Our protocol enables identification of larval neuronal populations encoding nutritional choices related to the control of the intake levels of two major macronutrients, proteins and carbohydrates. The simplicity and relatively high throughput of our method allows the quantification of food intake for several genotypes exposed to different nutritional conditions. To genetically cross the parental lines, set up 60 millimeter embryo collection cages with L3 rearing diet plates supplemented with some active yeast paste.
Transfer the adult TripA1 female virgins and Janelia GAL4 males aged five to eight days to the embryo collection cages and allow the mating to occur for 24 to 48 hours at 25 degrees Celsius with 60%humidity and a 12, 12 light dark cycle. After the mating period, transfer the mated adult flies to fresh L3 rearing diet plates and allow the egg-laying to occur for three to four hours at 25 degrees Celsius. Make sure that all plates are labeled with the genotype and date of the egg lay.
When the egg-laying period is finished, remove the plates from the cages and estimate the number of eggs per plate by counting the number of embryos in one quarter of the plate. Keep the larval density to a maximum of 200 embryos per plate and cover the plates with plastic lids. Incubate the L3 rearing plates at 18 degrees Celsius, 60%humidity, and a 12, 12 light dark cycle for nine days.
After the incubation, collect three groups of 10 L3 from each of the genotypes to be tested, as well as one group of 10 L3 for the zero dye food control. Use number five or featherweight forceps performing the larvae collection as gently as possible. Transfer the collected larvae to plastic dish weight boats containing one milliliter of water.
Set up a 30 degree Celsius incubator and keep humidity levels high by adding a tray filled with water to avoid larval dehydration during the assay. Equilibrate the temperature of the assay plates by warming them to 30 degrees Celsius prior to starting. Make sure that all plates are labeled with the genotype and protein to carbohydrate ratio of the diet.
Heat shock the experimental larvae for two minutes by directly transferring the plastic boats containing the animals to a water bath at 37 degrees Celsius. Set the required number of timers for one hour, then carefully drain the water from the plastic boats and transfer the L3 groups from the boats to the center of the asset plates. Cover the plates and start the timer.
Allow the larvae to feed for one hour at 30 degrees Celsius in the dark. Stop the feeding assay by transferring the plates to an ice bath. Prepare two milliliter microtubes for each L3 group tested containing enough 0.5 millimeter glass beads to fill the bottom portion of the microtube and 300 microliters of ice cold methanol.
Use a bench cooler to keep the microtubes cold. Carefully recover the L3 groups from the feeding assay plates and transfer them to the lids of the plates with some water. Rinse the larvae to remove food debris from their bodies, taking care to avoid any injuries.
Transfer the larvae to the prepared microtubes and lyse them with a tissue lyser to extract the food dye from the guts. Transfer the extracts to clean 1.5 milliliter microtubes by directly inverting the two milliliter microtubes onto the new tubes. Do so gently so that most of the glass beads stay at the bottom of the two milliliter tube.
Clear the cellular debris by centrifuging the extracts at a maximum speed and four degrees Celsius for 10 minutes, then transfer the supernatants to clean 1.5 milliliter tubes. To perform calorimetric quantification of food consumption, prepare standard solutions of blue dye by serially diluting it one to two in methanol. Transfer 100 microliters of the experimental samples, standards, and blank to the wells of a 96 well microplate and measure the absorbance at 600 nanometers.
This protocol was used to quantify the relative amounts of macronutrients consumed for animals under thermogenetic activation of specific neuronal populations in the larval nervous system. Activating distinct of populations of neurons significantly affected macronutrient balancing in third-instar larvae. The feeding pattern observed for the control line, here indicated in gray color, show an expected decrease of food intake in higher protein to carbohydrate ratio diets, demonstrating the effectiveness of the method.
The five phenotypic classes that were established for the experimental animals were eat a lot and compensate for protein dilution by overeating, eat a lot, but do not compensate, eat little, but compensate, eat little and do not compensate, and eat aberrantly. For each of these phenotypic classes and genotypes, the GFP patterns in the central nervous systems of third-instar larvae are shown. When attempting this protocol, it is important to generate developmentally synchronized early third-instar larvae in order to minimize the variation associated to animal behavior.
Following this protocol, radio labeling of the foods would allow the confirmation and further dissection of the hits found using this methods.
