Materials and Reagents

Clean paper towels
Intruder (experimental) adult male Sprague-Dawley rats
Resident (aggressor) adult male retired breeder Long-Evans rats
Scoring sheet (Table 1)
70% ethanol

Disinfectant wipes for equipment (for example, Caviwipes Surface Disinfectant Wipes, Metrex item# 13-1100)

Table 1. Scoring sheet.
This is a standard scoring sheet that can be useful for the social defeat procedure. After submission, or if any criteria (a-c) are met, the Phase 1 session ends and the intruder rat is placed in the wire mesh enclosure. The Phase 2 session (separated by wire mesh enclosure) ends after 15 minutes. See completed sheet below for more details (Table 2).

Resident Intruder Stress				Protocol:		Your Name:
Resident rat + cage is placed in procedure room
After at least 5 min, the Intruder rat is brought into the procedure room in a transport cage; the rat is placed in the cage with the Resident. Timer is started.
After submission, the Intruder is placed into a smaller wire mesh enclosure within the Resident cage for 15 min
END Phase 1 session after submission or: a. 15 min with no submission b. 10 attacks with no submission c. 5 min with no attacks END Phase 2 after: a. 15 min after placement in wire mesh enclosure
Intruder (Stressed) Rat Number	Date	Resident Rat Number	Start Time	Time to First Attack	Number of Attacks	Submitted? (Y or N)	Time Separated
















Comments:

Equipment

Resident rat housing cages: sanitized woodchip bedding, 21” × 11.5” × 8” height, clear polycarbonate, wire top
Rat transport cages: sanitized woodchip bedding, 12” × 6.5” × 5” height, clear polycarbonate, microisolator top
Smaller wire mesh enclosure (Figure 3): approximately 6” × 7” × 8” height, 3/4” square mesh, PVC coated
Digital stopwatch with time of day

Procedure

RSDS by the resident-intruder paradigm is a robust model of chronic psychological stress in rodents (Rygula et al., 2005; Berton et al., 2006; Liu et al., 2017). In our study, housing cages of rats were randomly assigned to the stressed and control groups.
Note: Most Long-Evans retired breeders are aggressive in our conditions, but some are not. You can screen these rats prior to experiments, using the same resident-intruder approach described below, to select more or less aggressive rats depending on your needs. If more aggressive resident behavior is required, pre-screening criteria may include short latency to attack and multiple attacks within the first minute of the session. In our experiments, the retired breeders displayed reduced attacks over the course of weeks-months. If a retired breeder displayed no attacks, it might be removed from the rotation of aggressive residents.
The housing cages of the resident aggressor Long-Evans rats, containing one Long-Evans rat per cage, were transported to a dimly lit procedure room free from noise within the animal facility on the morning of the experiments. They were left undisturbed for acclimation in the environment for 15-30 min.
Note: In our experience, a single experimenter can perform this procedure with up to 3 resident-intruder interactions at the same time (i.e., 3 resident cages in the procedure room). More than this becomes difficult to observe by one individual.
Experimental Sprague-Dawley rats were weighed daily in their housing room and their body weight was noted. Animal condition was also noted, with attention to porphyrin around the eyes, nose, or fur.
After weighing, the Sprague-Dawley rats were placed in clean transport cages.
1. Control rats (housed together) remained in transport cages for 30 min, then they were returned to their home cage.
2. Rats in the stress group were transported to the procedure room in clean transport cages separate from the controls.
PHASE 1 (Direct physical contact): Individual Sprague-Dawley rats were transferred to the housing cage of a resident Long-Evans rat in the procedure room and the lid of the cage was secured tightly. This allowed the intruder rat to stay in direct physical contact with the resident rat (Figure 1). The time of this event was noted. The rats were continuously observed for the entire duration of the interaction.
All scoring of a cohort of rats was performed by one trained experimenter. The experimenter was positioned at a distance that allows clear observation of the rats and rapid intervention, but far enough that rats do not direct attention towards the experimenter (approximately 1-2 meters). The measures can be reliably obtained by one experimenter, and we therefore did not routinely video sessions.

Figure 1. Setting up the resident intruder procedure. A. Photographs of the resident cage with the wire mesh enclosure (green) placed inside (left). Also shown with resident present (right) for scale. B. Phase 1 of the stress procedure, when resident and intruder can have direct physical contact. C. Phase 2 of the stress procedure, when resident and intruder are separated by the wire mesh enclosure.
During the period of direct physical contact, every attack made by the aggressor rat on the intruder was scored manually by a trained observer, including noting down the time of first attack (Table 2). A defeat was scored when the intruder rat submitted to the attack of the aggressor by lying down and exposing the ventral surface (abdomen; Figure 2C).
This direct physical contact was allowed for a maximum of 15 min each session. The experimental rat was separated with the wire mesh enclosure prior to 15 min if any of the following criteria was met: (i) submission of the intruder; (ii) ten attacks with no submission; (iii) 5 min with no attack; or (iv) any attack that severely wounded the experimental rat. Thus, it was possible for a rat to be removed without demonstrating submission each day. Parameters can be modified if experimental design calls for daily defeat.
PHASE 2 (Physical separation): The intruder was separated within the resident rat’s cage using a smaller wire mesh enclosure for an additional 15 min (Figure 1C, Figure 2C). The time of physical separation was noted.
Note: The wire mesh enclosure used for physical separation was spacious enough not to cause any form of physical restraint to the movements of either the intruder rat inside or the resident rat outside the mesh enclosure. This was done in order to remove only the physical component of the social stress in Phase 2, without affecting the sensory (visual, auditory, and olfactory) components associated with the stressors (aggressor rats).
All surfaces were thoroughly cleaned with 70% ethanol between experiments, including wire mesh enclosures.
This same procedure was repeated for five consecutive days, once per day for each intruder rat. Resident-intruder pairings were cycled such that each intruder experienced a novel resident each day.
Notes:
a. Resident rats were limited to three daily sessions. After three daily sessions, or after extended periods of time (weeks to months), resident retired breeder rats tend to display less aggression. It has also been noted that resident rats tend to be less aggressive on the day of changes to a clean housing cage.
b. If a rat were to get badly wounded during an attack (eye damaged, large wound) it would be removed and humanely euthanized. In some instances, a rat might receive a wound that is not severe (e.g. <2 cm to flank, without apparent muscle damage). If this occurs, the wound should be flushed with sterile saline followed by application of antibiotic+analgesic cream (e.g. Neosporin dual action cream). The rat should be monitored daily. If the wound does not close or otherwise show significant improvement within two days, the rat should be euthanized.
c. If your endpoint includes immunological measures, you might consider removal of any rat that has been scratched, and pre-screening resident rats for those that do not tend to cause any wounds.

Figure 2. Postures during attack and defeat. A. Examples of social interaction, followed by a common posture that signals an imminent attack: the resident rat rotates so it is flank-to-flank with the intruder and then lifts the hind leg adjacent to the intruder, to maintain position before it enters the next step. B. The next steps that escalate into the most common attack. The resident rat twists its body while keeping the hind leg close to the intruder, and then turns under the intruder. This flips the intruder over, with the resident on top. In other less common instances, the resident rat may initiate and continue an attack from the top position, for instance when both rats are in an upright boxing stance (not shown). After attacks, the intruder may avoid the resident rat. C. After the experience, the intruder rat shows a typical defeat posture. At this point in the protocol, the rats are separated with a wire mesh enclosure.

Table 2. Example of a completed scoring sheet for a five day repeated social defeat protocol of three intruder rats (rats 1-3) that were exposed to a rotation of resident rats (rats 1-3, and 6-9).

Instructions to make the wire mesh enclosure
There are commercially available enclosures for pets that may be suitable. We have never tried those options and have instead made our enclosures from supplies available at most hardware stores. There are a range of suitable options for materials to make a wire mesh enclosure. We chose a PVC-coated steel mesh, because it is easy to cut, bend to the desired shape, and can be readily cleaned. A mesh opening of 0.5-1 inch is suitable for our purposes, because this is too small for the resident rat to effectively attack through. We chose a mesh of 16 gauge because of its durability and weight. The final dimensions are approximately 6 × 7 × 8 inches.

Materials
Heavy duty wire cutters
Pliers
Coated wire mesh (for example, item 9259T11 from McMaster-Carr; www.mcmaster.com)
Zip ties (for example, item 6705K35 from McMaster-Carr; www.mcmaster.com)

Steps:
1. Cut the wire mesh to pattern (Figure 3).
2. Bend where indicated to form an open cube.
3. Fasten corners together with zip ties. Trim the ends of zip ties.

Figure 3. Photograph of completed wire mesh enclosure. This enclosure has lasted eight years of use with minimal damage. A template and instructions are shown here.

Data analysis

Exclusion criteria: The intensity of the repeated stress experience can be varied, depending on the experimental needs. In our studies, rats had to exhibit at least one instance of defeat, demonstrated by a passive defeat posture (see Figure 2). Experimental rats were excluded if they received a severe injury (see Step 10 Notes, under the “Procedure” section). Resident rats were excluded if they exhibited two sessions without attacks, or if they consistently attacked with a severity that produced injury.

During the period of direct physical contact, every attack made by the aggressor rat on the intruder was scored manually by a trained observer, including noting down the time of first attack and the time of physical separation (Table 2). The following parameters were analyzed: number of attacks made by the aggressor on the intruder rats, whether rats submitted, and if so, time of submission by the intruder rat (i.e., latency to submit, which will be the same as the “Time separated” in our score sheet), and the number of rats that submitted daily.

Conclusion

In this article, we discussed the protocol for the induction of RSDS in rats that we used in our study, showing that RSDS is able to induce robust immunological and behavioral changes in rats (Munshi et al., 2020). This builds on many prior studies that have successfully achieved social defeat in rats and mice.

Acknowledgments

The authors gratefully thank Matthew Anagnostopoulos and his team at the Biological Resource Facility at Rosalind Franklin University of Medicine and Science for taking care of the animals used in the study. The study was supported by the National Institutes of Health grants MH084970 and MH109484. The funding body had no role in the design of the study, collection and analysis of data, or decision to publish. The protocol is based on the original research article by Munshi et al. (2020) published in Brain, Behavior, and Immunity.

Competing interests

The authors declare no competing interests.

Ethics

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Rosalind Franklin University of Medicine and Science and were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

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