The overall goal of this automated rapid iterative negative geotaxis assay is to analyze Drosophila adult climbing deficits associated with neurodegeneration. This method can help to answer key questions in the neuroscience field such as the mechanism of locomotive deficits associated with neurodegeneration. The main advantage of this technique is that fly climbing activity can be analyzed in an automated and high proof profession.
The implications of this technique extends towards the therapy and diagnosis of neurodegeneration, because neurodegenerative diseases are frequently associated with a progressive loss of movement ability and reduced lifespan and age dependent neurodegeneration. We first had the idea for this method. We wanted to measure Drosophila adult's locomotive activity in a more accurate fashion.
To carry out fly collection, begin by maintaining the flies on standard fly food at 25 degrees Celsius, 70%humidity and to 12 hour light, 12 hour dark cycle. Collect UAS-Dicer2 DDC-GAL4 fly virgins under carbon dioxide anesthesia. Cross the virgins to two-day old adult male flies carrying the following genotypes, with a male/female ratio of one-to-two.
Separately collect newly occlosed males and females in three vials for each group per experiment, placing 10 flies in each regular fly vial with standard food. Then set the vials to 25 degrees Celsius. To perform the automated RING assay, transfer 10 collected unisex flies per genotype into each vial.
Then secure the vial with a screw. Analyze control flies and flies carrying different genotypes together for each set of experiments. Turn on the digital camera placed in front of the apparatus, and once the flies are loaded and ready, start recording.
The camera should be placed horizontally and the camera a proper distance from the RING apparatus to capture the positions in a relatively accurate locations of all the flies. After allowing one minute for the flies to settle in the vials, turn on the step controller that controls the step driver. This drives the small electric motor to control the attached lever, so that it consecutively rises and taps the apparatus four times in two seconds.
After tapping the apparatus, note that the flies begin to ascend the wall. With recording still on, repeat the synchronization as just demonstrated, in 60 second intervals for three to five consecutive trials. Repeat the experiment for five, 15, 25 and 35 day old flies.
Conduct at least three independent experiments for each group for each experiment, with at least 30 collected flies or three vials. To analyze the data, import the recorded video into the computer. For each trial, take a snapshot of the video at six seconds after tapping.
In the R-fly detection software, use the file menu to import the snapshot image. Next, using the baseline icon on the Toolbar, precisely set the upper and lower baselines of the vial and then use the cursor to mark the upper and lower baselines on the image. Within the Settings bar, in the Flies in Rect field, input the number of flies per vial, and in the Tube Height field, input the vial length.
Individual fly positions are detected and labeled with dots on the screen for each vial. Note that in a table on the right hand panel, the software automatically determines the climbing distance for each fly and displays the averaged values from 10 flies in each vial. Present the data as the mean plus or minus SEM.
Calculate the P-values of significance using a one-way ANOVA with a Bonferroni multiple comparison test. Analysis of Dicer2 designated flies using the automated RING assay presented in this video showed an age dependent decrease in the climbing distance in a six-second timeframe for both male and female flies, suggesting that aux expression in dopaminergic or DA neurons is crucial for fly locomotor activity. Climbing ability does not seem to be affected upon auxGFP over expression in DA neurons and co-expression of auxR and auxGFP in DA neurons rescued from the RNAi phenotype.
Once mastered, this can be done is two to three hours if it is performed properly. Having this development is now a way for researchers in the field of neuroscience to explore neurodegeneration in Drosophila melanogaster. Don't forget that working with electric RING apparatus can be extremely hazardous and each confirm should be fastened tightly and carefully checked before performing this procedure.
