Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a simple behavioral assay that allows for studying adverse associative learning and memory consolidation in flies, including effects of gene environment interactions. The assay presented here is a simple, reproducible, and cost effective procedure for studying memory mechanisms that can be easily assembled with a few available supplies. This assay could provide insight into basic mechanisms underlying learning and memory impairments resulting from various genetic, pharmacological, and dietary manipulations.

Demonstrating this procedure will be Morgan Tedder and Steven Bradley, undergraduate students from my laboratory. To begin, drill a four millimeter hole perpendicularly about four millimeters from the bottom within a 14 milliliter polypropylene culture tube. Remove the upper part of the culture tube to make a 45 millimeter bottom fragment utilized as the lower compartment.

Cut off the top of a 1000 microliter pipette tip to create a 12 millimeter fragment. Insert the fragment into the four millimeter hole of the culture tube as a loading dock to transfer the flies. Cut a 15 millimeter piece of transparent vinyl tubing and insert the lower and upper compartment from opposite ends into the tubing.

Attach the assembly vertically using a two-prong adjustable clamp. Locate the upper compartment vertically attached with shock tubes. Connect the shock tubes with an electrical stimulator to generate electric shocks.

Using an ice cold block, immobilize three-to-four-day-old flies and transfer them into separate vials with food 24 hours before the experiment. Assign a code to all the vials as described in the text manuscript. Gently suck one fly into the mouth aspirator by drawing air.

Deposit the fly by lightly blowing into the loading dock and immediately start a one-minute timer and stopwatch. Turn on the stimulator and deliver an electric shock when the fly enters the shock tube. Press the stopwatch to record the first latency.

If the fly reenters the shock tube, deliver additional shocks. Using a tally or an Arduino-based counter, record the number of shocks during a one-minute trial. Gently transfer the fly back into the vial at the end of a one-minute trial.

Record the latency, the number of received shocks, and any notable change in behavior. Using 70%ethanol, clean the lower and shock compartment. Wipe with a lint-free cleaning tissue and dry it using a hair dryer.

Repeat the trial with the next fly. Clean the lower compartment with water and odorless detergent after completing the experiment. Using 70%ethanol, wipe the lower and shock compartment and air dry overnight.

By repeating the above process after 24 hours, perform a second trial. In a similar sequence, test the flies. After performing a passive avoidance assay in Drosophila melanogaster male flies, results have shown that the latency increases, the number of shocks decreases, demonstrating that flies learned association between the upper compartment and an electric shock.

Frequency distribution revealed that most flies enter the upper compartment and receive one to three shocks in the first trial. By the fourth trial, most flies do not enter the upper compartment and receive zero shocks. In subsequent experiments, the trial duration was reduced to one minute because the frequency distribution for latencies clearly showed that if the fly does not enter the upper compartment within 60 seconds in the first trial, it usually does not enter at all.

The passive avoidance assay experiments were repeated in Drosophila simulans male and female flies. Results have shown that the flies were effective at learning the passive avoidance behavior as evident from the changes in frequency distributions for latencies and shocks in male and female flies. Comparison of latencies and number of shocks between male and female did not reveal any statistically significant differences.

However, female flies received slightly more shocks in each trial. Analysis of grooming bouts in trials two and three revealed that the total duration of grooming decreases significantly in female flies from trial one to trial three. This suggests that flies experience behavioral stress which parallels rodent anxiety-like behaviors.

Comparison of the effects of the Western diet and flight exercise on passive avoidance behavior show that the Western diet decreases the latency and increases the number of shocks indicating that the Western diet impairs aversive associative learning in flies. Conversely, flight exercise mitigated the negative effect of the Western diet. When attempting this procedure, remember not to stress flies.

Stressed flies would either enter the upper compartment too quickly or won't enter at all. All experiments should be performed under the same environmental conditions and at the same time of the day. This technique gives researchers a new simple assay that can be used to dissect intrinsic mechanisms of learning and memory consolidation in flies.