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 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. 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. Cut off the tip of 1,000 µL blue pip…

Representative 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 b…

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

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