The overall goal of this procedure is to quantitatively measure the food-intake of adult Drosophila, which provides a means to evaluate the effects of genetic manipulations such as thermogenetic and optogenetic activations of neurons. These measures can help answer key questions in the feeding field such as the neuro mechanics of hunger satiety and appetite. The main advantage of this technique is that it simultaneously quantifies food intake of multiple groups of flies, making it especially useful to identify abnormally feeding flies in genetic screens.
To be begin gather supplies to prepare the outside container. Suspend 0.5 grams of agarose and 50 milliliters of double-distilled water. Heat with frequent agitation, and boil to dissolve completely on a magnetic stirrer with a heat plate.
When the 1%agarose cools to about 60 degrees Celsius, transfer five milliliters to an empty outside container and keep the container open until the agarose has cooled down to room temperature to yield an agarose pad. Next, add 0.5 grams of agarose to 50 milliliters of double-distilled water. Heat with frequent agitation and boil to dissolve completely on a magnetic stirrer with a heat plate.
When the 1%agarose cools to about 60 degrees Celsius, add 1.71 grams of sucrose to the agarose to obtain 100 millimole of sucrose. Add 0.25 grams of blue dye to the agarose solution to obtain 0.5%weight per volume dyed labeled food. Then prepare the inside container.
Put the empty inside containers on a flat horizontal surface. Then add 750 microliter of blue dye labeled food to individual food containers. Let solidify for at least five minutes.
Transfer a filled inside container into a prepared outside container and position it at the center of the agarose pad. Prepare feeding chambers with dye-free food for background subtraction. Leave the feeding chambers open at room temperature for 40 minutes, until the wall of the outside container is free of condensation.
Before behavioral experiments, equilibrate the prepared feeding chambers at the experimental temperature of 30 degrees Celsius for two hours to ensure the temperature is relatively uniform in the feeding chambers. To start the feeding process, carefully transfer 20 flies from their home vial into a preheated feeding chamber by gentle tapping. Continue the feeding process at 30 degrees Celsius for 60 minutes.
Cease feeding by freezing the feeding chambers at negative 80 degrees Celsius for 30 minutes. Carefully transfer 20 flies from one vial into a feeding chamber by gentle tapping. And then place the chamber sideways on the illumination setup.
For optogenetic manipulation, use an array of orange LEDs as the source of light stimulation, and fix a horizontal plexiglass plate above the LEDs to support the feeding chambers during illumination. Keep the setup in an incubator at 25 degrees Celsius with 60%humidity. Stimulate the genetic control flies in parallel with the Taotie-GAL4 UAS CsChrimson flies, but in different feeding chambers.
Continue the feeding process for 60 minutes with back illumination by the orange light. Then freeze the feeding chambers at negative 80 degrees Celsius for 30 minutes. After ceasing the feeding, pour out all 20 flies from the feeding chamber on to a piece of weighing paper.
Then transfer them into a 1.5 milliliter tube using a soft brush. Add 500 microliters of PBST to the tube and homogenize the flies using a grinding mill at 60 hertz for five seconds. Then confirm sufficient homogenization by observation when there are no detectable fragments of body parts.
Spin the tubes with homogenates for 30 minutes at 1300 RPM to clear the debris. After centrifugation, transfer 100 microliters of supernatant to a well of 96 well plate. To avoid the miscalculation of absorbents caused by debris, gently take the tube used out of the centrifuge and carefully drop the supernatant without disturbing the pallet.
After all the samples are loaded, place the plate into a plate reader to measure the absorbents of the samples at 630 nanometers. The feeding response was quantified by visually inspecting and scoring flies with detectable dye in the gut. After activation of Taotie neurons, about 58%of Taotie dTRPA1 flies exhibited strong feeding behaviors.
And 23%of these showed mild feeding behaviors. Acute activated Taotie-GAL4 neurons by TRPA1 dramatically increased food intake, suggesting that the Taotie-GAL4 labeled neurons participate in regulation of food intake of adult Drosophila. After watching this video, you should have a good understanding of how to quantify the amount of food intake after manipulating the activity of neurons to investigate neuro mechanisms of feeding control in adult Drosophila melanogaster.
