The overall goal of this procedure is to assess neurodegeneration of dopaminergic neurons in transgenic drosophila. This is accomplished by first expressing the desired transgenes under the control of the tyrosine hydroxylase promoter via the GAL four UAS system. The second step of the procedure is to collect flies of the desired genotype, separate them by gender and age them.
The third step is to test the climbing activity of flies by negative geo axis as they age. The final step is to count the number of dopaminergic neurons from dissected brains collected at different ages. Ultimately, the results will show changes in the animal's locomotor behavior and in the size of their dopaminergic neuron population.
The implications of this approach extend toward the therapy of neurodegenerative diseases such as Parkinson's disease. This approach can help to identify neuroprotective genes and pharmacologic interventions To set up the assay, select age matched flies of the desired genotypes. Separate them by gender and randomly group them into cohorts of 20 to 30 animals.
Each cohort represents a statistical sample. Maintain the cohorts on a 12 hour light dark cycle to control the effects of circadian rhythms on the locomotor behavior, and at a temperature that can be replicated in the testing room. Change the cohort's vials every two to three days and test them weekly at a consistent time.
30 minutes before the test, anesthetize each cohort with carbon dioxide and transfer them to a labeled plastic tube made from empty food vials joined with tape. The flies should be awake after five to 10 minutes, but complete recovery from anesthesia varies with age and genotype and must be optimized.Now. Create a height scale to view behind the test vials.
Mark distances between one and 20 centimeters on a piece of white paper. Then secure the scale into position. Now align the tubes to be tested against the scale about 20 to 30 centimeters away.
Position a digital camera with a square view of the vials. Make sure the labels on the tubes and the scale are clearly in view. Once positioned, start the recording and start a digital timer.
Now tap a tube for a few seconds. All of the flies should collect the bottom of the tube. Be sure to do this step consistently with the same duration and same force throughout the whole experiment.
After a minute, repeat the knockdown again for the second of 15 consecutive trials. When the 15 trials are all completed, return the flies to fresh vials by visually inspecting the movies. For each of the 15 trials performed per cohort, count the number of flies above the two centimeter mark.
After 10 seconds have elapsed, the two centimeter mark is determined empirically, it is the distance that all of the controls cross after 10 seconds at many of the ages being tested. Take the average score from the 15 trials and express it as a percentage of cohort members that crossed the mark. The number of statistical repeats is equal to the number of cohorts tested for each group, not by the 15 trials.
Assess the statistical significance between different genotypes using a student's T-test, anova, or other appropriate methods of analysis. Put flies in a dissection dish with 70%ethanol for about one minute. To remove the cuticle wax.
Then transfer the flies to another dissection dish filled with ice cold PBS, and proceed with dissecting out the brain. Position the fly with the ventral side up and optionally remove the wings and legs. Use one set of forceps to hold onto the body, and another one to hold onto the cuticle under the eye.
Then pull the head away from the body. Next, remove the probos by pulling it off of the head. Then grasp the head cuticle on opposite edges of the new hole, and pull in opposite directions until the brain is removed from the head capsule.
Now delicately, remove the remaining cuticle from the brain surface. Then remove all of the air-filled tracheal tissue. This will prevent the brain from floating during the following incubation steps.
Prepare a brain handling pipette from a P 200 pipette tip. Cut off a few millimeters and equilibrate it with 0.1%Triton X 100 in PBS. Transfer the brains to a half milliliter tube completely filled with 4%PFA in PBS.
Fix the brains at room temperature for 20 minutes with gentle rotation. After 20 minutes, use a P 1000 pipette to replace the fixative with half a milliliter of washing buffer being 0.1%Triton X 100 in PBS. Mix by inversion and allow the brains to incubate for one minute before replacing the wash buffer.
After a minute, replace the wash buffer a second time and incubate the brains at room temperature for 20 minutes. Then replace the wash buffer for a third time and incubate the brains for another 20 minutes. Now, remove the washing buffer and let the brains incubate in half a millimeter of blocking buffer for one hour at room temperature.
After an hour, add a one to 100 dilution of antit antibody in blocking solution. Let it incubate for two days at four degrees Celsius with gentle rotation. To remove the block, repeat the washes used to remove the fix.
Then equilibrate the brains with half a milliliter of one to 200 dilution of secondary antibody in blocking solution for two days at four degrees Celsius with gentle rotation. A few days later, remove the secondary antibody using the same wash procedure. To prepare the mounting slides, add 2 25 microliter drops of mounting media to a cover glass.
Place two cover glasses on the mounting media with a gap in the middle. Allow the slides to dry. If the width of the mounting slide does not fit the stage of the confocal microscope, use nail polish to attach a 22 by 22 millimeter cover slip to the slide so it fits properly.
Now replace the solution that the brains are in with 50 microliters of mounting media. Equilibrate the brains with the mounting media by pipetting with a cut P 200 tip. Next, use a cut P 200 tip to transfer the brains to the trench.
In the mounting slide, orient the brains to view their anterior or posterior side. Then cover them with a number 1.5 cover slip and from one corner, fill the space between the cover glasses with mounting media to remove all of the air. Allow the slides to dry and then seal them with nail polish before their examination.
Human alpha-synuclein was expressed in DA neurons using the TH GAL four driver in the climbing assay. Males expressing this transgene exhibited an accelerated decline relative to age matched controls and to fly as coex expressing the NRF 2D NA binding partner. Math S females showed similar results comparing four week old male flies of different genotypes.
Th immunofluorescence based DA neuron counts were used to quantify the number of PPL one neurons. A small but significant loss of PPL one neurons was found in flies expressing alpha-synuclein. After watching this video, you should have a good understanding of how to assess neurodegeneration of the varg neurons in transgenic zoa by CLA assays and tyrosine aase immunofluorescence.
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