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

Summary

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

Abstract

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Introduction

Learning and memory is an evolutionarily ancient adaptation mechanism to the environment, conserved from Drosophila (D.) to human¹. The fruit fly is a robust model organism to study fundamental principles of learning and memory as it offers a wide range of powerful genetic tools to dissect intrinsic molecular mechanisms². The pioneering genetic screening studies, which identified rutabaga³, amnesiac⁴, and dunce⁵ genes critical for learning and memory², took advantage of olfactory conditioning as the fruit flies rely on their keen sense of smell to find food, potential mates, and to avoid predators⁶.

Olfactory conditioning has become a popular paradigm to study the mechanism of learning and memory, thanks to the introduction of olfactory T-maze by Tully and Quinn^7,8. Subsequently, other methods to measure various types of learning and memory have been proposed, including visual conditioning⁹, courtship conditioning¹⁰, aversive phototaxis suppression assay¹¹, and wasp-exposure conditioning¹². However, most of these assays have a complex setup that must be custom-built at a university workshop or purchased through a vendor. The paradigm described here is based on a simple behavioral assay to study aversive associative learning in flies that can be easily assembled with a few available supplies.

The described paradigm is equivalent to passive (or inhibitory) avoidance behavior in laboratory mice and rats in which animals learn to avoid a compartment where they have previously received electric foot shock¹³. In murids, the procedure is based on their innate avoidance of bright light and preference for darker areas¹⁴. On the first trial, the animal is placed into the bright compartment, from where the animal quickly exits, stepping into a dark compartment, where an electric foot shock is delivered. Usually, a single trial is sufficient to form a solid long-term memory, resulting in significantly increased latency 24 h later. The latency is then used as an index of the ability of the animal to remember the association between the aversive stimulus and the specific environment¹⁵.

This work describes an analogous procedure using D. as a model system which offers several advantages over rodent models including cost-effectiveness, larger sample size, the absence of regulatory oversight, and access to powerful genetic tools^16,17. The procedure is based on negative geotaxis behavior, which manifests in flies' urge to climb up when they are placed on a vertical surface¹⁸. The setup consists of two vertical chambers. On the first trial, a fruit fly is placed into a lower compartment. From there, it usually exits within 3-15 s, stepping into the upper compartment where it receives an electric shock. During a 1 min trial, some flies may occasionally re-enter the upper compartment, which results in an additional electric shock. During the testing phase, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first day indicating that flies formed aversive associative memory about the upper compartment. The latency, number of shocks, and the duration and frequency of grooming bouts are then used to analyze the animal behavior and the ability to form and remember the association between the aversive stimulus and the specific environment. The representative results reveal that exposure to the Western diet (WD) significantly impairs passive avoidance behavior in male flies, suggesting that the WD profoundly impacts the fly's behavior and cognition. Conversely, flight exercise alleviated the negative effect of the WD, improving passive avoidance behavior.

Protocol

1. Preparation of passive avoidance apparatus

1. Drill a 4 mm hole perpendicular to the wall surface of the 14 mL polypropylene culture tube and 8 mm away from the tube bottom.
   NOTE: Use an electric drill and 5/32 drill bit for best results.
2. Using a steel utility knife, cut off the upper part of the 14 mL polypropylene culture tube to create a 45 mm long tube bottom fragment. The bottom fragment serves as the lower compartment.
3. Cut off the tip of 1,000 µL blue pipette tip using a single edge razor blade to make the opening wide enough for a passage of a single fly. Cut off the narrowing part of the blue tip to create a 12 mm fragment. Insert this fragment firmly into a 4 mm hole of the lower compartment. This is used as a loading dock for transferring the flies.
4. Cut a 15 mm piece of transparent vinyl tubing 5/8" ID (see Table of Materials) to create a coupling. Insert upper and lower compartment into the coupling from opposite ends to securely attach the lower compartment to the upper compartment.
5. Using a 2-prong adjustable clamp, attach the assembly to a vertical stand. Orient the assembly vertically with the shock tube as an upper compartment.
6. Connect the shock tube wires to an electrical stimulator (see Table of Materials) to deliver electric shocks. The duration of the training period is 1 min.
   ​NOTE: To facilitate observation, position a piece of white paper behind the shock tube to serve as a white background of the apparatus. Put a lamp with a 75 W equivalent soft light bulb above the shock compartment. Place an adjustable arm magnifier lamp in front of the setup. A representation of the passive avoidance apparatus is shown in Figure 1.

2. Preparation of the flies for the passive avoidance procedure

1. Immobilize 3-4 day old D. melanogaster or D. simulans flies using ice-cold block and transfer them into individual vials with food 24 h before the experiment (1 fly per vial) following the procedure described previously¹⁹.
   NOTE: The experiments described here compared 3-4 day-old male vs. female flies in D. melanogaster and D. simulans.
2. Before the behavioral experiments, code all the vials. For this, assign each group a letter, for example, "A", "B", "C", etc., and each fly a number. Reveal this code only after all data have been recorded and analyzed. Use at least 20 flies per genotype or treatment to counter individual variations.
   ​NOTE: Performing the experiments and analyses "blind" allows excluding a bias in assessing the performance of the fly and data analysis.

3. Performing the first trial

1. Using a fly mouth aspirator (see Table of Materials) described previously^20, gently transfer a fly from the individual vial into the lower compartment via the loading dock. Gently suck one fly into the mouth aspirator by sucking air. Deposit the fly by lightly blowing into the loading dock.
   NOTE: Avoid stressing the animal during catching and loading.
2. Immediately after the fly is loaded into the lower compartment, start a 1 min timer and stopwatch.
   NOTE: The stopwatch is used to measure latencies and tally counter to count the number of shocks.
3. Press the stopwatch to record the first latency when the fly enters the shock tube by placing all paws on the grid. Turn on the stimulator to deliver an electric shock to the fly. The stimulation parameters are 120 volts, 1000 ms duration, 1 pulse/s (PPS), train duration 2000 ms.
4. Deliver additional shocks if fly re-enters shock tube. Record the number of received shocks during a 1 min trial with a tally counter or an Arduino-based counter (see Table of Materials). If using the Arduino-based counter, please follow the steps below.
   NOTE: An optional Arduino-based device AKM-007 (see Table of Materials) can be used to measure time, latency, the number of shocks, and the frequency and duration of grooming bouts for each animal by pressing and releasing the corresponding buttons on the device. The buttons on the device are assigned to measure latency, administer and record the number of shocks, and measure the frequency and duration of grooming bouts.
   1. Press the Start button at step 3.2., and press the Shock button at step 3.3.
   2. To record the duration of a grooming bout, press the Grooming button at the beginning of a grooming bout on the device and release this button at the end of the grooming bout.
      NOTE: The grooming bouts were measured throughout 1 min trial. Extensive grooming could be indicative of animal stress^21,22. The Arduino-based device saves all data as CSV file to a memory card.
5. At the end of a 1 min trial, gently transfer the fly back to an individual vial. Write down the latency, the number of received shocks, and any notable changes in the behavior.
6. Clean the lower and shock compartment with 70% ethanol, wipe with a lint-free cleaning tissue (see Table of Materials), and dry with the hairdryer. Repeat the trial with the next fly.
7. After the behavioral experiments, clean the lower compartment with water and odorless detergent. Wipe the lower compartment and the shock compartment with 70% ethanol, and air-dry overnight.

4. Performing the second trial

1. Perform the second trial by repeating the procedure described above (step 3) 24 h later. Test the flies in the same sequence as in the previous day.

5. Analysis of the results

1. Calculate the average latency, the average number of shocks, and duration of grooming bouts for trial 1 and trial 2 for each experimental group of animals. Perform student t-test for two-group comparison or ANOVA for multiple comparisons with post-hoc analyses using Tukey's test²³.

Results

The passive avoidance was studied in D. melanogaster (Canton-S) and D. simulans. The experiments compared the latencies and number of received shocks between consecutive trials. Initially, the experiments were performed with 3-4 day old male D. melanogaster flies. Flies were maintained on the standard Bloomington Formulation diet in a climate-controlled environment at 24 °C under a 12 h light-dark cycle, 70% humidity, and controlled population density. The density was controlled by kee...

Discussion

Avoidance of threatening stimuli is a crucial characteristic of adaptive behavior in various species from C. elegance to human³². Avoidance learning procedures which typically entail the escaping of an aversive event, are commonly used behavioral tasks to investigate learning and memory processes in laboratory rodents¹³ since the 1970's³². In active avoidance procedures, an indifferent stimulus or conditioned signal (CS) is followed by a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bloomington Formulation diet	Nutri-Fly	66-112	Available from Genesee Scientific Inc., San Diego, CA
1000 µL Blue tip	Fisher	NC9546243
17 x 100 mm 14 mL polypropylene culture tube	VWR	60818-689
Aduino-based Automatic Kontrol Module	In-house	AKM-007	This unit is optional. Complete description, schematics, wiring diagram and a code are provided at the ECU Digital Market - https://digitalmarket.ecu.edu/akmmodule
Dual-Display 2-Channel  Digital Clock/Timer	Digi-Sense	AO-94440-10	https://www.amazon.com/Cole-Parmer-AO-94440-10-Dual-Display-2-Channel-Jumbo-Digit/dp/B00PR0809G/ref=sr_1_5?dchild=1&keywords=Dual-Display+timer+jumbo&qid=1627660660&sr= 8-5#customerReviews
Electronic Finger Counter	N/A	N/A	https://www.amazon.com/gp/product/B01M8IRK6F/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1
Fisherbrand Sparkleen 1 Detergent	Fisher Scientific	04-320-4
Fly mouth aspirator	In-house		Prepared as described in reference 19.
Grass S88 stimulator	N/A	N/A	Could be replaced with any stimulator which can provide described parameters
Kim-wipes	Fisher Scientific	06-666	Kimberly-Clark Professional 34120
Metal block for fly immobilization	In-house		4 x 13 x 23.5cm aluminum block
Nutiva USDA Certified Organic, non-GMO, Red Palm Oil	Nutiva	N/A	https://www.amazon.com/Nutiva-Certified-Cold-Filtered-Unrefined-Ecuadorian/dp/B00JJ1E83G/ref=sxts_rp_s1_0?cv_ct_cx=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&dchild=1&keywords=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&pd_rd_i=B00JJ1E83G&pd_ rd_r=f35e9d2f-afe4-44b6-afc2-1c9cd705be18&pd_rd_w= R3Zb4&pd_rd_wg=eUv1m&pf_rd_ p=c6bde456-f877-4246-800f-44405f638777&pf _rd_r=M94N11RC7NH333EMJ66Y &psc=1&qid=1627661533&sr=1-1-f0029781-b79b-4b60-9cb0-eeda4dea34d6
Shock tube	CelExplorer	TMA-201	https://www.celexplorer.com/product_detail.asp?id=217&MainType=110&SubType=8
Stopwatch	Accusplit	A601XLN	https://www.amazon.com/gp/product/B0007ZGZYI/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1
Transparent vinyl tubing (3/4” OD, 5/8” ID)	Lowes		Avaiable from Lowes