Observational Fear as a Model of Affective Empathy in Mice

Summary

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.

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 behaviors^4,5,6,7,8,9. 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 primates^11,12, indicating that observational fear may indeed represent a fundamental feature of emotional empathy². Compared to other empathy assays^1,2,4,10, 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.

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

1. 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.
2. 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.
3. Setup for observational fear conditioning
   1. 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).
      1. 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.
   2. 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.
4. 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

1. Habituation Period
   1. Assign one animal as the observer and the other as the demonstrator, depending on which animal will be subjected to aversive foot shocks.
   2. 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.
   3. Run the programmed protocol using the software immediately after closing the chamber door.
      NOTE: Enter a protocol into the FreezeFrame software that delivers a 2 s foot shock to the demonstrator mouse every 10 s while allowing the camera to record.
   4. Allow the observer and demonstrator mice to freely move and explore in the chamber for 5 min during the habituation session.
2. Conditioning period
   1. Automatically deliver electrical foot shocks to the demonstrator mouse for 4 min after the habituation period.
   2. Ensure that the observer mouse witnesses the pain responses of the demonstrator mouse during the conditioning period.
   3. After each session, place demonstrators and observers in separate holding cages. Upon completion of testing, return each animal to its home cage.
   4. 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

1. Analyze the recorded behavior of the observer during both the habituation and conditioning periods¹⁵.
2. 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

1. After experiments, monitor the behavior and health of all animals to ensure they recover fully from any stressors.
2. Provide proper housing and care for animals, including food and water, immediately after the completion of testing.
3. Record any changes in behavior during and after the experiments to assess the impact of the conditioning procedures.

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 ...

Discussion

The contextual observational fear conditioning (OFC) test, described in this behavioral protocol, is employed to study affective empathy in mice^4,16. 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...

Materials

Name	Company	Catalog Number	Comments
0.5 mL Micro Centrifuge Tubes	Globe Scientific	111554A
96 Well Standard Black Microplate	Dot Scientific	4ti-0223
Biotinylated poly dT Oligonucleotide	IDT
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153-10G
Dithiothreitol (DTT)	Dot Scientific	DSD11000-10
Ethylenediaminetetraacetic acid (EDTA)	Dot Scientific	DSE57020-500
Hydrochloric Acid	Fisher Scientific	A144-500
Kimtec Science Kimwipes	Kimtech	34120
Octet N1 Software	Sartorius	1.4.0.13
Octet SA Biosensor	Sartorius	18-5019
PBS pH 7.2 (10x)	Gibco	1666711
Personal Assay Octet N1 System	Sartorius
Phosphoric Acid	Ward's Science	470302-024
Sodium Chloride (NaCl)	Dot Scientific	DSS23020-5000
Tris Base	Dot Scientific	DST60040-5000
Tween20	Bio-Rad	170-6531