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 preparation
<ol>
	<li>Group-house mice (3-4 per cage; maintained at 23-27 &#176;C; under a 12 h light/dark cycle; provided ad libitum access to food and water) until the start of the experiments.</li>
	<li>Individually house each mouse in a plexiglass cage (14 cm &#215; 21 cm &#215; 12 cm) for at least 3 days before undergoing this modified fear conditioning.</li>
</ol>
2. Setting up the tools/equipment
<ol>
	<li>Fear conditioning box (Figure 1A)
	<ol>
		<li>Utilize a fear conditioning chamber (25 cm &#215; 25 cm &#215; 25 cm) enclosed in a sound-attenuating box (67 cm &#215; 53 cm &#215; 55 cm) (see Table of Materials).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Illuminate the experimental box using an overhead white light-emitting diode (LED, 240 lux) (see Table of Materials).</li>
	</ol>
	</li>
	<li>Shocker
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Audio generator
	<ol>
		<li>Place a speaker (see Table of Materials) on the ceiling. All acoustic stimuli are amplified.</li>
		<li>Digitally modify and calibrate the overall amplitudes of each stimulus to yield sound pressure levels (SPL, re: 20 &#181; Pa) at the 5 cm front of the speaker with a &#188; 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.</li>
	</ol>
	</li>
	<li>Transducer
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Video camera
	<ol>
		<li>Position a CMOS camera (see Table of Materials) on the ceiling to track the subject&#39;s motions and record the sound in the conditioning box.</li>
	</ol>
	</li>
	<li>Triggering system
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Behavioral experiment
<ol>
	<li>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&#39;s purpose.
	<ol>
		<li>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.</li>
		<li>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&#39;s purpose. Previous reports have shown that stronger CS intensity triggers more active defensive behaviors than softer ones15. Conditioning days can also be extended.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>On day 1, expose subjects to 5 CS alone trials in context A.</li>
		<li>On days 2 and 3, condition subjects with 5 CS-US association trials in context B.</li>
		<li>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. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>To measure the percentage of freezing, use the transducer signal in time.
	<ol>
		<li>Preprocess transducer signals using a 20-500 Hz band pass filter.</li>
		<li>Calculate the root mean square amplitude of the transducer signal in time for each 50 ms bin.</li>
		<li>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.</li>
		<li>Manually measure the duration of freezing by watching the video.</li>
		<li>Adjust the threshold of signal amplitude for freezing by comparing the manually measured percentage of freezing and the percentage calculated from the transducer signal.</li>
	</ol>
	</li>
	<li>Count the number of jumps manually from video files. 
	&#8203;NOTE: Counting the number of darts will also be useful in assessing the flight response.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Set the statistical significance at p &#60; 0.05.</li>
	<li>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.</li>
</ol>

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 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.

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

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/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.

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 &#60; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66266/66266fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66266/66266fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66266/66266fig02.jpg" /> 
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 &#60; 0.05. The figure is modified from Furuyama et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66266/66266fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Behavior

758 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.

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

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a significant public health concern, particularly in children. Both of these conditions are highlighted by the repeated expression of meaningless behaviors. Individuals with OCD often show checking, frequent hand washing, and counting. Children with ASDs also engage in repetitive tapping, arm or hand flapping, and rocking. These behaviors can vary widely in intensity and frequency of expression. More intense forms of repetitive behaviors can even result in injury (e.g.&#160;excessive grooming, hand washing, and self-stimulation). These behaviors are therefore very disruptive and make normal social discourse difficult. Treatment options for repetitive behaviors in OCD and ASDs are somewhat limited and there is great interest in developing more effective therapies for each condition. Numerous animal models for evaluating compulsive-like behaviors have been developed over the past three decades. Perhaps the animal models with the greatest validity and ease of use are the marble burying test and the nestlet shredding test. Both tests take advantage of the fact that the target behaviors occur spontaneously in mice. In the marble burying test, 20 marbles are arrayed on the surface of clean bedding. The number of marbles buried in a 30 min session is scored by investigators blind to the treatment or status of the subjects. In the nestlet shredding test, a nestlet comprised of pulped cotton fiber is preweighed and placed on top of cage bedding and the amount of the nestlet remaining intact after a 30 min test session is determined. Presently, we describe protocols for and show movie documentation of marble burying and nestlet shredding. Both tests are easily and accurately scored and each is sensitive to small changes in the expression of compulsive-like behaviors that result from genetic manipulations, disease, or head injury.

Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...