Methods to Assay Drosophila Comportamento

1. Larval Crawling Assay

1. Larvae Collection

1. Set up an 8 ounce bottle of flies (10-15 males + 10-15 females).
2. Let flies lay eggs for 24 hours, then clear bottle of flies. (Transfer the adults into a new bottle and repeat as necessary).
3. Incubate bottle for 3-4 days, or until third instar larvae are visible.
4. Add 50 – 100 ml of 20% sucrose to the bottle with larvae and let sit for 20 minutes. Larvae will float to the top.
5. Collect larvae using a 25 ml serological pipette with the tip cut off, and place into a mesh basket.
6. Wash larvae in the mesh basket two times with deionized H₂O. Larvae are now ready for the experiments.

2. To treat larvae with drug

1. Use a brush to transport the desired number of larvae to a 5 ml beaker containing a solution 5% sucrose + drug.
2. Let larvae feed for at least 15 minutes.
3. Pour drug-treated larvae into a mesh basket and rinse. They are now ready to use.

3. Locomotor Assay (measuring total distance travelled or body wall contractions)

1. Use a brush to transport individual larva to a:
   1. 15 cm Petri dish containing 2% agarose (previously poured and allowed to harden) over graph paper with a 0.2 cm² grid.
      1. Count number of grid lines crossed in 1 minute.
   2. Well of a glass dissection dish containing a dilute yeast paste solution.
      1. Count peristalsis contractions (full anterior to posterior movement = 1 contraction) in one minute while observing under a dissection microscope.
2. Repeat until the desired numbers of larvae have been counted.

2. Rapid Iterative Negative Geotaxis (RING) Protocol

This assay was originally described by Gargano et al⁵.

1. Collect newly emerged adult male flies under light CO₂ anesthetization and place into a standard vial containing food (or food + drug).
2. Maintain flies at room temperature (on the bench top. ~22 °C) for 2-3 days to allow for recovery from CO₂ (and accumulation of steady-state drug levels if appropriate).
3. Transfer about 25 flies without anesthetizing to prepared polystyrene vials.
4. Assemble vials with flies into the RING apparatus (Figure 1).
5. Allow flies to acclimate to the environment, undisturbed, for 15-20 minutes.
6. During this time place digital camera ~1 m in front of the apparatus (on a platform if necessary to align the center of the lens at mid-height of the vials), focus and zoom the camera onto the apparatus, and set a timer to 3.0 seconds.
7. Carefully take hold of the RING apparatus with your left hand so as not to disturb the flies, and hold the timer with your right hand.
8. Sharply tap the apparatus down on the surface of the bench three times, ensuring that the tap is hard enough to knock down all the flies to the bottom of the vials.
9. Simultaneously with completion of the third tap, start the 3 second countdown timer.
10. At three seconds take a picture.
11. Reset the timer for 1 minute and start. During this time reset the camera and focus on the apparatus, and set another channel of the timer for three seconds.
12. After 1 minute, repeat steps 1.7-1.10
13. After a total of 5-6 trials, upload images onto a computer and use your favorite image viewer to open, and score the average height climbed for each vial.
14. Perform statistical analysis on your different groups comparing the mean height climbed.

3. Courtship and Mating Assay

1. First thing in the morning, clear well-producing bottles of flies to be used.
2. Over the course of the day (every 3-4 hours), collect newly emerged sexually naive males and females:
   1. Place males individually in vials or tubes with medium.
   2. Place 5-6 females together per vial/tube.
   3. Isolate collected flies at 25 °C under 12 hr light/dark for 5 days.
   4. Transfer one female into the chamber of a mating wheel.
   5. Transfer one male into the chamber of a mating wheel.
   6. Observe pair under a dissection microscope for the following behaviors:
      1. Orientation (the male orients towards the female)
      2. Tapping (the male taps the female)
      3. Wing song (the male extends and vibrates one wing)
      4. Licking (the male licks the female genitalia)
      5. Curling (the male curls its abdomen under itself)
      6. Copulation attempt (Curling activity while attempting to mount the female)
3. Observe for 10 minutes or until successful copulation, noting the time at which each behavior occurs (latency), the total time engaged in courtship behavior until copulation (to calculate the courtship index), as well as the number of pairs that successfully perform a given behavior (frequency). 100% of wild type pairs will generally mate within 5 minutes.
4. Calculate a courtship index (CI) by dividing the time spent in courtship divided by the total time until copulation. For wild type pairs this usually ranges between 0.6-0.8.

4. Representative Results

Crawling assay

Normal wild type larva will wander ~3 cm/minute, and exhibit ~40-50 body wall contractions in one minute. We have recently developed a Drosophila model of FUS/TLS-related amyotrophic lateral sclerosis that shows larval crawling defect, reduced life span and adult climbing impairment⁶. We targeted expression of wild type and mutant forms of FUS/TLS to the motor neurons (OK-371-gal4 driver) and performed a larval crawling assay. As shown below, wild type larvae crawl up to 12 cm, whereas expression of wild type FUS decreased the larval crawling ability to approximately 6 cm. Animals expressing the ALS-causing mutation R521C in FUS/TLS demonstrate a very severe impairment in their crawling movement (Figure 1), crawling only about 1 cm/minute.

Negative geotaxis RING assay

Young wild-type adult flies should have an average climbing height of ~4-5 cm in a 3-second time period (the time can be adjusted from 3 seconds to accommodate different strains or activity levels to define a given average height for a given strain/treatment). Flies that remain at the bottom are assigned a value of 0. It is not advised to use more than 25 flies per vial because it then becomes difficult to determine the location of each individual fly to measure height. No desensitization has been observed at up to the 6 consecutive trials spaced 1 minute apart that we have employed. It is critical NOT to reuse the polystyrene testing vials in this assay after the initial sets of data are gathered because new flies placed into used vials will not climb to the same extent as in fresh vials.

Figure 1. Setup for the RING Assay. The digital camera is placed ~1 m in front of the apparatus containing flies in the polystyrene vials; focus and zoom the camera onto the apparatus; and set a timer to 3.0 seconds.

Figure 2. Representative data from the larval crawling assay using flies ectopically expressing UAS-FUS WT, and UAS-FUS R521C under the control of a motor neuron driver (OK371-gal4).