Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

The overall goal of this procedure is to quantify the climbing ability of wild type flies and flies mutant for the genes involved in locomotion. This is accomplished by first transferring the flies into a glass graduated cylinder. The second step is to displace the flies to the bottom of the cylinder by lightly tapping the cylinder on foam padding.

Next flies crossing the target line are recorded using a video camera for a duration of two minutes. The final step is to analyze the videos and plot the number of flies above the target line every 10 seconds. Ultimately, the climbing assay is used to identify defects in climbing ability of mutant lines of fruit flies.

The main advantage of this technique, or existing techniques, such as the wild method, is that it has the ability to detect minor differences in climbing ability. This method can help answer key questions in the field of neurodegenerative disorders, such as the role of specific genes on hereditary spastic paraplegia on progressive gait impairment. The implication of this technique is tend to toward the development of therapy.

IP for neurological disorders because it provides with a simple quantitative assay for testing drug response and mutants for candidate neurodegenerative genes Begin by anesthetizing 20 flies using carbon dioxide and place all the flies in a 25 millimeter by 95 millimeter collection vial that contains food. Then store the vial containing the flies horizontally in a cardboard tray to avoid trapping them in any liquid that may accumulate in the bottom of the vial. Place the flies in an incubator for at least 21 hours under a 12 hour light dark cycle the following morning, set up the climbing assay by transferring the flies from the vial into a 250 milliliter glass graduated cylinder, and seal the top with wax film to prevent the escape of any flies.

Conduct the experiment in ambient light at 22 degrees Celsius and 40%humidity. Always perform experiments at the same time of day to avoid confounds with sleep cycles. Also, mark the location of the cylinder on the benchtop to keep it constant every day.

Count the number of dead flies at the bottom of the cylinder and in the food vials, record this number as the mortality. Next, set up the video camera on a tripod. Focus the camera on the 190 milliliter line of the 250 milliliter graduated cylinder very lightly.

Tap the cylinder five to 10 times against a closed cell foam pad with enough force to displace the flies to the inner bottom surface. Then use the other hand to press record on the camera. Then tap the cylinder six times in a distinct non rhythmic pattern while the camera is recording.

Conduct each trial for two minutes from the time the flies are last tapped. Finally, dispose of the flies in 95%ethanol. Once the trial is complete, repeat this climbing assay 10 times using 20 fresh flies each time in order to detect small differences in locomotion between groups.

Wash the cylinders in a dishwasher and dry overnight to be reused once the experiment has been completed to analyze fly performance, begin by watching videos of each fly trial record. The total number of flies that pass the target line every 10 seconds. If a fly climbs back down or falls, record that fly as minus one and count the next fly to cross the target line as the same number as the fly that climbed back down or fell.

For example, if the 15th fly falls below the target line, the next fly to cross the line is considered the 15th fly and not the 16th. Then subtract the mortality from the total number of flies to obtain the number of flies that remain in the trial plot. The percentage of flies above the target line at each time point when working with severe mutations, different degrees of climbing defect can be seen using different climbing assays.

Here, the number of eight day old flies that climbed to the top of an empty food vial after 18 seconds was recorded showing no significant difference in climbing behavior between the two groups. On the other hand, by using the climbing assay from this protocol, the Bastin 17 dash seven over TM three mutant flies show a deficit in performance compared to the controls. Since many locomotive disorders are progressive, it is important to observe evolution of the flies over time.

At two days old, there is a significant difference in the number of flies that climb above the target line. Compared to controls. This climbing performance continues to decrease as the mutant flies age from two to days.

Once mastered, this experiment and analysis can be completed in three hours. After watching this video, you should have a good understanding on how to quantify locomotive defects in dola mutant models for neurological disorders.