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 conditionin....

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 ....

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

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