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.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Observational Fear as a Model of Affective Empathy in Mice
In This Article
Summary
Abstract
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.
Introduction
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.
Access restricted. Please log in or start a trial to view this content.
Protocol
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 °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
- 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.
- 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. - Setup for observational fear conditioning
- 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).
- Include two chambers partitioned by a porous (six holes with a radius of 3 mm), transparent Plexiglas divider (18 cm wide × 32.2 cm long) in the middle. Place a grid consisting of multiple steel rods on the chamber floor to administer electrical foot shocks.
- 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.
- 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).
- 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's behavior.
2. Observational fear conditioning procedure
- Habituation Period
- Assign one animal as the observer and the other as the demonstrator, depending on which animal will be subjected to aversive foot shocks.
- 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.
- 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. - Allow the observer and demonstrator mice to freely move and explore in the chamber for 5 min during the habituation session.
- Conditioning period
- Automatically deliver electrical foot shocks to the demonstrator mouse for 4 min after the habituation period.
- 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's pain responses to electric shock, essential social cues for inducing emotional state alterations in observer mice. - After each session, place demonstrators and observers in separate holding cages. Upon completion of testing, return each animal to its home cage.
- 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.
3. Analysis of vicarious freezing responses
- Analyze the recorded behavior of the observer during both the habituation and conditioning periods15.
- 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.
4. Follow-up procedures
- After experiments, monitor the behavior and health of all animals to ensure they recover fully from any stressors.
- Provide proper housing and care for animals, including food and water, immediately after the completion of testing.
- Record any changes in behavior during and after the experiments to assess the impact of the conditioning procedures.
Access restricted. Please log in or start a trial to view this content.
Results
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 ...
Access restricted. Please log in or start a trial to view this content.
Discussion
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' emotional responses, including familiarity, social isolation, social hierarchy, prior shock experience, and the strength of the foot sho...
Access restricted. Please log in or start a trial to view this content.
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Precision animal shocker | Coulbourn Instruments | H13-15 | |
| Grid floor | Coulbourn Instruments | H13-15 | |
| Ribbon cable for grids | Coulbourn Instruments | H93-01-25 | |
| FreezeFrame | ACTIMETRICS | ACT-100A | |
| FreezeView software | ACTIMETRICS | ||
| Stimulator | ACTIMETRICS | ACT-712 | |
| Observational fear chamber | Coulbourn Instruments | height 32.7 cm × width 19.1 cm × length 37.8 cm | |
| Video recording system | ACTIMETRICS | ||
| Sound attenuating cubicle | |||
| 70% ethanol | Widely available. | ||
| Sanitizer | Widely available. | ||
| paper towel | Widely available. | ||
| Plexiglas divider | Custom | N/A | width 18 cm × length 32.2 cm |
| GraphPad Prism 8 | GraphPad Software Inc. |
References
- De Waal, F. B. M., Preston, S. D. Mammalian empathy: Behavioural manifestations and neural basis. Nat Rev Neurosci. 18 (8), 498-509 (2017).
- Panksepp, J., Panksepp, J. B. Toward a cross-species understanding of empathy. Trends Neurosci. 36 (8), 489-496 (2013).
- Decety, J. Dissecting the neural mechanisms mediating empathy. Emotion Rev. 3 (1), 92-108 (2011).
- Jeon, D., et al. Observational fear learning involves affective pain system and Cav1.2 Ca2+ channels in ACC. Nat Neurosci. 13 (4), 482-488 (2010).
- Langford, D. J., et al. Social modulation of pain as evidence for empathy in mice. Science. 312 (5782), 1967-1970 (2006).
- Burkett, J. P., et al. Oxytocin-dependent consolation behavior in rodents. Science. 351 (6271), 375-378 (2016).
- Zhang, M., Wu, Y. E., Jiang, M., Hong, W. Cortical regulation of helping behaviour towards others in pain. Nature. 626 (7997), 136-144 (2024).
- Wu, Y. E., et al. Neural control of affiliative touch in prosocial interaction. Nature. 599 (7884), 262-267 (2021).
- Bartal, I. B. A., Decety, J., Mason, P. Empathy and prosocial behavior in rats. Science. 334 (6061), 1427-1430 (2011).
- Haaker, J., Golkar, A., Selbing, I., Olsson, A. Assessment of social transmission of threats in humans using observational fear conditioning. Nat Protoc. 12 (7), 1378-1386 (2017).
- Olsson, A., Nearing, K. I., Phelps, E. A. Learning fears by observing others: The neural systems of social fear transmission. Soc Cogn and Affect Neurosci. 2 (1), 3-11 (2007).
- Kleberg, J. L., Selbing, I., Lundqvist, D., Hofvander, B., Olsson, A. Spontaneous eye movements and trait empathy predict vicarious learning of fear. Int J Psychophysiol. 98 (3), 577-583 (2015).
- Jeon, D., Shin, H. S. A mouse model for observational fear learning and the empathetic response. Curr Protoc in Neurosci. 57 (1), 8.27.1-8.27.9 (2011).
- Keum, S., et al. Variability in empathic fear response among 11 inbred strains of mice. Genes Brain Behav. 15 (2), 231-242 (2016).
- Keum, S., et al. A missense variant at the Nrxn3 locus enhances empathy fear in the mouse. Neuron. 98 (3), 588-601.e5 (2018).
- Keum, S., Shin, H. S. Neural basis of observational fear learning: A potential model of affective empathy. Neuron. 104 (1), 78-86 (2019).
- Gonzalez-Liencres, C., Juckel, G., Tas, C., Friebe, A., Brüne, M. Emotional contagion in mice: The role of familiarity. Behav Brain Res. 263, 16-21 (2014).
- Yusufishaq, S., Rosenkranz, J. A. Post-weaning social isolation impairs observational fear conditioning. Behav Brain Res. 242 (1), 142-149 (2013).
- Keum, S., Shin, H. S. Rodent models for studying empathy. Neurobiol Learn and Mem. 135, 22-26 (2016).
- Sakaguchi, T., Iwasaki, S., Okada, M., Okamoto, K., Ikegaya, Y. Ethanol facilitates socially evoked memory recall in mice by recruiting pain-sensitive anterior cingulate cortical neurons. Nature Commun. 9 (1), 1-10 (2018).
- Park, J., Ha, S., Shin, H. S., Jeong, J. Experience of a hierarchical relationship between a pair of mice specifically influences their affective empathy toward each other. Genes Brain Behav. 21 (5), e12810(2022).
- Panksepp, J. B., Lahvis, G. P. Differential influence of social versus isolate housing on vicarious fear learning in adolescent mice. Behav Neurosci. 130 (2), 206-211 (2016).
- Chen, Q. L., Panksepp, J. B., Lahvis, G. P. Empathy is moderated by genetic background in mice. PLOS One. 4 (2), e4387(2009).
- Keysers, C., Knapska, E., Moita, M. A., Gazzola, V. Emotional contagion and prosocial behavior in rodents. Trends Cogn Sci. 26 (8), 688-706 (2022).
- Carrillo, M., et al. Emotional mirror neurons in the rat's anterior cingulate cortex. Curr Biol. 29 (8), 1301-1312.e6 (2019).
- Silverstein, S. E., et al. A distinct cortical code for socially learned threat. Nature. 626 (8001), 1066-1072 (2024).
- Allsop, S. A., et al. Corticoamygdala transfer of socially derived information gates observational learning. Cell. 173 (6), 1329-1342.e18 (2018).
- Terranova, J. I., et al. Hippocampal-amygdala memory circuits govern experience-dependent observational fear. Neuron. 110 (8), 1416-1431.e13 (2022).
- Choi, J., et al. ARNT2 controls prefrontal somatostatin interneurons mediating affective empathy. Cell Rep. 43 (9), 114659(2024).
- Pisansky, M. T., Hanson, L. R., Gottesman, I. I., Gewirtz, J. C. Oxytocin enhances observational fear in mice. Nature Commun. 8 (1), 2102(2017).
- Panksepp, J. B., Lahvis, G. P. Sociability versus empathy in adolescent mice: Different or distinctive. Learn Motiv. 83, 101892(2023).
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
