Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Summary

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Abstract

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience and its behavioral impact on mice should be comparable to humans. Chronic social defeat (CSD) utilizes a type of social stress involving physical attacks and sensory threats to induce mental dysfunctions resembling human affective disorders. To strengthen the psychosocial component of CSD, a 10-day CSD protocol was applied in which daily physical attacks are standardized to three 10 s episodes followed by a 24 h sensory phase. After the 10^th sensory phase, the CSD protocol is followed by a refined behavioral assay called the social threat-safety test (STST). Post-stress behavioral assays need to determine how and to what extent the social stressor has influenced behavior. The STST allows chronically socially defeated male mice to interact with 2 novel male individuals (social targets): one social target from the attacking strain encountered during the CSD days and the other from a novel strain. Both are presented simultaneously in different compartments of a three-chambered test arena. The test enables a simultaneous assessment of social avoidance development to measure successful aversive conditioned learning and social threat-safety discrimination ability. The development of social avoidance towards both strains reflects a generalized aversive response and thus, a measurement of stress susceptibility. Meanwhile, the development of social avoidance towards only the attacking strain reflects threat-safety discrimination and thus, a measurement of stress resilience. Finally, the absence of social avoidance towards the attacking strain reflects impaired aversive conditioned learning. The protocol aims to refine the currently used mouse models of stress susceptibility/resilience by including translational criteria, specifically threat-safety discrimination and aversive response generalization, to categorize a single group of chronically socially defeated animals into resilient and susceptible subgroups, eventually advancing future translational approaches.

Introduction

Stress is defined as the disruption of homeostasis caused by physical or psychological stimuli¹. Stress is a well-known major risk factor for the development of mental disorders such as post-traumatic stress disorder, depression, and anxiety^2,3. In particular, social stress is considered a major risk factor for the development of stress-related mental disorders⁴. One type of social stress that has gained particular importance in research is social subordination stress⁵. Mice, like humans, are capable of a rich set of social behavior⁶, rendering them suitable for investigations involving social stress. In the laboratory setting, when adult mice are group housed, they establish a social structure involving the formation of ranks⁷. Accordingly, the colony model was designed to study the effects of naturally established social hierarchies in mixed-sex groups of mice⁸. Over the years, variations of the colony model have been developed to utilize social subordination stress, including same-sex group models, the social instability model, and the intruder-colony model. In recent years, however, one particular variant known as the male resident-intruder model has been popularized in literature, simplifying the social complexity to two mice: a resident and an intruder. The animal of interest, known as the intruder, is placed into the cage of a larger, older, and retired breeder, known as the resident or aggressor. The resident then physically attacks the intruder as a method of confrontation, establishing a social hierarchy wherein the resident is dominant and the intruder is subordinate. When confrontations are one-time events, they classify as "acute" (the "acute social defeat model"), whereas repeated confrontations lasting over several days (usually 10) are known as "chronic" (the "chronic social defeat model"). In the chronic social defeat (CSD) model, attacks are intermittent and typically confined to a period of 5-10 min⁹, termed the physical phase. Following the physical phase, the intruder and resident are kept overnight in the same cage, separated in half with a mesh wall, allowing for all forms of interaction except physical contact. This configuration, known as the sensory phase, induces stress through the continuous appearance of threat instead of direct physical confrontation. In 2018, van der Kooji and colleagues introduced a modified chronic social defeat treatment to focus on the psychosocial component of the model by standardizing and strictly limiting the physical phase¹⁰. The modified model limits physical attacks to three 10 s episodes with different residents, occurring in 15 min inter-episode intervals of the sensory phase. Following the third physical episode, the sensory phase lasts overnight. This cycle repeats for 10 consecutive days with new residents per episode. The modified treatment enhances the translational validity of the chronic social defeat model as physical harm of the intruder is minimized, and outcome variability from differential durations of physical attacks is reduced.

Since the CSD model is utilized to study stress-related illness (e.g., depression, anxiety, post-traumatic stress disorder), post-behavioral assays are chosen, including, but not limited to, behavioral assays of aggression, memory, and anhedonia. In recent years, post-CSD behavioral assays in mice often evaluate how and to what extent sociability is affected⁹. Sociability is defined as the innate preference of mice to socially interact rather than socially avoid a conspecific. Since sociability is subject to stress effects, assays that solely assess social avoidance development were established. Stress-induced social avoidance has a translational relevance as it represents one of the main behavioral symptoms of social anxiety and depression in humans¹¹. Similar to humans, not all mice develop social avoidance following CSD treatment, suggesting the presence of individuality in stress responsiveness. Cohen and colleagues have proposed cut-off behavioral criteria to be a promising approach for studying the neurobiology of individuality¹². Selection of animals based on behavior results in group division, underlining the basis for gene-environment studies. Subsequently, different subgroups often show distinct enrichment of specific genetic variants/modifications, which in turn can be investigated under different environmental conditions¹³. Accordingly, individuality in the development of social avoidance was utilized to divide the single group of chronically socially defeated male mice into two subgroups: stress susceptible (socially avoidant) and stress resilient (socially non-avoidant^9,14). However, the interpretation of the social avoidance phenotype in mice as a maladaptive or adaptive behavior should be considered in the overall context of both the treatment (here CSD) and post-treatment behavioral assay. Additionally, the post-treatment behavioral assay of choice would ideally assess other facets of sociability and not solely social avoidance development. Our recent work revealed the involvement of conditioned learning in CSD-induced social avoidance¹⁵. Specifically, CSD-induced social avoidance is an aversive conditioned response towards the characterizing traits of the residents' strain serving as the conditioned stimulus to the unconditioned stimulus, namely the attacks by the residents. Moreover, within the socially avoidant subgroup, some individuals can discriminate between the traits of the aversive residents' strain and those of other safe novel strains, while other individuals show generalized social avoidance to both strains. We propose here a refined behavioral post-CSD assay: the Social Threat-Safety Test (STST)¹⁵. Unlike other social interaction tests⁹, the STST enables a simultaneous assessment of social avoidance development as a measurement of the correct aversive conditioned response (i.e., successful conditioned learning) and social threat-safety discrimination ability, both of which are utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice. The assessment of social threat-safety discrimination versus aversive response generalization extends the translational criteria used to classify the single group of chronically socially defeated animals into resilient and susceptible subgroups.

Protocol

All procedures were performed in accordance with the European Communities Council Directive regarding the care and use of animals for experimental procedures and were approved by local authorities (Landesuntersuchungsamt Rheinland-Pfalz). Figure 1 represents a schematic timeline.

1. Treatment

1. Animals of interest: Obtain C57BL6/J male mice at 7 weeks of age, and upon arrival, single-house in a temperature- and humidity-controlled facility on a 12 h light-dark cycle (lights on: 8:00; lights off: 20:00; 23 °C; 38% humidity) with food and water ad libitum.
2. Chronic social defeat (CSD)
   1. Treatment group
      1. Following 1 week of habituation, perform CSD treatment for 10 consecutive days using the CD-1 strain as the resident's strain (for a detailed protocol, refer to chronic social defeat⁹ and the modified chronic social defeat treatment¹⁰).
      2. Introduce the C57BL6/J mouse into the cage of the CD-1 mouse and count 10 s of physical attack. Repeat this episode three times, each with a different CD-1 mouse, and separate by 15 min inter-episode intervals.
      3. Place a mesh wall between the C57BL6/J mouse and CD-1 mouse during these intervals, allowing only sensory contact. Following the third episode, house the C57BL6/J mice overnight in the cages of the CD-1 mice, separating both by a mesh wall. Repeat for 10 days.
         NOTE: CD-1 mice number = C57BL6/J mice number + 1. If the number of C57BL6/J treated mice is below 10 then a minimum of 10 CD-1 mice is still needed to ensure that every day the last sensory phase (lasting overnight) is with a new CD-1 mouse throughout the 10 days of treatment.
      4. Carefully assess the physical well-being of the animals throughout the 10 days. If an animal is severely wounded, exclude it from the experiment for ethical and scientific (mobility/activity during the post-treatment test) reasons. Table 1 provides a well-being checklist.
   2. Control group
      1. Upon arrival, maintain same-age mice in the same conditions as the treatment group.
      2. Following 1 week of habituation, introduce the control animals for 90 s in an empty cage and then return them to individual cages (single-housed) separated in half by mesh walls identical to those used for the treatment group. Perform this daily in parallel to the 10 treatment days.
         NOTE: It is advisable to keep the control group and the treatment group housed in different rooms.
3. After the last (10^th) sensory phase, single-house all mice in new cages in similar conditions to those described upon arrival and leave them to rest overnight.
   NOTE: The last sensory phase should last 24 h, then animals are single-housed.

2. Post-treatment test: Social threat-safety test (Figure 2)

1. Following CSD treatment, single-house all mice (treated and control groups) in new cages in similar conditions to those described upon arrival and leave them to rest overnight.
2. During the morning hours (8:00-13:30), clean the three-chambered arena (rectangle in shape with a total size of 60 cm x 40 cm, made of transparent acrylic walls and smooth floors) with 5% ethanol and place it under the camera with light conditions of 37 lux. Ensure that the entire arena is visible.
3. Clean the mesh enclosures (cage-like made from metal or acrylic) with 5% ethanol and position them as shown in the corners in Figure 1A.
4. Habituation phase: Introduce the animal of interest at the center of the arena, allow for exploration for 6 min, and then return them to their home cage.
5. Place the novel (unknown) CD-1 social target (conspecific) under one mesh enclosure and the novel 129/Sv social target under the other mesh enclosure.
   NOTE: It is important to use an unknown 129/Sv conspecific to avoid a familiarity bias. Preferably have 4 mesh enclosures per arena: 2 attributed to the habituation phase and 2 to the testing phase.
6. Testing phase: Immediately re-introduce the animal of interest at the center of the arena and allow exploration for 6 min.
7. Return all animals to their home. Clean the arena and mesh enclosures with 5% ethanol between tests of different animals, but never during the observation of the same animal, i.e., never between habituation and testing phases.
8. Alternate the location of the mesh enclosures between animals (and never between the two phases within the same animal) to control for possible location preferential bias.

3. Scoring and analysis

NOTE: Only the post-stress treatment test, i.e., the STST is scored and analyzed (and not the CSD stress treatment).

1. Define the interaction zone as 2 cm around the mesh enclosures' boundaries.
2. Score the duration spent exploring the mesh enclosures during the habituation phase when the animal's nose is within the interaction zone.
3. Score the duration spent interacting with the social targets during the testing phase when the animal's nose is within the interaction zone.
   NOTE: Detection can be achieved either manually (using a timer or software for manual scoring) or automatically. Regardless of the detection method, take the nose point for exploration and social interaction measurements and the center point of the body for activity-related measurements (e.g., distance moved).
4. Calculate the social interaction index as follows: time spent exploring each social target during the testing phase / average time spent exploring the two empty mesh enclosures during the habituation phase (Figure 2B).
5. Divide the treatment group into 3 subgroups as follows: Animals with a social interaction index ≥1 with the CD-1 social target are non-avoiders, animals with a social interaction index <1 with both social targets are indiscriminate-avoiders, animals with a social interaction index ≥1 only with the 129/Sv social target are discriminating-avoiders (Figure 2C-D).
   NOTE: The number of animals within each of the three subgroups can differ between different animal batches (about 1/3 of all animals that undergo the CSD treatment will display the phenotypic characteristics of one of the three subgroups).
6. Assess the stress effect by statistically analyzing the social interaction index with the CD-1 social target between the treatment and control groups (either parametric two-sample t-test or nonparametric Mann-Whitney test).

Results

Social interaction index as a measurement of aversive conditioned response
A social interaction index ≥1 reflects greater social interaction with the respective social target compared to the exploration of the empty mesh enclosures. Under baseline conditions, defined here as having neither appetitive nor aversive experience with the characterizing traits of a specific strain (here both social targets to the control group and the 129/Sv social target to the treatment group), intact sociability...

Discussion

The behavioral protocol here describes the Social Threat-Safety Test, used to divide a single group post-CSD treatment into three different subgroups, serving as a method to investigate the underlying biology of stress susceptibility and resilience and to test potential therapies. The biological context and technical details need to be carefully considered to guide a thorough experimental design.

Different housing conditions can alter aggression sociability levels, potentially influencing resu...

Materials

Name	Company	Catalog Number	Comments
Arenas	Noldus, Sociability cage, Wageningen, the Netherlands	https://www.noldus.com/applications/sociability-cage	Three-chambered, rectangle in shape with a total size of 60 cm x  40 cm, made of acrylic transparent walls and smooth floors
Camera for video recording	Basler AG, Germany An der Strusbek 60-62 22926 Ahrensburg	ace Classic acA1300-60gc	If using automatic detection program, make sure cameras are compatible
Camera objective	KOWA Kowa Optimed Deutschland GmbH Fichtenstr. 123 40233 Duesseldorf: LMVZ4411 | 1/1.8" 4.4~11mm Varifokal Objektiv	Part-No. 10504
Detection program/Timer	Noldus, EthoVision-XT, Wageningen, the Netherlands	https://www.noldus.com/ethovision-xt	Detection can be achieved either manually (using a timer or a software for manual scoring) or automatically
Housing cages	ZOONLAB GmbH, Hermannstraße 6, 44579 Castrop-Rauxel	3010010	Type 2 cages: 265 mm x 205 mm x 140 mm (l x w x h) i.e. 360 cm² bottom area. Made of Polycarbonate (Makrolone©) and Polysulfone. Lids are made of stainless steel. European standard cages for up to 5 mice (20–25 g). Autoclavable up to 134 °C
Mesh enclosures	Part of the Arena Package: Noldus, Sociability cage, Wageningen, the Netherlands	https://www.noldus.com/applications/sociability-cage	Small acrylic or metal cage-like with a diameter of 100 mm and a height of 200 mm with openings of a 10 mm in size. Two mesh enclosures per arena would work but four is preferable (see point 2.5 in protocol)
Mesh wall	selfmade	N/A	Acrylic or metal, one for each cage. Size depends on cages used. The walls must not allow the two animals to have a physical contact
Social targets: Mice of the strains CD-1 and 129/Sv; retired male breeders	Mice provided by Charles River: Strain name: CD-1®IGS Mouse 129S2/SvPasCrl	Crl:CD1(ICR); 129S2/SvPasCrl	CD-1 and 129/Sv retired male breeders, single-housed, novel (unknown) conspecifics to the animals of interest. If retired male breeders are not available then males older than 1 year from both strains would suffice