Name: Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research
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This research is supported by the Collaborative Research Center 1193, Subproject Z02, funded by the German National Research Foundation (SFB1193, Neurobiology of Resilience) and the Boehringer Ingelheim Foundation (grant to Leibniz Institute for Resilience Research and Individual Phenotyping and High-Resolution Automated Behavioural Analysis). We would like to thank Dr. Konstantin Radyushkin and Mrs. Sandra Reichel for their technical assistance as well as Mrs. Hanna Kim for her English language support. The funding sources had no involvement in the model design; collection, analysis, and interpretation of data; in the writing of the protocol; and in the decision to submit the protocol for publication.

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
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	<li>Animals of interest: Obtain C57BL6/J male mice at 7 weeks of age, and upon arrival, single-house in a temperature- and humidity-controlled facili...

Threat-Safety Test for Stress Susceptibility or Resilience in Mice Models

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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...

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 anxiety2,3. 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 &#34;acute&#34; (the &#34;acute social defeat model&#34;), whereas repeated confrontations lasting over several days (usually 10) are known as &#34;chronic&#34; (the &#34;chronic social defeat model&#34;). 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-avoidant9,14). 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&#39; 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&#39; 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.

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			<td>Arenas</td>
			<td>Noldus, Sociability cage, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/applications/sociability-cage</td>
			<td>Three-chambered, rectangle in shape with a total size of 60 cm x&#160; 40 cm, made of acrylic transparent walls and smooth floors</td>
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			<td>Camera for video recording</td>
			<td>Basler AG, Germany 
			An der Strusbek 60-62 
			22926 Ahrensburg</td>
			<td>&#160;ace Classic 
			acA1300-60gc</td>
			<td>If using automatic detection program, make sure cameras are compatible</td>
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			<td>Camera objective</td>
			<td>KOWA Kowa Optimed Deutschland GmbH 
			Fichtenstr. 123 
			40233 Duesseldorf: LMVZ4411 | 1/1.8&#34; 4.4~11mm Varifokal Objektiv</td>
			<td>Part-No. 10504</td>
			<td></td>
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			<td>Detection program/Timer&#160;</td>
			<td>Noldus, EthoVision-XT, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/ethovision-xt</td>
			<td>Detection can be achieved either manually (using a timer or a software for manual scoring) or automatically</td>
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			<td>Housing cages</td>
			<td>ZOONLAB GmbH, Hermannstra&#223;e 6, 
			44579 Castrop-Rauxel</td>
			<td>3010010</td>
			<td>Type 2 cages: 265 mm x 205 mm x 140 mm (l x w x h) i.e. 360 cm&#178; bottom area. Made of Polycarbonate (Makrolone&#169;) and Polysulfone. Lids are made of stainless steel. European standard cages for up to 5 mice (20&#8211;25 g). Autoclavable up to 134 &#176;C</td>
		</tr>
		<tr>
			<td>Mesh enclosures&#160;</td>
			<td>Part of the Arena Package: Noldus, Sociability cage, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/applications/sociability-cage</td>
			<td>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)</td>
		</tr>
		<tr>
			<td>Mesh wall</td>
			<td>selfmade</td>
			<td>N/A</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Social targets: Mice of the strains CD-1 and 129/Sv; retired male breeders</td>
			<td>Mice provided by Charles River: 
			Strain name: CD-1&#174;IGS Mouse 
			129S2/SvPasCrl&#160;</td>
			<td>Crl:CD1(ICR); 129S2/SvPasCrl&#160;</td>
			<td>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</td>
		</tr>
	</tbody>
</table>

social threat-safety test uncovers psychosocial stress-related phenotypes

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 10th 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.

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Social interaction index as a measurement of aversive conditioned response 
A social interaction index &#8805;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...

Watch this Scientific Journal Video about Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research at JoVE.com

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.

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience ...

Our research seeks to uncover the underlying mechanisms of stress resilience. By understanding those mechanisms, we can employ them to develop better treatment strategies for stress related mental disorders and prevention. Our research has developed a new mouse model with high face validity that allows stratifying a single group of stressed mice into resilience and susceptibility associated phenotypes that are based on the fear circuitry.Also, we provide evidence for the presence of global resilience mechanisms. Our study on stressed mice revealed that they exhibited characteristics that are indicative of both resilience and susceptibility in humans. Specifically, they show different abilities in threat safety discrimination and responsiveness to extinction, which are key features associated with resilience.The resilience and susceptibility associated phenotypes that we identified in mice have face validity and are based on the fear circuitry. This circuitry is highly conserved across species and is arguably one of the best studied circuits. In turn, this allows mechanistic investigations that have the potential to contribute to treatment, development and prevention measures.We'll use our new model to dig deeper into neurobiological mechanisms of resilience and understand how we can actively promote resilience. In addition, we will employ longitudinal analysis to decifer how individual resilience develops over time.