Name: Observational Fear as a Model of Affective Empathy in Mice (Video) | JoVE Journal
Uploaded: 2024-11-22T14:00:00.000+00:00
Duration: 4 min 14 s
Description: Explore observational fear conditioning to study affective empathy in mice. This method reveals how the anterior cingulate cortex processes emotional information and generates vicarious freezing behaviors.

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5 minutes

This work was supported by the Institute for Basic Science (IBS), Center for Cognition and Sociality (IBS-R001-D2), and the Korea Brain Research Institute (KBRI) basic research program through KBRI, funded by the Ministry of Science, ICT, and Future Planning (23-BR-03-04).

All procedures using live animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute for Basic Science (IBS). Male or female C57BL/6J mice (observers and demonstrators), aged 10-12 weeks, were housed separately. Mice were housed 2-5 animals per cage and maintained at a constant temperature of 23-25 &#176;C with a 12-h light/dark cycle. The mice were provided with mouse chow and water ad libitum. No handling was performed prior to testing. Details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Preparation for the observational fear conditioning
<ol>
	<li>Housing conditions: House siblings together (2-3 mice/cage) after weaning. House non-sibling mice in separate cages13. For experiments involving mating pairs, prepare mating cages with one male and two female mice. Co-house the males and females for at least 10 weeks.</li>
	<li>Room habituation: Transfer the animals in their home cages to the waiting area at least 30 min before behavioral testing begins to allow the mice to acclimate to the testing environment. 
	NOTE: Maintain light intensity in the experimental room at approximately 90 lux and in the waiting area for habituation at 35 lux.</li>
	<li>Setup for observational fear conditioning
	<ol>
		<li>Apparatus configuration: Assemble the apparatus for observational fear conditioning, measuring 32.7 cm in height, 19.1 cm in width, and 37.8 cm in length (Figure 1A).
		<ol>
			<li>Include two chambers partitioned by a porous (six holes with a radius of 3 mm), transparent Plexiglas divider (18 cm wide &#215; 32.2 cm long) in the middle. Place a grid consisting of multiple steel rods on the chamber floor to administer electrical foot shocks.</li>
		</ol>
		</li>
		<li>Ensure that the protocol for observational fear conditioning consists of two periods: a 5 min habituation period followed by a 4 min conditioning period, during which a 2 s foot shock (1 mA) is delivered every 10 s to the demonstrator mouse. Program this protocol using FreezeFrame software. 
		NOTE: As previously described14, ensure the foot shock intensity of 1 mA to reliably induce the full range of behavioral responses in the demonstrator animals, including running, vocalization, and jumping. Avoid reducing this intensity, as it may lead to inconsistent responses in both demonstrators and observers. However, consider using a lower shock intensity or fewer shock deliveries if a full range of behavioral responses from the demonstrator can be induced under specific experimental settings.</li>
	</ol>
	</li>
	<li>Video recording: Turn on the video camera and ensure that the testing chamber is fully visible within the frame for subsequent analysis. 
	NOTE: Install the video camera inside the sound-attenuating cubicle, positioning it directly above the animal to ensure a clear recording of the animal&#39;s behavior.</li>
</ol>
2. Observational fear conditioning procedure
<ol>
	<li>Habituation Period
	<ol>
		<li>Assign one animal as the observer and the other as the demonstrator, depending on which animal will be subjected to aversive foot shocks.</li>
		<li>Place the demonstrator mouse in the chamber where the foot shock will be administered first, and then position the observer mouse in the opposite chamber, separated by a transparent Plexiglas divider.</li>
		<li>Run the programmed protocol using the software immediately after closing the chamber door. 
		NOTE: Enter a complete protocol into the FreezeFrame software that includes both a habituation period (without foot shocks) followed by a conditioning phase that delivers a 2 s foot shock to the demonstrator mouse every 10 s while allowing the camera to record throughout the entire procedure.</li>
		<li>Allow the observer and demonstrator mice to freely move and explore in the chamber for 5 min during the habituation session.</li>
	</ol>
	</li>
	<li>Conditioning period
	<ol>
		<li>Automatically deliver electrical foot shocks to the demonstrator mouse for 4 min after the habituation period.</li>
		<li>Ensure that the observer mouse witnesses the pain responses of the demonstrator mouse during the conditioning period. 
		NOTE: Analgesics were withheld to preserve the demonstrator&#39;s pain responses to electric shock, essential social cues for inducing emotional state alterations in observer mice.</li>
		<li>After each session, place demonstrators and observers in separate holding cages. Upon completion of testing, return each animal to its home cage.</li>
		<li>Clean the apparatus and the grid on the floor using 70% ethanol and paper tissues. 
		NOTE: Thoroughly clean the apparatus with sanitizer between repeated experiments to eliminate any residual olfactory cues from previous subjects and maintain consistency across trials. Additionally, wipe with 70% ethanol after all experiments are completed.</li>
	</ol>
	</li>
</ol>
3. Analysis of vicarious freezing responses
<ol>
	<li>Analyze the recorded behavior of the observer during both the habituation and conditioning periods15.</li>
	<li>Run the software to automatically detect motionless bouts of the observer mouse lasting more than 0.75 s, which are considered freezing. 
	NOTE: Conduct analysis either manually or automatically for scoring freezing responses15.</li>
</ol>
4. Follow-up procedures
<ol>
	<li>After experiments, monitor the behavior and health of all animals to ensure they recover fully from any stressors.</li>
	<li>Provide proper housing and care for animals, including food and water, immediately after the completion of testing.</li>
	<li>Record any changes in behavior during and after the experiments to assess the impact of the conditioning procedures.</li>
</ol>

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

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</ol>

The authors have nothing to disclose.

The contextual observational fear conditioning (OFC) test, described in this behavioral protocol, is employed to study affective empathy in mice4,16. To successfully execute this paradigm, careful consideration should be given to experimental conditions and practical details, particularly factors that influence the subjects&#39; emotional responses, including familiarity, social isolation, social hierarchy, prior shock experience, and the strength of the foot shocks delivered to demonstrators4,17,18,19,20,21,22. In OFC, the demonstrator&#39;s behavioral reactions to foot shocks serve as social cues that trigger emotional contagion, manifested as vicarious freezing in the observer mice. As demonstrated by previous research4, a foot shock protocol consisting of a 2 s foot shock (1 mA) with a 10 s interval for a 4 min conditioning period reliably and consistently induces behavioral reactions, such as jumping, running, and distress vocalizations in demonstrator mice.
Consequently, this protocol elicits high and stable levels of vicarious freezing in na&#239;ve observer mice. When protocols with lower shock intensity were utilized, demonstrators exhibited inconsistent behavioral responses, such as running, vocalization, and/or jumping, without consistently displaying all three behaviors4. Additionally, when demonstrators were subjected to protocols with longer shock intervals, na&#239;ve observers showed reduced levels of vicarious freezing during OFC4. In addition to the experimental paradigm utilizing na&#239;ve observers without prior shock exposure16,23, other well-established OFC protocols employ observers with prior shock experience24,25,26, which may also be useful depending on the psychological components or brain areas of interest and/or the contingencies of your institutional IACUC approval process. Mice with a single priming foot shock experience displayed significantly augmented observational fear responses that may be influenced by previous aversive experiences20,27,28,29. Despite similarities in behavioral outcomes, it is crucial to acknowledge the significant distinctions in the underlying neural mechanisms regulating OFC in na&#239;ve observers compared to observer mice with previous shock experience16,24.
One crucial experimental condition that should also be carefully controlled is the elimination of potential sources of auditory disturbance. It is essential that the observers devote their full attention to observing the distress response exhibited by the demonstrator without any extraneous distractions compromising the observation. Even white noise, commonly employed in rodent behavioral tests, has the potential to interfere with this focused observation. Experiments should be conducted without the presence of white noise to minimize any potential disturbances.
Social conditions, especially those affected by the observer&#39;s litter size and rearing environment, play a crucial role in the empathic freezing response in observer mice. Socially challenging circumstances, such as crowded housing or social isolation, can impair the development of sociality and observational fear, highlighting the significance of social context in modulating empathic behaviors21. Furthermore, alterations in social conditions, such as the size of the litter or bedding, immediately before testing can serve as potential stressors. Therefore, it is advisable to avoid changing the housing environment within 7 days prior to commencing the behavioral paradigm. Familiarity is another significant factor, as the vicarious freezing response of the observer mouse is positively influenced by the animal&#39;s familiarity or kinship with the demonstrator4,17. When the demonstrator is a sibling or a long-term mating partner, it tends to elicit a higher fear response in the observer. This dependence on familiarity appears to be more prominent in male observer mice30. Additionally, the effects of social hierarchy on empathic response in the context of familiarity need to be considered, as recent evidence has demonstrated a negative correlation between the relative status of social dominance and the observational fear response of the observer21. Overall, understanding the multifaceted influences on observational fear responses provides valuable insights into the complex nature of empathy-like behaviors in rodents16,20,24,28,31.
Although substantial cross-species parallels and conservation exist, the study of affective empathy in rodents faces limitations due to the lack of corroborating evidence regarding mice&#39;s self-awareness of emotional states, unlike humans, who can provide self-reports on their emotional and affective states. Nevertheless, given that empathy manifests through bottom-up processes preserved across species, such as motor mimicry and emotional contagion, observational fear is considered a basic and primitive form of affective empathic processes1,3,24. Therefore, the observational fear paradigm is a suitable method for studying the underlying neural mechanisms of affective empathy in animal models.

Empathy -- the capacity to recognize and share the feelings of others -- is crucial for our emotional and social interactions1. This protocol aims to investigate affective empathy in rodents through the observational fear conditioning assay, designed to assess vicarious fear responses in an observer rodent. Empathic ability has deep neurobiological underpinnings, with evolutionary continuity from rodents to humans2. Converging lines of evidence demonstrate that rodents exhibit significant affective sensitivity to the emotional states of their social counterparts3 and display empathy-related behaviors, including observational fear, consolation-like allogrooming, emotional contagion of pain, and prosocial helping behaviors4,5,6,7,8,9. This continuity has important implications, as it enables the use of animal models to gain deeper insights into the neurobiological mechanisms underlying human empathy10.
Observational fear, in particular, serves as a valuable behavioral model for assessing empathic fear responses, as rodents can vicariously experience fear by observing conspecifics receiving aversive stimuli4. Studies have shown that stronger vicarious fear responses are positively associated with trait measures of empathy in humans and primates11,12, indicating that observational fear may indeed represent a fundamental feature of emotional empathy2. Compared to other empathy assays1,2,4,10, observational fear conditioning is advantageous as it allows the measurement of emotional contagion without subjecting the observer to direct aversive stimuli. This provides a less invasive yet reliable measure of affective empathy, making it suitable for repeated measures and in-depth studies on empathy-related neural circuits.
In observational fear conditioning, a mouse (observer) is vicariously conditioned for context-dependent fear by watching another mouse (demonstrator) receive repetitive foot shocks, without experiencing direct aversive stimuli. The observational fear conditioning assay employs a dual-compartment fear conditioning apparatus, which is divided by a transparent Plexiglas divider13. This setup allows the observer in one chamber to clearly see the demonstrator mouse in the opposite chamber. Additionally, smells and sounds can be transmitted between the chambers through the rod floor.In one compartment, the demonstrator mouse, which will receive the aversive stimulus of a foot shock, is positioned, while in the other compartment, the observer mouse is situated to witness the distress of the demonstrator mouse through the transparent middle partition. This paradigm consists of two sessions: a 5-min habituation followed by a 4-min conditioning period. The freezing behavior displayed by the observer mouse occurs when the demonstrator exhibits typical responses to aversive foot shocks, including pain-induced squeaks, jumping, running, and freezing, during the conditioning period. This socially evoked vicarious freezing response in observers is considered a measure of emotional contagion, a basic form of affective empathy in rodents. The development of the observational fear assay in rodents has enabled researchers to investigate the neural circuits and genetic underpinnings that mediate affective empathy.

<table><tbody><tr><td>Precision animal shocker</td><td>Coulbourn Instruments</td><td>H13-15</td><td></td></tr><tr><td>Grid floor</td><td>Coulbourn Instruments</td><td>H13-15</td><td></td></tr><tr><td>Ribbon cable for grids</td><td>Coulbourn Instruments</td><td>H93-01-25</td><td></td></tr><tr><td>FreezeFrame</td><td>ACTIMETRICS</td><td>ACT-100A</td><td></td></tr><tr><td>FreezeView software</td><td>ACTIMETRICS</td><td></td><td></td></tr><tr><td>Stimulator</td><td>ACTIMETRICS</td><td>ACT-712</td><td></td></tr><tr><td>Observational fear chamber</td><td>Coulbourn Instruments</td><td></td><td>height 32.7 cm &amp;times; width 19.1 cm &amp;times; length 37.8 cm&amp;nbsp;</td></tr><tr><td>Video recording system</td><td>ACTIMETRICS</td><td></td><td></td></tr><tr><td>Sound attenuating cubicle</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td>Widely available.</td></tr><tr><td>Sanitizer</td><td></td><td></td><td>Widely available.</td></tr><tr><td>paper towel</td><td></td><td></td><td>Widely available.</td></tr><tr><td>Plexiglas divider</td><td>Custom</td><td>N/A</td><td>width 18 cm &amp;times; length 32.2 cm&amp;nbsp;</td></tr><tr><td>GraphPad Prism 8</td><td>GraphPad Software Inc.</td><td></td><td></td></tr></tbody></table>

observational fear as a model of affective empathy in mice

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

As described in the behavioral diagram (Figure 1A), the session starts with habituation (5 min), followed by an observational fear conditioning period (4 min). In this experiment, the demonstrator mice in the observational fear (OF) group were subjected to aversive foot shocks during the conditioning period, while those in the control (Con) group did not receive foot shocks. Only the observer mouse in the OF group witnessed the distress responses of a demonstrator mouse receiving repetitive foot shocks. Mice in the OF group exhibited a significantly elevated level of vicarious freezing during the conditioning period compared to the Con group (Figure 1B). The total freezing level (in seconds) during conditioning showed a marked difference between the OF and Con groups (Figure 1C).
<img alt="Observational fear study in mice, includes conditioning diagram and freezing behavior graphs." class="xfigimg" src="/files/ftp_upload/67130/67130fig01.jpg" data-altupdatedat="1788840098000" data-alt="Figure 1"> 
Figure 1: Observational fear paradigm. (A) This panel outlines the behavioral paradigm for observational fear conditioning. The Observer (OB) and Demonstrator (DM) mice are placed individually in the chamber and allowed a 5 min exploration period for habituation. Following this, the OB mouse observes the DM mouse receiving foot shocks through a transparent partition during a 4 min conditioning period. (B) Vicarious freezing percentages of OB mice in both groups were measured at 1 min intervals. Mice in the OF group showed a marked increase in observational fear (Con, n = 15; OF, n = 11; two-way repeated measure (RM) ANOVA followed by Šídák's post hoc test, F(1, 24) = 16.02, p &lt; 0.001). (C) Total freezing time (in seconds) during the 4 min conditioning period was significantly increased in the OF group (Con, n = 15; OF, n = 11; ***p &lt; 0.001 by Student's t-test). Data are presented as means ± SEM. <a href="https://www.jove.com/files/ftp_upload/67130/67130fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The vicarious freezing response was measured in different inbred mouse strains. The 129S1/SvlmJ (129S1) strain exhibited a marked increase in observational fear compared with the B6J strain, while mice of the AKR/J (AKR) strain14,15 showed a significantly lower level of vicarious freezing compared to B6J mice (Figure 2A).
Optogenetic manipulation could be employed in this paradigm to investigate the underlying circuit mechanisms for observational fear. The previously published data suggested that optogenetic suppression of somatostatin (SST)-expressing neurons drives enhanced vicarious fear responses15. To determine whether activation of SST+ interneurons affects the level of observational fear, we injected an AAV expressing Cre-dependent channelrhodopsin (AAV-DIO-ChR-YFP) into the right anterior cingulate cortex (ACC) of SST-Cre mice to selectively label SST+ interneurons (Figure 2B). Optogenetic stimulation of SST+ interneurons in the right ACC resulted in the abolishment of vicarious freezing acquisition.
<img alt="Freezing behavior graphs; optical stimulation effect on mice (A: B6J, AKR, 129S1; B: SST-YFP, SST-ChETA)." class="xfigimg" src="/files/ftp_upload/67130/67130fig02.jpg" data-altupdatedat="1788840098000" data-alt="Figure 2"> 
Figure 2: Measurements of observational fear under various experimental conditions. (A) This panel compares the vicarious freezing response percentages across different inbred mouse strains. The vicarious freezing response was highly variable among strains. The level of observational fear response in 129S1 observer mice was significantly higher compared to that observed in B6J mouse strains (B6J, n = 11; 129S1, n = 15; two-way repeated measure (RM) ANOVA followed by Šídák's post hoc test, F(1, 24) = 11.94, p = 0.0021). AKR mice exhibited significantly decreased observational fear compared with B6J mice (B6J, n = 11; AKR, n = 14; two-way repeated measure (RM) ANOVA followed by Šídák's post hoc test, F(1, 23) = 25.07, p &lt; 0.0001). (B) Vicarious freezing percentages were inhibited by optogenetic activation of SST+ interneurons in the anterior cingulate cortex (ACC) (SST-YFP control, n = 8; SST-ChETA, n = 10; two-way repeated measure (RM) ANOVA followed by Šídák's post hoc test, F(1, 16) = 15.56, p &lt; 0.01). The stimulation parameters are as follows: 241 s duration, 20 Hz, 5 ms pulse width, 0.5-1.0 mW at the fiber tip. Data are presented as means ± SEM. Significance levels are indicated as *p &lt; 0.05, ***p &lt; 0.001, ****p &lt; 0.0001. This figure has been modified from Keum et al.15. <a href="https://www.jove.com/files/ftp_upload/67130/67130fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

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Explore observational fear conditioning to study affective empathy in mice. This method reveals how the anterior cingulate cortex processes emotional information and generates vicarious freezing behaviors.

Observational Fear as a Model of Affective Empathy in Mice

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