Modified Fear Conditioning for Inducing Flight Behaviors in Mice

Summary

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

Abstract

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Introduction

The appropriate selection of defensive behaviors under threatening circumstances is crucial for the survival of all animals. Defensive behaviors gradually shift from one to another based on threat proximity, such as the transition between freezing and flight behaviors^1,2,3. Dysregulation of these behaviors is often observed in various mental disorders⁴. Post-traumatic stress disorder (PTSD) is one such disorder characterized by exaggerated defensive behaviors, like panic responses to non-threatening stimuli⁴.

Protocol

This protocol was conducted in accordance with the guiding principles of the Physiological Society of Japan and received approval from the Animal Care Committee of Kanazawa Medical University (2021-32). All procedures were conducted in compliance with the ARRIVE guidelines. Adult male C57BL/6J mice (3-6 months old) were utilized for the study, and it was previously confirmed that these mice exhibited the flight behaviors described in this manuscript¹⁵.

1. Animal preparation

1. Group-house mice (3-4 per cage; maintained at 23-27 °C; under a 12 h light/dark cycle; provided ad libitum acce....

Results

Results obtained with the modified fear conditioning in male mice (C57BL/6J; 3-6 months old) are presented, following the schedule shown in Figure 1C. The experiment was designed to investigate how the conditioned context influences the expression of flight behaviors. Two groups were assigned: Group 1 (n = 10) and Group 2 (n = 10). A CS (95 dB SPL) and a US (0.9 mA) were used in this experiment.

On day 1, all mice underwent exposure to 5 conditioned stimulus (CS) .......

Discussion

The modified fear conditioning protocol introduced in this article is a stable method for investigating flight behaviors in a fearful context. By employing this protocol, we have found that the flight behaviors of mice in the fearful context are triggered by salient stimuli and depend on the context. The characteristics of flight behavior were not well-investigated, as there was no suitable protocol to observe flight behaviors. This protocol will be one of the suitable methods for studying active defensive behaviors in a.......

Materials

Name	Company	Catalog Number	Comments
Audio speaker	Fostex	FT17H
Amplifier	Sony	TA-F500
CMOS camera	Sanwa Supply Inc.	CMS-V43BK
Fear conditioning chamber	Panlab S.L.U.	LE116
Food pellets	Nosan	Labo MR standard
LED	Yamazen	LT-B05N
Microphone	ACO	type 4156N
Scramble shocker	Panlab S.L.U.	LE 100-26
Sound card	Behringer	UMC202
Sound software	Syntrillium Software	Cool Edit 2000
Transducer	Panlab S.L.U.	LE 111