Modified Fear Conditioning for Inducing Flight Behaviors in Mice

Takafumi Furuyama; Ryo Yamamoto; Nobuo Kato; Munenori Ono

doi:10.3791/66266

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Modified Fear Conditioning for Inducing Flight Behaviors in Mice

Published: December 15, 2023

doi:

10.3791/66266

Takafumi Furuyama*¹, Ryo Yamamoto*¹, Nobuo Kato¹, Munenori Ono¹

¹Department of Physiology,Kanazawa Medical University

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 behaviors¹^,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.

Classical fear conditioning in rodents is commonly used as a model for PTSD⁵^,⁶^,⁷, but rodents do not express flight (panic-like) behaviors in this model⁸. Consequently, the classical fear conditioning model, often referred to as the 'rodent PTSD model,' lacks face validity for PTSD in humans, particularly in capturing flight or panic-like symptoms, which have not been well-studied.

Recently, several modified fear conditioning protocols have successfully demonstrated that rodent subjects exhibit flight behavior during these procedures. For instance, repetitive associations of a conditioned stimulus (CS) and an unconditioned stimulus (US) seven times in a day allowed female rats to exhibit darting behaviors similar to flight behaviors⁹. In two-day fear conditionings using serial compound stimuli (SCS; composed of tone followed by noise), mice began showing flight behaviors during the noise part of SCS presentations¹⁰^,¹¹^,¹². The detailed description of the SCS method is provided in a protocol report¹³. A three-day fear conditioning with SCS also worked for rats to induce flight behaviors¹⁴. However, these new protocols have some limitations. For example, the use of serial cue presentation does not allow for the exclusion of the influence of proximity estimation on defensive behavior. In the case of seven times association of CS-US in rats, the majority of flight responses were observed in females rather than males.

In light of these considerations, we introduce a modified fear conditioning protocol for mice to investigate flight behaviors in a fearful context. Male mice consistently exhibit flight behavior during our modified fear conditioning. In this protocol, the salient tone is used as the CS instead of SCS. Additionally, a minimum of five pairings of CS-US in a day for at least two days, along with fear potentiation by the conditioned context, is required. The protocol provides another option for investigating flight behaviors, complementing previous protocols, depending on the research purpose.

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

Group-house mice (3-4 per cage; maintained at 23-27 °C; under a 12 h light/dark cycle; provided ad libitum access to food and water) until the start of the experiments.
Individually house each mouse in a plexiglass cage (14 cm × 21 cm × 12 cm) for at least 3 days before undergoing this modified fear conditioning.

2. Setting up the tools/equipment

Fear conditioning box (Figure 1A)
1. Utilize a fear conditioning chamber (25 cm × 25 cm × 25 cm) enclosed in a sound-attenuating box (67 cm × 53 cm × 55 cm) (see Table of Materials).
2. Two contexts (A and B) are required. For context A, create black and white stripes on the walls by attaching white plastic cardboard with black tapes (3 cm width, 4 on a board). Use a white smooth plastic board for the floor.
3. Wipe the walls and floor with Heptanol (1%) before each session.
  NOTE: No active ventilation system is used. Cleaning with alcohol at the end of the session diminishes the Heptanol odor.
4. For context B, make the appearance of walls entirely black by removing the board used in context A. The floor is a grid floor.
  NOTE: No specific odor is presented other than a slight alcohol odor for cleaning.
5. Illuminate the experimental box using an overhead white light-emitting diode (LED, 240 lux) (see Table of Materials).
Shocker
1. Connect a scramble shocker (see Table of Materials) to a grid floor composed of stainless-steel rods. This is used to provide foot shocks. The foot shock intensity was set at 0.9 mA following the common methods of fear conditioning¹⁰^,¹¹^,¹²^,¹³.
Audio generator
1. Place a speaker (see Table of Materials) on the ceiling. All acoustic stimuli are amplified.
2. Digitally modify and calibrate the overall amplitudes of each stimulus to yield sound pressure levels (SPL, re: 20 µ Pa) at the 5 cm front of the speaker with a ¼ inch microphone. Present a continuous tone burst through this speaker.
  NOTE: Calibrating the sound speaker is crucial to examine the fine impact of sound stimulus on defensive behaviors during this modified fear conditioning.
Transducer
1. Place the floor of the test chamber on a transducer (see Table of Materials) for the detection of vibration.The signal from the transducer is transmitted to a sound card with an 8 kHz sampling frequency to record the behavioral vibrations.
Video camera
1. Position a CMOS camera (see Table of Materials) on the ceiling to track the subject's motions and record the sound in the conditioning box.
Triggering system
1. Use sound software (see Table of Materials) for triggering tones or foot-shocks at scheduled timings.
  NOTE: Any commercially available stimulator will work for this.

3. Behavioral experiment

Plan for four days of fear conditioning procedures: habituation (1 day, 5 trials), conditioning (2 days, 5 trials each), and test/extinction sessions (1 day, 5 or 15 trials). The intertrial intervals varied between 60-75 s (Figure 1B).
NOTE: Preferably, include ten or more subjects in a group to obtain reliable behavioral tendencies. Two or three groups are required depending on the study's purpose.
1. During the conditioning session, present the unconditioned stimulus (US) (1 s, 0.9 mA) immediately after the termination of the conditioned stimulus (CS) (continuous tone burst, 8 kHz, 20 s, 95 dB SPL) as shown in Figure 1B. Deliver five CS-US pairs in a conditioning day.
2. After the termination of the 5th foot-shock, leave the subject in the context for 1 min before returning it to the home cage. Wipe the chamber with 70% alcohol for cleaning following each behavioral test.
  NOTE: CS and US intensity can be modified depending on the study's purpose. Previous reports have shown that stronger CS intensity triggers more active defensive behaviors than softer ones¹⁵. Conditioning days can also be extended.
Induce flight behaviors during CS presentations following the schedule mentioned below.
NOTE: A CS (95 dB SPL) and a US (0.9 mA) are used in this experiment.
1. On day 1, expose subjects to 5 CS alone trials in context A.
2. On days 2 and 3, condition subjects with 5 CS-US association trials in context B.
3. On day 4, expose subjects to 5 CS alone trials for the recall session in context B. In case of testing memory extinction, expose subjects to 15 CS alone trials.
  NOTE: To test memory stability, extending extinction sessions for 2-3 days will help. Also, testing memory one week later instead of on day 4 can provide additional confirmation of memory stability.

4. Analysis of defensive behaviors

NOTE: Motion, percentage of freezing, and the number of jumps during CS presentations are analyzed. Details are described below. If possible, analyzing in a double-blind manner would be better.

Synchronize the timings of events in the video and the timings of stimuli (CS and US) by using the tone onset recorded in the video.
Utilize a custom-made code to calculate both the averaged and total motions of mice based on the difference in the center of mass of the subject silhouette across frames.
NOTE: An arbitrary unit is used for this measurement since the speed of motion depends on the sampling rate of the movie.
To measure the percentage of freezing, use the transducer signal in time.
1. Preprocess transducer signals using a 20-500 Hz band pass filter.
2. Calculate the root mean square amplitude of the transducer signal in time for each 50 ms bin.
3. Set a threshold for the signal amplitude to detect the immobility period. The duration of immobility is the signal period lower than the threshold for more than 1 s.
4. Manually measure the duration of freezing by watching the video.
5. Adjust the threshold of signal amplitude for freezing by comparing the manually measured percentage of freezing and the percentage calculated from the transducer signal.
Count the number of jumps manually from video files.
NOTE: Counting the number of darts will also be useful in assessing the flight response.

5. Statistical analysis

Set the statistical significance at p < 0.05.
For comparisons between multiple groups and multiple factors, conduct a multiway ANOVA followed by post-hoc tests. If a specific day of the conditioning schedule is tested, perform multiple comparison tests or permutation tests.

Representative 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) alone trials in context A. Following this, all mice were conditioned with 5 CS-unconditioned stimulus (US) trials in context B on days 2 and 3. On day 4, Group 1 experienced 5 CS-alone trials for the recall session in context B, while Group 2 was tested in context A.

Subjects in Group 1 displayed pronounced flight behaviors, such as jumping or short darting, particularly during CS presentations on days 3 and 4 (see Figure 2A,B). Both the total motions and the number of jumps during CS presentations increased with the progression of conditioning (Figure 2A,B). Freezing during CS presentations showed an increase on day 2 and remained relatively constant in subsequent trials (Figure 2B). The subjects exhibited heightened movements at the onset of the CS presentation and consistently demonstrated flight behaviors throughout the CS presentation (Figure 2A).

Subjects in Group 2 exhibited robust flight behaviors almost identical to those in Group 1 on days 2 and 3 (see Figure 2A). However, in context B on day 4, which was the unconditioned context, subjects in Group 2 did not display any flight behaviors during CS presentations (Figure 2A,B). Comparisons of motions during CS on day 4 showed that Group 1 possessed a significantly greater amount of motion than Group 2 (see Figure 2C; permutation test; G1 vs. G2, p = 0.014). Additionally, comparisons of freezing during CS on day 4 showed statistically significant differences between the two groups (see Figure 2D; permutation test; G1 vs. G2, p < 0.000). Regarding jumps on day 4, Group 1 exhibited more jumps than Group 2 (see Figure 2E; permutation test; G1 vs. G2, p = 0.034). These findings suggest that flight behaviors triggered by the tone during fear conditioning are context-dependent.

Figure 1: The design of the modified fear conditioning experiments. (A) Schematic representations of the experimental contexts A and B are shown. (B) The composition of CS and US presentations. The CS was an 8 kHz continuous tone burst (20 s) and the US (foot shock, 1 s) was delivered immediately after the CS termination. Inter-trial intervals were 60-75 s. (C) The schedule of modified fear conditioning experiments. The figure is modified from Furuyama et al.¹⁵. Please click here to view a larger version of this figure.

Figure 2: The contexts essential for the expression of flight behaviors. (A) Averaged motions of each condition around the CS presentation by day are shown. The gray-shaded periods represent CS presentations, and the red lines indicate US presentations. Gray lines indicate the standard error of means of each trace. On day 3, motions increased during CS presentations in G1 and G2. On day 4, the motion was increased during CS presentations in G1. (B, Motions) Averaged total motions during the CS presentation of each trial are plotted. (B, Freezing) Averaged percentages of freezing during the CS presentation of each trial are plotted. (B, Jumps) Averaged jumps during the CS presentation of each trial are plotted. G1 jumped during CS presentations on day 4. (C) Comparison of motions on day 4. G1 moved more than G2. (D) Comparison of percentage freezing on day 4. G2 showed more freezing than G1. (E) Comparison of the total number of jumps on day 4. G1 jumped more than G2. The horizontal red bars indicate the averages, and the vertical red bars indicate the SEM of each group in panels (C–E). *p < 0.05. The figure is modified from Furuyama et al.¹⁵. Please click here to view a larger version of this figure.

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 fearful context.

Recently, several protocols have been introduced in addition to the present protocol. Multiple days of conditioning with SCS stably induce flight behaviors during cue presentations in mice and rats¹⁰^,¹¹^,¹²^,¹³^,¹⁴. Also, seven repetitive CS-US associations in a day let female rats exhibit darting, a kind of flight behavior⁹. These protocols are all reliable, same as this protocol introduced here, although the protocols, including the present one, have advantages and disadvantages depending on the purpose of each study. For example, the subject can estimate the proximity of threats with the SCS presentation, which is composed of two serial stimuli followed by foot shock. If a study aims to investigate the pure effect of CS features on the expression of flight behaviors, the SCS protocol is not the best. However, with the SCS protocol, the transition between freeze and flight always occurs in a short period (in 20 s). Therefore, for a study that focuses on the transition from passive defensive behavior to active defensive behavior and vice versa, the SCS protocol works best. The protocol using seven times CS-US association works best for the study of the active defensive behaviors of female rats, while for male rats, some modification would be required.

This protocol uses salient pure tone presentation instead of the SCS; thus, this protocol is suitable for investigating the effect of various CS (tone with various envelopes or tone shapes such as ramping/damping) for triggering flight behaviors. We have demonstrated that at least the tone intensity, one of the CS characters, has a critical influence on the expression of flight behaviors¹⁵. Then, it is expected that various CS features would have different effects on flight behaviors. The most important point of our protocol is the calibration of the speaker for presenting tone stimuli. Often, commercially available speakers in fear conditioning boxes are not well calibrated, and the parameters are not reliable. It is strongly recommended to use a reliable speaker with fine calibration for this experiment. Regarding the conditioning days, it is possible to extend the number of training days by reducing the number of trials in a day. For example, the protocol introduced here used a schedule of five trials per day for two days. Instead of this, four trials per day for three days also works. The schedule could be modified depending on the purpose of each study.

Finally, the active defensive behaviors introduced in these protocols are different from the active defensive behaviors observed in the active avoidance (shuttle avoidance) experiment. The escape behavior during active avoidance is more habit-like, and once it is learned, the subject keeps escaping³^,¹⁶^,¹⁷, while the flight in this protocol looks like a panic behavior, and the subject stops exhibiting flight behaviors once it notices that no US follows CS¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵. Also, these panic-like flight behaviors are distinct from behavioral suppression reported in licking suppression in a fearful context¹⁸^,¹⁹, while both of those are fear-induced defensive behaviors. These panic-like flight behaviors have been overlooked and not well-studied. By using new protocols⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵, including the present one, the neural correlate for panic behaviors will be elucidated.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported partly by KAKENHI Grants JP22K15795 (to T.F.), JP22K09734 (to N.K.), JP21K07489 (to R.Y.), Kanazawa Medical University (C2022-3, D2021-4, to R.Y.) and The Naito Foundation (to T.F.).

Materials

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

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Furuyama, T., Yamamoto, R., Kato, N., Ono, M. Modified Fear Conditioning for Inducing Flight Behaviors in Mice. J. Vis. Exp. (202), e66266, doi:10.3791/66266 (2023).