Paradigm for Social Recognition in Mice for Distinguishing Short- and Long-Term Relationships

Summary

Here, we outline a thorough procedure for measuring social recognition between novel, short-term, and long-term relationships using a Four Cups behavioral experimental paradigm. We outline the benefits and potential uses of this paradigm, along with important information for accurately interpreting the outcomes. 

Abstract

Social memory is the ability to remember and recognize conspecifics. It is a fundamental aspect of mammalian behavior, with demonstrated survival advantages in highly sociable mammals such as mice and humans. Rodents, particularly mice, are an established animal model extensively used to study social memory and its underlying mechanisms. Most existing social memory paradigms primarily assess short-term social recognition, wherein the relationship duration and familiarity level between the subject and an agent is between 5–15 min. Therefore, existing paradigms and past studies do not capture social memories from long-term relationships and focus on the novelty vs. short-term familiarity dichotomy, leaving a large gap in our mechanistic understanding of social memories. To fill this gap, we designed a simple assay that captures mouse responses to varying levels of familiarity, placing complex social memory recall in an observable, quantifiable context. In the four cups paradigm, a subject mouse freely explores an arena containing four perforated cups placed in each corner. These cups house three agent mice of varying familiarity levels (social stimuli): a long-term familiar (cagemate; 1 month co-housing), a short-term familiar (10 min of prior exposure), a novel conspecific, and an empty cup as a control. CD-1 male mice show a preference for investigating the novel and long-term familiar mouse over investigating short-term familiar mice in this paradigm. This task demonstrates recognition of familiarity level and minimizes aggressive behaviors while still controlling for exposure history. This novel assay provides a platform for directly comparing the neural mechanisms of social memory across long-term, short-term, and novel individuals. The four cups paradigm serves as a novel measurement for long-term familiarity recognition that will complement existing short-term recognition assays. 

Introduction

Social memory is a core feature of mammalian social behavior and is particularly well developed in highly social species such as mice and humans. In rodents, recognition of others underlies behavioral dynamics critical for survival within family and conspecific units, including group cohesion, dominance hierarchies, mating behaviors, and parental care^1,2. In addition to its evolutionarily advantageous role in animal survival, social memory disruption in humans is a pervasive hallmark across multiple neuropsychiatric and neurological disorders, including autism spectrum disorder, schizophrenia, and Alzheimer's disease^3,4,5,6. Because social recognition is fundamental to the daily functioning of social species and has clear clinical importance, developing behavioral assays that accurately capture its multidimensional nature and neural underpinnings is vital. While existing paradigms measure some aspects of social recognition in mice, most assays are designed to assess novelty preferences by comparing novel versus short-term relationships formed over minutes or hours, rather than quantifying recognition of long-term social relationships.   

Existing approaches, such as the three-chamber paradigm or the habituation-dishabituation paradigms, assess novelty preference following only brief prior exposure, often limited to 5–15 minutes^7,8,9,10,11. These methods have provided substantial insight into the neural mechanisms of these short-term social memories in mice^12,13. However, naturalistic social memories and social function must incorporate the distinct mechanisms at play in long-term relationships, built via cohabitation, long-term social hierarchies, or mating partners. While some studies do use the three-chamber paradigm with littermates¹⁴ or familiar mice co-housed with the subject for up to 72 h^15,16, the paradigm's primary use still focuses on the simple novel vs. familiar dichotomy. This limits comparisons across multiple different familiar mice. With established social recognition paradigms biased towards studying short-term relationships, we identify a clear gap in our knowledge of the neural mechanisms underlying social recognition of long-term relationships.   

To address this limitation, we developed the four cups paradigm: a simple, flexible assay designed to differentiate recognition of multiple social familiarity levels within a single testing session. In this paradigm, a subject mouse explores four corners of an open arena, each containing a perforated cup holding a conspecific or a control empty cup. The cups contain a long-term familiar conspecific (cagemate), a short-term familiar conspecific with limited prior exposure (10 min), a novel mouse, and an empty control. Investigation time serves as a quantitative index of social recognition and social preference. This configuration allows simultaneous comparisons across familiarity levels while minimizing direct physical interaction and potential aggressive encounters. 

Protocol

All procedures involving animals must be performed in accordance with institutional guidelines and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). 

1. Animal preparation and housing  

1. Use adult same-sex group-housed mice for all experiments.  Handle mice for at least 2 days to habituate to handling and decrease anxiety prior to experimental testing.
2. House the mice in standard laboratory cages under a reverse light-dark cycle with ad libitum access to food and water. 
   NOTE: Adult CD-1 and C57BL/6 mice (3–4 months of age) were used for representative experiments. Mice were housed in groups of 3–4 animals per cage. Representative datasets included 10 male CD-1 mice, 12 female CD-1 mice, and 31 male C57BL/6 mice. The behavior should be run in the subject's active phase (dark phase). Mice included in this study ran between 9 am–6 pm and were kept in a reverse light-dark cycle (dark phase at 8 am–8 pm).
3. Define a long-term familiar agent as a same-strain and same-sex cagemate that has cohabited with the subject mouse in the same cage for a minimum of 3–4 weeks prior to testing. 
4. Select short-term familiar and novel conspecific agents from the same strain and sex as the subject mouse. 
5. Ensure that novel mice have had no prior physical or olfactory exposure to the subject mouse. 
6. Select a novel agent and a short-term familiar agent from different home cages to avoid similar olfactory cues in those two agents.
   NOTE: Record approximate ages for all animals used and minimize reusing the same novel and short-term agents multiple times a day. If the agents are to be reused, provide >2 h of break in homecage between test trials to avoid stressed agents.

2. Arena setup 

1. Place a clean, square open-field arena (49.5 cm x 49.5 cm x 49.5 cm; Table of Materials) in a quiet behavioral testing room.
2. Illuminate the area using a single tall or overhead lamp adjusted to approximately 8–20 lux.  
3. Place four identically weighed perforated plastic cups in each corner of the arena (Figure 1A).
   1. Use visually identical cups whenever possible. If different colored cups are used, counterbalance cup assignments and locations across trials.
   2. Press two sides of each cup flush against the arena walls to restrict access, minimizing hiding behavior and standardizing investigatory angles. 
   3. Use cups that are sufficiently heavy to prevent escape and sufficiently tall to prevent climbing.
      NOTE: In this study, the representative data cups and agent location were pseudorandomized across subjects by rotating the colored, plastic cups clockwise while agent type was moved counterclockwise across trials.
4. Place the camera high enough that the entire assay box is in frame to avoid blind spots during behavioral coding. Maintain minimal glare and consistent illumination across the box floor (Figure 1A).
5. Clean the arena floor and cups thoroughly between trials using an appropriate disinfectant, followed by water to remove residual odors. 
6. Allow the arena to dry completely before reintroducing animals. 

3. Habituation to arena on Day 0

1. Place the subject and its cagemates into the arena containing four empty perforated cups and allow free exploration for 20 min.
   NOTE: Habituate subjects individually for tethered experiments. Additional habituation sessions may be required. It is recommended to conduct multiple habituation days if mice do not fully explore the entire arena after one day.
2. Remove all mice from the arena and return to the home cage.
3. Place all social agents under cups for 10 min to habituate them to being restrained. Habituate agents from the same home cage simultaneously, one per cup (Figure 1B).
4. Clean the arena and cups prior to introducing mice from a new home cage.
   NOTE: Allow mice to rest for at least 15 min after transport to the room before beginning the session.

4. Baseline 

1. Set up the arena according to section 2. Maintain the same arena configuration across all experimental days. 
2. Place the subject into the arena containing four empty cups and allow free exploration for 10 min.
   NOTE: Do not pause the recording at any time, allowing it to continuously record throughout all stages of the current trial.  

5. Short-term acquisition 

1. Use a weighted perforated cup to confine the subject while placing the designated "short-term familiar" agent. Alternatively, expose the subject to the short-term agent in a clean cage.
2. If choosing to do the arena exposure, confine a designated short-term familiar agent under one perforated cup in the arena, leaving the other three cups empty. 
3. Release the subject and allow free exploration for 10 min (Figure 1B).
   NOTE: If using the method of exposure to a short-term agent in an arena, alternate the corner used for the short-term familiar agent across subjects to avoid spatial bias. 

6. Inter-trial interval (ITI) 

1. Retrieve the short-term familiar agent and place it back in its home cage. Allow the subject mouse to remain alone in the arena and move freely for 10 min. 
2. Gather novel and long-term familiar agent mice for the test phase.  
   NOTE: Return the short-term familiar agent to the same cup used during short-term acquisition (section 5). This is recommended to avoid novelty in spatial location if doing short-term acquisition in an arena.

7. Social recognition phase 

1. Place an additional weighted perforated cup on the subject mouse.
2. Place one mouse under each of the three weighted perforated cups: a long-term familiar (cagemate), a short-term familiar, and a novel conspecific. Leave a designated fourth cup empty as a control.  
   NOTE: Label cup tops to identify agent locations during behavioral scoring.
3. Remove the perforated cup from the subject mouse in the center of the arena and allow free exploration for 10 min (Figure 1A).
4. Remove all mice at the end of the 10 min interval and stop recording.
5. Return all mice to their home cages. 
6. Clean the arena and cups thoroughly before the next trial.  
   NOTE: Keep replacement cups available in case of damage.

8. Behavioral scoring

1. Import all recorded videos into behavioral analysis software capable of manual annotation, such as Behavioral Observation Research Interactive Software (BORIS).
   NOTE: Behavioral scoring was not blinded to cagemate location, given that labels with agent ID were located above each cup and visible in the videos, and cage number could be inferred. Scorers were not actively paying attention to visible labels and were blinded to the identity of the novel and short-term agent. All scorers followed predefined operational definitions of investigatory behavior and standardized scoring criteria.
2. Define the behavioral matrix before scoring.
3. Define an investigation bout as the subject mouse positioning its nose within 1 cm of a cup while exhibiting sustained investigatory posture. 
   NOTE: Include chewing and gnawing directed at the cup as investigation bouts.
4. Manually annotate the total investigation duration for each cup across the 10-min social recognition test phase. Score baseline and acquisition as desired.
   NOTE: Automated pose tracking may be used with a 1 cm semicircular region of interest surrounding each cup.
5. Export annotated data for subsequent statistical analysis.

Results

Following the four cups paradigm protocol, CD-1 male mice exhibited differential social investigation behavior across the long-term familiar (cagemate), short-term familiar, and novel conspecifics (Figure 2). For each agent, a discrimination index (DI) was calculated based on seconds spent investigating in that agent vs the empty cup, calculated as (agent − empty)/(agent + empty) during the 10-min social recognition phase. This index was normally distributed based on the Shapiro-Wilk test; t...

Discussion

There are key considerations for applying the four cups paradigm to effectively and reliably measure these social dynamics in other laboratory settings. Habituation on Day 0 reduces non-social novelty-driven exploration of the arena and cups, which can otherwise mask social investigation during testing^7,8,9. Precise control of the short-term familiarization duration is also essential, as small differences in exposure time can si...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Behavioral Scoring Software	Behavioral Observation Research Interactive Software	Version 9.12.7	Software for manual event annotation
Open-field arena	Rafferty Machine and Tool	Custom made	Structural FRP fiberglass sheet, square arena (49.5 cm x 49.5 cm x 49.5 cm); Non-transparent walls, if arena transparent, cover outside walls with paper to minimize distractions for animal
Overhead camera	Logitech	Logitech C920x HD Pro PC Webcam	Camera capable of continuous video recording; Mounted above center of arena
Perforated cups	Chinco Store	ONG-Chinco-0279	dimensions of cups: 4.01"L x 4.01"W x 4.52"H; Perforated plastic or metal cups, could be square or cylindrical
Prism	Graph Pad	Version 10.6.0	Software for data analysis; Could be done with other software