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

<ol>
	<li>De Waal, F. B. M., Preston, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+empathy:+Behavioural+manifestations+and+neural+basis.">Mammalian empathy: Behavioural manifestations and neural basis.</a> Nat Rev Neurosci. 18 (8), 498-509 (2017).</li><li>Panksepp, J., Panksepp, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+cross-species+understanding+of+empathy.">Toward a cross-species understanding of empathy.</a> Trends Neurosci. 36 (8), 489-496 (2013).</li><li>Decety, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+neural+mechanisms+mediating+empathy.">Dissecting the neural mechanisms mediating empathy.</a> Emotion Rev. 3 (1), 92-108 (2011).</li><li>Jeon, D., 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=Observational+fear+learning+involves+affective+pain+system+and+Cav1.2+Ca2++channels+in+ACC.">Observational fear learning involves affective pain system and Cav1.2 Ca2+ channels in ACC.</a> Nat Neurosci. 13 (4), 482-488 (2010).</li><li>Langford, D. 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. E., Jiang, M., Hong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+regulation+of+helping+behaviour+towards+others+in+pain.">Cortical regulation of helping behaviour towards others in pain.</a> Nature. 626 (7997), 136-144 (2024).</li><li>Wu, Y. E., 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=Neural+control+of+affiliative+touch+in+prosocial+interaction.">Neural control of affiliative touch in prosocial interaction.</a> Nature. 599 (7884), 262-267 (2021).</li><li>Bartal, I. B. 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. 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+is+moderated+by+genetic+background+in+mice.">Empathy is moderated by genetic background in mice.</a> PLOS One. 4 (2), e4387 (2009).</li><li>Keysers, C., Knapska, E., Moita, M. A., Gazzola, V. <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+and+prosocial+behavior+in+rodents.">Emotional contagion and prosocial behavior in rodents.</a> Trends Cogn Sci. 26 (8), 688-706 (2022).</li><li>Carrillo, M., 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=Emotional+mirror+neurons+in+the+rat's+anterior+cingulate+cortex.">Emotional mirror neurons in the rat's anterior cingulate cortex.</a> Curr Biol. 29 (8), 1301-1312.e6 (2019).</li><li>Silverstein, S. E., 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+distinct+cortical+code+for+socially+learned+threat.">A distinct cortical code for socially learned threat.</a> Nature. 626 (8001), 1066-1072 (2024).</li><li>Allsop, S. A., 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=Corticoamygdala+transfer+of+socially+derived+information+gates+observational+learning.">Corticoamygdala transfer of socially derived information gates observational learning.</a> Cell. 173 (6), 1329-1342.e18 (2018).</li><li>Terranova, J. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+enhances+observational+fear+in+mice.">Oxytocin enhances observational fear in mice.</a> Nature Commun. 8 (1), 2102 (2017).</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=Sociability+versus+empathy+in+adolescent+mice:+Different+or+distinctive.">Sociability versus empathy in adolescent mice: Different or distinctive.</a> Learn Motiv. 83, 101892 (2023).</li></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 sho...

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

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 ...

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

Research

JoVE Journal

Behavior

466 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 ...

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 ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Neuroscience

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

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 ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

Studying Active Defensive Behaviors in Mice with Modified 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 defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

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

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...