogaster larval brain, select the late-third instar larvae from a vial of cultured Organ R flies. Place the larva in the well of a clean dissecting spot plate containing 500 microliters of 1x PBS.
Next, wash the larva by gently shaking it in the well and transfer it to another clean well. Then, place the spot plate under the dissecting microscope. Grasp the larva at its midsection with one pair of tweezers while grasping the eye hooks with a second pair of tweezers.
Gently and smoothly pull the larva in opposite directions using the two sets of tweezers. Visualize the brain which typically stays attached to the eye hooks and commonly has eye antennal discs attached to it. Using the eye hooks to hold the brain in place, carefully remove additional tissues.
Lastly, separate the eye hooks from the brain. Then, use tweezers to transfer the dissected brain to a new well containing 1x PBS. To add the dissected brains to a 96-well metabolic assay plate, first add 50 microliters of assay media to all the wells, including the experimental, control, restraint-only, and background wells.
Then, carefully place one brain in each well. Under the dissection microscope, use a bent-needle microprobe to press the brains to the bottom of the well. Gently position the brains in the middle of the three raised spheres using the probe.
To add a micro-tissue restraint to the 96-well metabolic assay plate, first place it on the edge. Under a microscope, orient it with the plastic ring facing down and the mesh on top. Next, remove the plate from the microscope.
Use the tweezers to grasp the restraint on both sides and gently drop it into the well. Use a bent-needle microprobe to press the restraint down into the well. The micro-tissue restraints must be positioned properly over the centered brain in each well for this technique to provide meaningful results.
Under the microscope, verify that the dissected brain can be seen through the tissue restraint and that it is centered in the well and repeat the procedure for all of the wells that will be used in the assay. Carefully add 130 micro-liters of assay media to each of the experimental, control, and restraint-only wells. Verify that the brains and micro-tissue restraints have not moved while adding the media.
Then, add 180 micro-liters of assay media to the four corner wells for use as background control. In this procedure, remove the utility plate and add the cell plate without the lid in the same orientation to the metabolic analyzer. Select Load Cell Plate"for the instrument to take in the cell plate and close the tray and then begin the assay.
When the assay is complete, remove the ejected cell plate and cartridge. Using a dissecting microscope, verify that the brains and the micro-tissue restraints are still positioned properly in the well after the assay has completed and exclude any wells with abnormalities from the analysis. When the optimized conditions are followed, the assays with the larval and adult brains result in readings of OCR slightly exceeding 150 picomoles per minute at the stabilized sixth time point, about 25 minutes into the assay.
This rate is maintained for at least 30 minutes and up to two hours. The ECAR is slightly lower in adult brains than in larval brains at the sixth time point and is maintained for at least 30 minutes. This finding corresponds to an increase in glycolysis during the larval stages to support growth.
An injection with a mitochondrial inhibitor, such as Oligomycin, lowers the OCR readings to reveal the ATP-dependent respiration and further treatment with Rotenone and Antimycin A lowers the OCR to reveal non-mitochondrial oxygen consumption. While attempting this procedure, it's important to remember to center the brains and ensure the micro-tissue restraint is secured in place before running the assay and during the analysis. Following this procedure, other methods like quantitative PCR and western blot analysis can be performed in order to answer additional questions about how gene expression contributes to the observed metabolic phenotype.
