May 12, 2026
Modified Juvenile Reciprocal Social Interaction (JRSI) Protocol (cross-group dyads)
- 1Indian Institute of Technology Indore
Protocol Citation: Siddharth Singh 2026. Modified Juvenile Reciprocal Social Interaction (JRSI) Protocol (cross-group dyads). protocols.io https://dx.doi.org/10.17504/protocols.io.261geyy4dv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 12, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 316878
Keywords: standard mouse juvenile reciprocal social interaction, juvenile mice, total social interaction time, metrics such as total active social time, interaction test, interaction symmetry ratio, social behavior, relative engagement, total active social time, effects models with dyad type, jrsi, initiation count
Abstract
This protocol further develops the standard mouse Juvenile Reciprocal Social Interaction (JRSI) assay to compare social behavior in same- versus mixed-treatment dyads. Juvenile mice (postnatal days ~23-25) are paired (same-sex, unfamiliar) in a neutral arena for a 10‑min free-interaction test. Each partner’s actor and receiver behaviors are scored independently using a directional ethogram, yielding metrics such as total active social time, initiation counts, and an Initiation Symmetry Index (ISI) and Interaction Symmetry Ratio (ISR) that quantify each dyad member’s relative engagement. A custom Point Metrix system assigns weighted points for actions (approach, sniffing, grooming, etc.), with mutual events (nose-to-nose sniff, etc.) shared equally. Key derived indices (ISI, ISR, other asymmetry measures) are defined by simple ratio formulas. Data are captured at the event level and summarized in individual- and dyad-level tables. Blind scoring by trained observers (target ≥80% agreement) is recommended, with optional automated tracking as a supplement. Statistical analysis employs linear mixed-effects models with dyad type as a fixed effect and litter as a random effect, including relevant covariates (weight, locomotion). Planned comparisons focus on “within-” versus “across”-group pairs, and primary endpoints include total social interaction time and symmetry indices.
Materials
Apparatus and Environment
- Arena: Use a neutral open-field box or an automated social arena of ~30×30×30 cm (for juveniles). Cover the floor with a thin layer (~1 cm) of clean corn-cob bedding (no home-cage bedding, to avoid familiar scents). For the 3-chamber apparatus, clear walls 20–30 cm wide can be used (with partitions removed for RCI). Ensure the arena is non-reflective and free of distracting odors.
- Lighting: Test under dim diffuse light (∼30-100 lux at floor) to minimize anxiety. No strong directional lights or shadows.
- Recording: Mount a high-resolution video camera directly overhead (top-down view). Record continuously at ≥30 frames/sec to capture quick juvenile interactions. Ensure the full body of both mice is visible; white or light-colored floor provides contrast.
- Cleaning: Sanitize the arena between trials to remove odors. Wipe floors and walls with 70% ethanol (or a neutral unscented disinfectant) and let dry. Use separate bedding for each trial.
Objective
To quantify juvenile social reciprocity and engagement when one versus both mice in a dyad have received different treatments. By comparing same-group and mixed-group dyads, we can test if an experimental treatment alters social initiation, response, and avoidance relative to controls. Outcomes include total active social time (sniffing, following, play) and symmetry/asymmetry metrics between partners.
Experimental Design
Groups and Dyad Types: Mice are raised in >3 litters per group, with balanced sex and age. Dyads (same-sex, same-age, but different subjects) are formed as all pairwise combinations: same-group and mixed-group. Assign each subject to a single dyad to avoid repeated testing effects. Littermates must be spread evenly across dyad types to avoid litter confounds. Include randomization by assigning pups from each litter to different groups/dyads.
Sample Size: Aim for ≥10–15 dyads per comparison (ideally, power>0.8 based on estimated effect sizes). Conduct an a priori power analysis (α=0.05, β=0.8) using an estimated moderate effect size. Mixed-effects models include litter as a random effect to account for intra-litter similarity.
Sex and Age: Use one sex (male or female) to reduce variance, or sex-balance, and include sex as a covariate/random effect. Mice should be in the juvenile period (~P23-25). This avoids the immediate weaning stress at P21 and aligns with the established play peak. All mice should be in good health and in a similar weight range (weigh and record prior to testing).
Animal Preparation and Pairing
Housing Pre-test: Keep dyad partners in separate home cages (different litter) until testing. Immediately before the test, singly house each mouse for ~30-60 min (brief habituation) to minimize novelty stress. Avoid long isolation, as extended social deprivation itself alters juvenile play.
Identification: Mark mice with non-toxic fur dye or tail marks for ID. Wear gloves and handle gently during marking. Ensure the scorer is blinded to group identity.
Pairing Rules: Form dyads with unfamiliar mice that have never met. Use each mouse only once (one dyad per subject) to prevent learning or familiarity effects. Prefer that each dyad comes from different litters if possible. Test in a counterbalanced order of dyad types across days.
Pre-test Handling and Habituation
Handling: Acclimate mice to the experimenter and transport. On the day of testing, handle each mouse gently for a few minutes before isolation.
Habituation to Arena: Optionally, allow each subject a short (2-5 min) exploration of the empty arena alone prior to pairing (then remove and wait a few minutes) to minimize novelty effects, or simply use the isolation cage as a habituation environment. Avoid extensive habituation or multiple exposures to keep the
test maximally social-motivated.
10-Minute Trial Procedure
Set up: Place clean bedding in the arena. Turn on the camera and check the view.
Isolation: After habituation, place Subject A alone in the arena for 1 min to further acclimate (optional). Then briefly remove A.
Pair Introduction (t=0): Gently introduce Subject B into the arena at a standardized location opposite to where A was placed (or release simultaneously). Start video recording immediately.
Interaction (t=0–10 min): Allow subjects to freely interact. Do not intervene unless necessary. Record for a fixed 10-min session. Use a stopwatch synced with video.
Termination Criteria: If aggressive behavior occurs (defined as prolonged fighting with bites or injuries), immediately stop the session and safely separate the mice. Note aggression events. Typically, with juvenile same-sex pairs, this is rare, but monitor them closely.
End of Test: At 10 min or on termination, remove both mice. Return each to its home cage.
Cleaning: Clean the arena as above and allow it to dry before the next trial.
Ethogram and Scoring (Directional)
Behaviors are scored using a directional ethogram: one mouse is the actor (initiator) of an event, and the other is the receiver. Table 1 below list typical social behaviors and the points assigned to actor and receiver in our Point Metrix system. Non-social behaviors - e.g., self-grooming, rearing, digging – are noted as controls but not given social points.
Short-Event Rule: Very brief interactions (<0.5 sec) are counted as a minimum duration of ~0.1 sec to avoid undercounting. Points are awarded per bout, regardless of duration, but we accumulate duration-weighted scores by multiplying point value by seconds of engagement.
Mutual Events: Only nose-to-nose sniffing is scored as mutual. (If desired, side sniffing could be split similarly, but here we treat it as directional.)
| Actor Behavior | Points to Actor | Notes | |
| Approach from front | 1 | A moves toward B’s front/body | |
| Nose-to-nose sniffing | 0.5 | Mutual; each mouse gets 0.5 | |
| Anogenital sniffing | 1 | Sniffing partner’s rear/side | |
| Side (flank) sniffing | 1 | Sniffing partner’s lateral body | |
| Partner grooming | 1 | Grooming actions directed at partner | |
| Following | 1 | Pursuing partner, maintaining contact | |
| Pushing/Crawling under-over | 1 | Pushing under or crawling over partner | |
| (others, e.g. self-grooming, exploration) | 0 | Not counted as social (control measures) | |
| Receiver Behavior | Points to Receiver | Notes | |
| Nose-to-nose sniffing | 0.5 | Mutual (see above) | |
| (all other events) | 0 | Passive partner (no points) |
Table 1. Directional ethogram and point scoring for actor and receiver. These categories follow established JRSI scoring. Each observed bout is scored for its initiator and recipient. Note that nose‑to‑nose sniffing is treated as a mutual event (each receives 0.5). All other social actions are directional (actor=1 point).
Point Metrix and Derived Indices
From the scored data, define key metrics:
Total Active Social Time (T): Sum of durations of all social interactions (all scored events) per subject. This measures overall social engagement.
Total Social Points (S): Sum of points earned (actor points from Table 1) by a subject. This is weighted by behavior type and roughly correlates with social activity.
Initiation Count (I): Total number of events initiated by the subject (any scoring event where it was an actor.
Initiation Bias: IA - IB, or fraction (IA-IB)/(IA+IB), indicating which subject initiated more.
Avoidance Duration (A): Cumulative time a subject spends avoiding the partner (e.g., turning away, freezing on contact, or breaking contact). (Observers must code avoid behaviors when the partner approaches.) We assign 1 point per second of avoidance.
Symmetry/Asymmetry Indices
From these, compute symmetry/asymmetry indices for each dyad (label subjects as A and B):
Interaction Symmetry Index (ISI): ISI = (SA - SB)/(SA + SB) where SA,B are the total social points for each subject. ISI ranges from -1 to +1 (0 = equal scoring).
Initiation Symmetry Ratio (ISR): ISR = (IA - IB)/(IA + IB), analogously using initiation counts.
Non-social Asymmetry Index (NAI): As an example metric, one may compute (points in non-social behaviors) contrast: NAI = (GA - GB)/(GA + GB) where GA,B are points for self-grooming/exploration (control behaviors) for each subject. (This highlights asymmetry in non-social activity.)
Duration Ratio Index (DRI): For total active social time: DRI = (TA - TB)/(TA + TB) using durations TA,B. This is analogous to a discrimination ratio used in social preference tests.
Avoidance Asymmetry Index (AAI): AAI = (AA - AB)/(AA + AB), comparing avoidance times.
All formulas are simple difference-over-sum measures, yielding values in [–1,+1] (with 0 indicating symmetry).
Reporting and Outcomes
Primary outcomes (e.g., TotalSocialTime, ISI, ISR) should be highlighted and justified in advance. Secondary outcomes (specific sniffing or grooming counts) can support mechanistic interpretation. Include negative controls (self-grooming, exploration) to rule out nonspecific effects. Tables should summarize group means (±SEM) and statistical comparisons.
| Dyad Type | Primary Measure | Hypothesis/Comparison | Recommended Outcome | |
| C–C vs A–A | Total social time, ISI | Does A treatment alter sociality? | Primary (social duration) | |
| C–C vs H–H | Total social time, ISI | Does H treatment alter sociality? | Primary | |
| A–A vs A–H | ISR (initiation bias) | Does A mice interact with H mice differently than with A? | Secondary (asymmetry) | |
| C–C vs C–A | DRI, nose-sniff count | Does one A mouse affect a C mouse? | Secondary | |
| (etc.) |
Table 2. Example dyad comparisons and endpoints.