A standardized protocol for repeated social defeat stress in mice

Abstract

A major impediment to novel drug development has been the paucity of animal models that accurately reflect symptoms of affective disorders. In animal models, prolonged social stress has proven to be useful in understanding the molecular mechanisms underlying affective-like disorders. When considering experimental approaches for studying depression, social defeat stress, in particular, has been shown to have excellent etiological, predictive, discriminative and face validity. Described here is a protocol whereby C57BL/6J mice that are repeatedly subjected to bouts of social defeat by a larger and aggressive CD-1 mouse results in the development of a clear depressive-like syndrome, characterized by enduring deficits in social interactions. Specifically, the protocol consists of three important stages, beginning with the selection of aggressive CD-1 mice, followed by agonistic social confrontations between the CD-1 and C57BL/6J mice, and concluding with the confirmation of social avoidance in subordinate C57BL/6J mice. The automated detection of social avoidance allows a marked increase in throughput, reproducibility and quantitative analysis. This protocol is highly adaptable, but in its most common form it requires 3-4 weeks for completion.

Figure 1

Picture of the standard hamster cage used in repeated social defeat stress experiments. The resident aggressor is permanently housed on one side of the perforated divider, and all defeats are performed within that compartment while rotating C57BL/6J intruders across defeat days so that these experimental animals do not habituate to a single aggressor. Note that care must be taken to ensure that the divider is firmly secure, thereby preventing mice from escaping their overnight compartments. Social defeat experiments must follow all governmental and institutional guidelines for care and use of laboratory animals.

Figure 2

Schematic of the social interaction arena and representative front and side pictures of a wire-mesh enclosure. (a) Top-down view of the social interaction arena, delineated with zones and dimensions. (b) Picture of wire-mesh enclosure used to secure the target CD-1 during the `target present' portion of the social interaction test. Note that this is custom designed, and, assuming that dimensions are held constant, it can be fabricated with some flexibility in construction materials. Of importance is the ability for visual and olfactory cue transmission, with physical separation of the target aggressor and defeated mouse. On the left is a front view, and on the right a side view, of the same wire-mesh enclosure.

Figure 3

Repeated social defeat stress induces avoidance behavior in susceptible mice. (a) Repeated social defeat stress results in a spectrum of avoidance behavior, divided between susceptible and resilient phenotypes as a function of their social interaction (SI) ratio score. This is the ratio of time a mouse spends in the interaction zone in the presence of a target CD-1 compared with the absence of a target CD-1. (b,c) Susceptible mice spend significantly more time in the corner zone than in the interaction zone, whereas resilient mice spend comparable amounts of time in the interaction zone to control mice that have never undergone a defeat procedure. Both control and resilient mice spend significantly more time in the interaction zone when a target is present. (d,e) Social avoidance behavior can also be expressed as a social interaction ratio. In this panel, the same data are shown in both manners, for comparison. Error bars represent means ± s.e.m. *P < 0.05, multivariate ANOVA/ANOVA. All data shown were collected while conforming to governmental and institutional guidelines for care and use of laboratory animals.