Social Defeat Stress (SDS) in Mice: Using Swiss Mice as Resident

[Abstract] Due to the high prevalence and great economic impact of depression, studies with animal models have been increasingly used to identify neurobiological mechanisms associated with this disorder. However, many animal models use stressful conditions that are not consistent with what we observe in the modern human world. Examples are the chronic unpredictable stress and the electric shock model used in rodents. It’s well established the social stress as the major cause of depressive disorder in human, in this way a social defeat stress model was recently standardized and can induce depressive-like behavior of social avoidance, a typical human depressive behavior. In this model, mice are exposed on consecutive days to an aggressor mouse, suffering brief periods of physical aggression followed by longer periods of visual and olfactory (sensory) contact and, as a consequence, a relationship of social submission is characterized. Thus, the objective of this work is to describe a social defeat stress protocol using swiss mice as resident, also describing valuable procedural suggestions that will help researchers to reproduce the model easily.

to evaluate anhedonia and despair depressive-like behaviors which are commonly analyzed by the sucrose preference and forced swimming test, respectively.
In this scenario, the social defeat stress model was recently standardized and can induce depressivelike behavior of social avoidance in mice (Golden et al., 2011). In this model, mice are exposed on consecutive days to an aggressor mouse, suffering brief periods of physical aggression followed by longer periods of visual and olfactory (sensory) contact. After this period a relationship of social submission is characterized, typically observed in human relations. As a result, stressed mice develop several physiological and behavioral changes, including altered levels of corticosterone, cardiac hypertrophy, circadian rhythm disturbances, widespread pain, anxiety, despair behavior, anhedonia and social avoidance (Krishnan et al., 2007;Pagliusi et al., 2018). Social interaction level is used to assess depressive-like behavior and, interestingly, some mice submitted to this social defeat stress protocol are resistant and do not develop social avoidance, similar to what occurs in humans, where not all individuals exposed to some form of chronic social stress develops psychopathologies (Krishnan et al., 2007).
Another important feature of this model, which makes it much more reliable than the oldest animal models of depression and depressive-like behavior tests, is the fact that the social avoidance behavior can be reversed only to chronic, and not acute, treatments with antidepressant drugs, as also is observed in humans Pollak et al., 2010). In addition, mice submitted to the SDS protocol exhibit neurobiological changes very similar to those found in humans with depression, including brain monoamines reduction, hypothalamic-pituitary-adrenal axis dysfunction, BDNF reduction in the hippocampus and BDNF increase in the nucleus accumbens (Hatzinger, 2000 Thus, the objective of this work is to describe a social defeat stress protocol adapted from that described by Golden et al. (2011), since many laboratories can't use the same materials and mice strain as those described in the original protocol. Therefore, we will describe how to use the Swiss mouse as an aggressor, since it is quite evident the natural aggressive behavior of this strain under the experimental conditions used in this protocol (Miczek et al., 2001). We will also describe valuable procedural suggestions that will help researchers to easily reproduce the model. 1. Purchase at least 40 Swiss mice that had already performed several cycles of mating and had been retired (retired breeder). It's necessary a large number of mice because on average only 40% are selected as good aggressors and at least 10 is required. The Swiss age should range from 6 to 12 months, which increases the percentage of mice selected as good aggressors.

Materials and Reagents
Prior to screening, keep them for a week single housed with free access to food and water for habituation to their new colony facility.
2. Start screening 10 days before the initiation of the SDS sessions and, during the screening, always use the Swiss mouse home cage (with wood chip bedding). Place a C57BL/6J mouse in the Swiss home cage for 3 min (use the timer) and write down the time elapsed until the first attack (latency) and the number of attacks (use the mechanical tally counter) performed by the Swiss mouse. At this moment, the C57BL/6J is called "screener mouse" and will not be used in social defeat stress sessions. Note that the same experimenter must perform all screening sessions, using the same criteria to consider what is an attack. In order to facilitate the reproducibility, we consider attack as any aggressive action with physical contact that the Swiss mouse performs against screener mouse.

Notes:
Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC. different Swiss on the same day, otherwise, the screener mouse may become very submissive and the Swiss mouse may not attack. Therefore, to perform the screening of 40 Swiss you will need at least 8 screener mouse.

Repeat
Steps A2-A3 two more days (totaling 3 days of screening), each day placing a different screener mouse for each Swiss, i.e., no Swiss defeats the same screener mouse twice. In this way, the Swiss mouse will not be habituated to the screener mouse.
5. Select Swiss mice that attacked the screener mouse on at least two consecutive days with a latency less than 1 min. Discard Swiss mice that do not meet these criteria. Among those who meet these criteria, use the number of attacks to choose which Swiss mice will be used in subsequent social defeat experiments, i.e., choose the Swiss mice that most attack during the 3 days of screening. Swiss mice that met the criteria but did not attack as much as the chosen ones may remain as reserves. That way, if a chosen mouse stops attacking during a defeat session you can exchange it for a reserve mouse. 6. Following screening, single house the aggressors with free access to food and water. Before each new social defeat experiment do at least one day of rescreening, ensuring that the Swiss mice continue to behave aggressively and dominantly.
B. Chronic social defeat stress 1. Assemble the laboratory rat breeding cage (at least 10 cages-depending on how many Swiss you have selected) as shown in Figure 1, positioning the perforated acrylic divider and making sure there is free access to water and food for both sides of the cage. Always use wood chip bedding; otherwise, it will be difficult for Swiss mice to attack and for C57BL/6J to escape the attacks. At this moment the laboratory rat breeding cage will be called defeat cage.  They will be continuously separated by the perforated acrylic divider paired with another C57BL/6J control mouse. 5. On the next day (preferably at the same hour as the previous day), place the defeat cages side by side on a bench or table. Take the C57BL/6J mouse from the first defeat cage and place it on the left side (same side of the resident mouse) of the next defeat cage. Perform this procedure with all defeat cages, placing the last C57BL/6J mouse on the left side of the first defeat cage (see Figure 2). After 10 min of social defeat, separate the mice by placing the C57BL/6J on the right side of the defeat cage. Rotate the C57BL/6J control mice in a way that they are not paired with the same control mouse from the previous day.

Repeat
Step B5 another 8 times, totaling 10 days of social defeat stress. On the last day of social defeat put the C57BL/6J mice in a standard mouse cage with free access to water and food (single housed). On the next day, perform the social interaction test.      Figure 5A). On the other hand, mice presenting SI-Z equal or higher than 1 are classified as resilient, i.e., even submitted to the social defeat stress they don't show depressivelike behavior of social avoidance (see Figure 5A). Also, it is possible to obtain the social interaction Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC. 9 www.bio-protocol.org/e3197 Bio-protocol 9(06): e3197. DOI:10.21769/BioProtoc.3197 ratio in the corners (SI-C) which is calculated by dividing the time spent in the corners in the target session by the time spent in the corners in the no-target session (see Figure 5B). SI-C higher than 1 means that mice spent more time avoiding the interaction zone in the "target" session, which can corroborate the SI-Z data. Our experience with the model shows that 20% to 40% of stressed mice are expected to become resilient, which means that up to 80% of the stressed mice show depressive-like behavior of social avoidance. It is important to mention that the depressive-like behavior of social avoidance induced by the SDS model can last up to 4 weeks (our group did not test a longer period than this), which means that these mice can be used for chronic experiments without problems. The results can also be shown using the total time spent in the interaction zone and corners (see   Measures for perforated transparent acrylic divider (X and Y depend on the size of your rat breeding cage and A, B and C depend on the size of your grid that covers the rat breeding cage-perforations should be no more than 1 cm in diameter).