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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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+modulation+of+pain+as+evidence+for+empathy+in+mice.">Social modulation of pain as evidence for empathy in mice.</a> Science. 312 (5782), 1967-1970 (2006).</li><li>Burkett, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin-dependent+consolation+behavior+in+rodents.">Oxytocin-dependent consolation behavior in rodents.</a> Science. 351 (6271), 375-378 (2016).</li><li>Zhang, M., Wu, Y. 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A., Decety, J., Mason, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empathy+and+prosocial+behavior+in+rats.">Empathy and prosocial behavior in rats.</a> Science. 334 (6061), 1427-1430 (2011).</li><li>Haaker, J., Golkar, A., Selbing, I., Olsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+social+transmission+of+threats+in+humans+using+observational+fear+conditioning.">Assessment of social transmission of threats in humans using observational fear conditioning.</a> Nat Protoc. 12 (7), 1378-1386 (2017).</li><li>Olsson, A., Nearing, K. I., Phelps, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+fears+by+observing+others:+The+neural+systems+of+social+fear+transmission.">Learning fears by observing others: The neural systems of social fear transmission.</a> Soc Cogn and Affect Neurosci. 2 (1), 3-11 (2007).</li><li>Kleberg, J. L., Selbing, I., Lundqvist, D., Hofvander, B., Olsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+eye+movements+and+trait+empathy+predict+vicarious+learning+of+fear.">Spontaneous eye movements and trait empathy predict vicarious learning of fear.</a> Int J Psychophysiol. 98 (3), 577-583 (2015).</li><li>Jeon, D., Shin, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+observational+fear+learning+and+the+empathetic+response.">A mouse model for observational fear learning and the empathetic response.</a> Curr Protoc in Neurosci. 57 (1), 8.27.1-8.27.9 (2011).</li><li>Keum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+empathic+fear+response+among+11+inbred+strains+of+mice.">Variability in empathic fear response among 11 inbred strains of mice.</a> Genes Brain Behav. 15 (2), 231-242 (2016).</li><li>Keum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+missense+variant+at+the+Nrxn3+locus+enhances+empathy+fear+in+the+mouse.">A missense variant at the Nrxn3 locus enhances empathy fear in the mouse.</a> Neuron. 98 (3), 588-601.e5 (2018).</li><li>Keum, S., Shin, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+basis+of+observational+fear+learning:+A+potential+model+of+affective+empathy.">Neural basis of observational fear learning: A potential model of affective empathy.</a> Neuron. 104 (1), 78-86 (2019).</li><li>Gonzalez-Liencres, C., Juckel, G., Tas, C., Friebe, A., Br&#252;ne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotional+contagion+in+mice:+The+role+of+familiarity.">Emotional contagion in mice: The role of familiarity.</a> Behav Brain Res. 263, 16-21 (2014).</li><li>Yusufishaq, S., Rosenkranz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-weaning+social+isolation+impairs+observational+fear+conditioning.">Post-weaning social isolation impairs observational fear conditioning.</a> Behav Brain Res. 242 (1), 142-149 (2013).</li><li>Keum, S., Shin, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+studying+empathy.">Rodent models for studying empathy.</a> Neurobiol Learn and Mem. 135, 22-26 (2016).</li><li>Sakaguchi, T., Iwasaki, S., Okada, M., Okamoto, K., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+facilitates+socially+evoked+memory+recall+in+mice+by+recruiting+pain-sensitive+anterior+cingulate+cortical+neurons.">Ethanol facilitates socially evoked memory recall in mice by recruiting pain-sensitive anterior cingulate cortical neurons.</a> Nature Commun. 9 (1), 1-10 (2018).</li><li>Park, J., Ha, S., Shin, H. S., Jeong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+a+hierarchical+relationship+between+a+pair+of+mice+specifically+influences+their+affective+empathy+toward+each+other.">Experience of a hierarchical relationship between a pair of mice specifically influences their affective empathy toward each other.</a> Genes Brain Behav. 21 (5), e12810 (2022).</li><li>Panksepp, J. B., Lahvis, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+influence+of+social+versus+isolate+housing+on+vicarious+fear+learning+in+adolescent+mice.">Differential influence of social versus isolate housing on vicarious fear learning in adolescent mice.</a> Behav Neurosci. 130 (2), 206-211 (2016).</li><li>Chen, Q. L., Panksepp, J. B., Lahvis, G. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal-amygdala+memory+circuits+govern+experience-dependent+observational+fear.">Hippocampal-amygdala memory circuits govern experience-dependent observational fear.</a> Neuron. 110 (8), 1416-1431.e13 (2022).</li><li>Choi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARNT2+controls+prefrontal+somatostatin+interneurons+mediating+affective+empathy.">ARNT2 controls prefrontal somatostatin interneurons mediating affective empathy.</a> Cell Rep. 43 (9), 114659 (2024).</li><li>Pisansky, M. T., Hanson, L. R., Gottesman, I. I., Gewirtz, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#215; width 19.1 cm &#215; length 37.8 cm&#160;</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 &#215; length 32.2 cm&#160;</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

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="Figure 1" class="xfigimg" src="/files/ftp_upload/67130/67130fig01.jpg" /> 
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 &#352;&#237;d&#225;k&#39;s post hoc test, F(1, 24) = 16.02, p &#60; 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 &#60; 0.001 by Student&#39;s t-test). Data are presented as means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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="Figure 2" class="xfigimg" src="/files/ftp_upload/67130/67130fig02.jpg" /> 
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 &#352;&#237;d&#225;k&#39;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 &#352;&#237;d&#225;k&#39;s post hoc test, F(1, 23) = 25.07, p &#60; 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 &#352;&#237;d&#225;k&#39;s post hoc test, F(1, 16) = 15.56, p &#60; 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 &#177; SEM. Significance levels are indicated as *p &#60; 0.05, ***p &#60; 0.001, ****p &#60; 0.0001. This figure has been modified from Keum et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67130/67130fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Observational Fear as a Model of Affective Empathy in Mice

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 ...

observational-fear-as-a-model-of-affective-empathy-in-mice

Nanyang Technological University

Research

JoVE Journal

Behavior

379 Views.  Institute for Basic Science (IBS). 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.

Video: Observational Fear as a Model of Affective Empathy in Mice

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 ...

Assessment of Age-related Changes in Cognitive Functions Using EmoCogMeter, a Novel Tablet-computer Based Approach

The main goal of this study was to assess the usability of a tablet-computer-based application (EmoCogMeter) in investigating the effects of age on cognitive functions across the lifespan in a sample of 378 healthy subjects (age range 18-89 years). Consistent with previous findings we found an age-related cognitive decline across a wide range of neuropsychological domains (memory, attention, executive functions), thereby proving the usability of our tablet-based application. Regardless of prior computer experience, subjects of all age groups were able to perform the tasks without instruction or feedback from an experimenter. Increased motivation and compliance proved to be beneficial for task performance, thereby potentially increasing the validity of the results. Our promising findings underline the great clinical and practical potential of a tablet-based application for detection and monitoring of cognitive dysfunction.

We tested the usability of a tablet-computer-based application (EmoCogMeter) in investigating the effects of age on cognition. Results show an age-related cognitive decline, thereby proving the usability of our application. Findings underline the great clinical and practical potential of a tablet-based application for detection and monitoring of cognitive dysfunction.

The main goal of this study was to assess the usability of a tablet-computer-based application (EmoCogMeter) in investigating the effects of age on ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

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 ...

Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic &#946;-Blocker

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a &#946;-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.

Disrupting reconsolidation is a promising approach to dampen the behavioral expression of fear memory in patients with anxiety disorders or posttraumatic stress disorder. In a series of human fear conditioning studies we showed that disrupting reconsolidation by the noradrenergic &#946;-blocker propranolol is very effective in erasing conditioned fear responding.

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

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 ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...