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.
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.
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.
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
2. Setting up the tools/equipment
3. Behavioral experiment
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.
5. Statistical analysis
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.
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.
The authors have nothing to disclose.
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.).
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 |