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.).

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 p...

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

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The authors declare no competing interests.

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...

<a href="https://app.jove.com/t/6598">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66272">10.3791/66266</a>

An erratum was issued for: <a href="https://www.jove.com/t/66266">Modified Fear Conditioning for Inducing Flight Behaviors in Mice</a>. The Abstract section was updated.

Erratum: Modified Fear Conditioning for Inducing Flight Behaviors in Mice

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 &#39;rodent PTSD model,&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Audio speaker</td>
			<td>Fostex</td>
			<td>FT17H</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Sony</td>
			<td>TA-F500</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Sanwa Supply Inc.</td>
			<td>CMS-V43BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Fear conditioning chamber</td>
			<td>Panlab S.L.U.</td>
			<td>LE116</td>
			<td></td>
		</tr>
		<tr>
			<td>Food pellets</td>
			<td>Nosan</td>
			<td>Labo MR standard</td>
			<td></td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Yamazen</td>
			<td>LT-B05N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>ACO</td>
			<td>type 4156N</td>
			<td></td>
		</tr>
		<tr>
			<td>Scramble shocker</td>
			<td>Panlab S.L.U.</td>
			<td>LE 100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound card</td>
			<td>Behringer</td>
			<td>UMC202</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound software</td>
			<td>Syntrillium Software</td>
			<td>Cool Edit 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>Panlab S.L.U.</td>
			<td>LE 111</td>
			<td></td>
		</tr>
	</tbody>
</table>

modified fear conditioning for inducing flight behaviors in mice

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.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

1 We are investigating the neural correlation<v>2 for various defensive behaviors 3 in fear learning and extinction. 4 The present protocol could aid in the development 5 of new treatment for anxiety disorder 6 and post-traumatic stress disorder. 7 Our protocol uses two sound pressure levels 8 of conditioning stimuli to reset 9 freezing, darting, and flight.10 So our technique can assess various defensive behaviors 11 better than classical fear conditioning. 12 Our laboratory focuses on neural correlates 13 for fear learning and extinction. 14 The neural mechanisms of various defensive behaviors 15 to threats could support the development of new treatment 16 for anxiety disorder and PTSD.

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) ...

Watch this Scientific Journal Video about Modified Fear Conditioning for Inducing Flight Behaviors in Mice at JoVE.com

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 ...

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Research

JoVE Journal

Behavior

Induction of Modified Fear Conditioning in Mice for the Analysis of Flight Behavior

1 To begin, set up a fear conditioning chamber<v>2 enclosed in a sound-attenuating box. 3 Next, create two contexts:A and B.4 For context A, attach black stripes 5 on a white plastic board. 6 For context B, make the wall entirely black.7 Use an overhead white light-emitting diode 8 to illuminate the experimental box. 9 Then, connect a scramble shocker to a grid floor 10 with stainless steel rods. 11 Now fix a speaker onto the box's ceiling.12 Then, digitally modify and calibrate the amplitudes 13 of each stimulus. 14 Place the floor of the test chamber on a transducer 15 to detect vibrations. 16 Then place a CMOS camera on the chamber's ceiling 17 to track the subject's motion and record sound.18 Set up the sound software to trigger tones or foot shocks 19 at scheduled time intervals. 20 Plan four days of fear conditioning, 21 with the first day assigned to habituation, 22 the next two days allocated to conditioning, 23 and the last day assigned to test or extinction sessions. 24 For the conditioning session, 25 present the unconditioned stimulus 26 immediately after terminating the conditioned stimulus.27 After ending the fifth shock, 28 leave the subject in the context for one minute 29 before returning to its home cage. 30 To induce flight behaviors, on day one, 31 expose the subjects to five conditioned stimuli only 32 in context A.33 On days two and three, condition the subjects 34 with five conditioned and unconditioned 35 stimuli association trials in context B.36 On the last day, expose the animal to five trials 37 of only conditioned stimuli in context B.38 The group one mice displayed pronounced flight behaviors 39 on days three and four. 40 The total motion increased with increased conditioning.41 Freezing during conditioned stimuli increased on day two 42 and remained constant in subsequent trials. 43 The animals demonstrated increased movements 44 at the onset of the condition stimuli 45 and were consistent throughout. 46 The group two mice showed flight behaviors 47 almost identical to group one on days two and three.48 However, the animals did not show any flight behavior 49 during conditioned stimuli presentations in context A.50 The group one mice displayed significantly higher motion 51 and total number of jumps relative to group two. 52 However, the group two mice froze more.

562 Views.  Kanazawa Medical University.  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.

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

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