The overall goal of this procedure is to demonstrate methods of examining drosophila motor sensory and coordination behaviors. This is accomplished by first assessing lava motor function, using the crawling assay, assessing adult motor function, using the ring assay, and assessing adult coordination and sensory abilities with the courtship assay. Ultimately, this panel of behavioral assays can show how genetic and external factors like drug treatments can influence activity and coordination.
Intra sophala. The main advantage of this assay over existing methods like negative geo axis, is that the ring assay is higher throughput and more sensitive. This method can help answer key questions in the neurodegenerative diseases field, such as early stage detection of locomotive defects.
Demonstrating this assay will be Dr.Jamie Becknell, a postdoc for my laboratory. The larval crawling asay is used to measure the effect of genetic and environmental factors on motility to begin set up a cross of 10 to 15 males and 10 to 15 virgins in a standard bottle. After 24 hours, healthy adults will have laid enough eggs to populate the bottle to keep that population aged matched to 24 hours.
Clear the adults to a fresh bottle and repeat the process. Now incubate the bottle until third in star larvae are observed in the food to the bottle. Add 50 to 100 milliliters of 20%sucrose and let it sit for 20 minutes.
The larvae will float to the top, collect the larvae using a 25 milliliter serological pipette with the tip cutoff and place into a mesh basket. Wash the larvae in the mesh basket twice with deionized water. They can now be experimented upon.
To treat the larvae with a drug, use a brush to transport the desired number of larvae to a five milliliter beaker containing a solution of 5%sucrose and the drug of interest. Allow them to feed on the drugged sugar solution for 15 minutes, then pour them back through the mesh basket, rinse them, and proceed with measuring the drug effect on motility. Use a brush to transport an individual lava to a 15 centimeter Petri dish containing 2%aros.
Set over a sheet of graft paper. Now collect the data. Simply count the number of grid lines the lava crosses in one minute.
Next, use the brush to transfer the lava to the well of a glass dissection dish containing a dilute yeast paste solution under a dissection microscope over a one minute period, count the Percys contractions. A single contraction is counted as a full posterior to anterior movement. Repeat until the desired numbers of larvae have been counted.
The ring protocol measures locomotive speed of adult animals collect newly emerged adult male flies under light anesthesia and transfer them into a standard file containing normal or drugged food. Put approximately 25 flies per vial. Maintain flies at room temperature for two to three days for a full anesthesia recovery and accumulation of steady state drug levels.
Then transfer about 25 flies without anesthesia to polystyrene vials with molded plugs, and assemble the vials into one or more ring apparatus. Allow the flies to rest for 15 to 20 minutes while aligning a digital camera to photograph the vials from a side view, have a timer ready and set it to the desired duration for the assay. Typically three seconds to perform the assay.
Hold the ring apparatus in one hand without disturbing the flies and hold the camera timer in the other hand sharply. Tap the apparatus on the bench three times to knock down the flies and simultaneously start the camera's countdown timer. Repeat the essay once per minute until five or six trials have been performed.
It is critical not to reuse the polystyrene testing vials after the initial datasets have been gathered. Because new flies placed into used vials will not climb to the same extent as flies placed into fresh vials. To collect the data using an image viewer score the mean height, climb by the different groups.
This assay requires prepared genetic crosses in bottles that are producing abundant progeny to collect flies for the assay. Within a couple of hours of the animal's subjective, dawn clear the bottles to collect all adults at three to four hour intervals. Collect newly emerged sexually naive males and females house the males individually in vials or tubes with food house the females in groups of five to six.
In similar enclosures, equal numbers of males and females are required for the assay. After isolating the collected flies for five days, prepare to load them into a mating wheel and conduct the assay. Next, carefully transfer one female and one male into the chamber of a mating wheel under a dissection microscope.
Observe the pair for 10 minutes or until they successfully copulate during that time. Record when each of several behaviors is first observed. Record when the male first turns towards the female record.
When the male first touches the female with its leg record, when the male first extends and vibrates a wing record when the male first licks the female genitalia record When the male first curls its abdomen under itself. Record when the male first attempts to mount the female email. Finally, record the total time engaged in courtship behavior until copulation occurs.
For wild type animals, this is almost always within five to 10 minutes, collect data by three different parameters. First, the latency to a behavior is the time it takes before it is first observed. Second, the frequency of a behavior is whether or not it is observed.
And third, the courtship index is the time spent in courtship divided by the total time and till copulation in an experiment with animals. Harboring mutations in F-U-S-T-L-S related a myotrophic laterals sclerosis. Gene mutants with an insertion center showed half the crawling ability of wild type controls left and an R 5 21 C missense mutation dropped crawling speed to only about one centimeter per minute.
Ring testing of young wild type adult flies resulted in an average height, climbed between three and five centimeters over a three second time period. In this assay, no desensitization was observed as up to the five consecutive trials spaced one minute apart While attempting the ring assay. It's important to remember to be as precise as possible with regards to timing.
Once mastered, the larval crowding essay can be done in 30 to 40 minutes to observe 10 trials for one genotype if it is performed properly. After watching this video, you should also have a good understanding of how to observe and score courtship behaviors in the fly.
