Social Defeat Stress Model for Adolescent C57BL/6 Male and Female Mice

Published: March 15, 2024

doi:

Andrea Harée Pantoja-Urbán, Samuel Richer, Michel Giroux, Dominique Nouel, Cecilia Flores^3,4,5

¹Integrated Program in Neuroscience,McGill University, ²Douglas Mental Health University Institute,McGill University, ³Department of Psychiatry,McGill University, ⁴Department of Neurology and Neurosurgery, Faculty of Medicine,McGill University, ⁵Ludmer Centre for Neuroinformatics & Mental Health,McGill University

Summary

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Abstract

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Introduction

The chronic social defeat stress paradigm is widely used to model social stress in adult postnatal day (PND) >65 male rodents. This paradigm is based on the natural aggressive behavior of an adult male rodent when an intruder invades its territory. This model is used in a variety of rodent species, including rats, hamsters, and mice¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹, and consists of a combination of physical aggression and psychological stress lasting about 10 days, during which an intruder rodent experiments a few min of physical aggression from the resident rodent. The two rodents remain in the resident's home cage, separated by a divider that allows sensory but not physical contact⁷. In mice experiments, the most commonly used resident/aggressor mice are retired breeders Swiss CD-1 mice, which show robust territorial behavior against intruder mice⁶^,⁷. For the intruder mice, the best characterized strain is the inbreed C57BL/6 strain²^,⁴^,⁵. The defeated mouse is exposed to a new aggressor every day to prevent habituation to the aggressor. Control mice are housed with a different conspecific each day. At 24 h after the last defeat session, experimental mice are tested in a social interaction test (SIT) in which they can explore an open field in the absence (no target), or presence of a novel CD-1 mouse (target). Control mice spend more time in the interaction zone with the target versus the non-target part of the task. Defeated mice are classified as susceptible (ratio<1) or resilient (ratio>1) according to a social interaction ratio (time spent in the interaction zone with aggressor present/time spent in the interaction zone with aggressor absent). This procedure provides a useful tool to study individual differences in response to stress.

Until recent years, the chronic social defeat stress model has been used mostly in adult male mice because the accentuated male dominance hierarchies involve fighting against males but not against females⁶^,⁷. Furthermore, male rodents typically do not attack females; instead, they engage in mating behaviors¹⁰. Notwithstanding these obstacles, different strategies have been developed to adapt the chronic social defeat stress paradigm for adult female mice. For example, the California mouse model of social defeat is based on the natural aggression of this monogamous species from both sexes when defending their territory⁹^,¹¹. Other approaches focus on inducing aggressive behavior of the CD-1 mice by stimulating their ventromedial hypothalamus to have consistent aggressive behavior¹⁰^,¹², or by applying male urine in the experimental adult female mice to receive attacks from CD-1 aggressors¹³. This heightened and consistent aggression of the CD-1 mice is critical for the experimental intruder mouse to show clear behavioral signs of subordination towards the repeated attacks by the aggressor during length of the interaction⁶.

Adapting the chronic social defeat model to use in adolescent C57BL/6mice
Adolescence is a period marked by substantial psychosocial maturation, which unfolds in parallel with changes in the micro and macro architecture of the brain, particularly in the prefrontal cortex. In both humans and rodents, there is little consensus regarding the specific onset and end of the adolescent period¹⁴^,¹⁵. Furthermore, there are critical windows of vulnerability within adolescence for experience-induced disruption of ongoing brain and cognitive development¹⁶^,¹⁷^,¹⁸^,¹⁹. Puberty and adolescence occur at the same time, but these terms are not synonymous. Puberty signifies the onset of sexual maturation, whereas adolescence represents a broader phase characterized by the gradual shift from a juvenile state to achieving independence²⁰. Different groups have suggested that adolescence in mice spans from weaning (PND 21) until adulthood (PND 60)²¹. Particularly, early adolescence can be referred to as the first and second weeks postweaning (PND 21-34) and mid-adolescence as the PND 35-48 period. These ranges encompass discrete developmental periods regarding for example the development of the dopamine system²²^,²³^,²⁴, vulnerability to drug effects on developing neuronal networks¹⁷^,²⁵^,²⁶^,²⁷, and distinct behavioral characteristics¹⁶^,²⁰^,²⁸^,²⁹^,³⁰.

Fighting behavior from the resident mouse is required for the social defeat protocol. However, as with female mice, males do not typically engage in aggressive interactions with early adolescent mice, possibly because they do not perceive them as a threat. Most studies exploring the effects of chronic social defeat in adolescent C57BL/6 mice have been performed during the mid-adolescent period³¹^,³²^,³³^,³⁴^,³⁵; others do not specify the postnatal day of adolescent exposure³⁶^,³⁷, or extend the days for the defeat to early adulthood³⁸ or do not allow sensory contact³⁹; other studies on adolescent mice use different strains⁴⁰^,⁴¹. The characteristics of these studies using chronic social defeat stress in adolescent male mice are summarized in Table 1.

Our research group is interested in targeting specific adolescent windows of exposure, including early adolescence, in C57BL/6 mice. Because of the short duration of the different adolescent periods, a modified version of the accelerated version of the chronic social defeat stress paradigm was designed⁴². This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Previous work shows that there are significant sex differences in sensitivity to social stress in adolescence in rats⁸^,²⁶^,⁴³^,⁴⁴^,⁴⁵, as well as in the harmful effects of social stress on mental health trajectories in humans⁴⁶^,⁴⁷^,⁴⁸^,⁴⁹^,⁵⁰^,⁵¹^,⁵²^,⁵³^,⁵⁴^,⁵⁵^,⁵⁶. The AcSD model works effectively for adolescent female mice, too, allowing them to investigate potential sex-specific consequences as well as explore the neurobiological foundation.

Table 1: Studies using social defeat stress paradigms in adolescent male mice. Strain and species: California Mouse: Peromyscus californicus. C57BL/6: Mus musculus black 6 inbred mouse strain. C57BL/6J: M. musculus black 6 inbred mouse model provided by Jackson's laboratories. CD-1: M. musculus from Swiss outbred albino mouse strain. ICR: M. musculus Institute of Cancer Research outbred albino mouse strain. OF1: M. musculus Oncins France 1 outbred albino mouse strain.
Abbreviations: wk = week; PND = Postnatal day; res = resilient; sus = susceptible; unsus = unsusceptible. Please click here to download this Table.

Protocol

Experimental procedures were performed in accordance with the guidelines of the Canadian Council of Animal Care and approved by the McGill University and Douglas Hospital Animal Care Committee (animal experiment approval number: 2005-5084). All mice were housed in a temperature- and humidity-controlled (21-22 °C; 60%) colony room and on a 12 h light-dark cycle (light on at 8:00 h) at the Neurophenotyping Centre of the Douglas Mental Health University Institute. The mice had ad libitum access to food and wat…

Representative Results

A total of four different experiments were performed using the chronic social defeat stress model in adolescent C57BL/6 male mice (PND 21). However, this model presented important limitations for its use in early adolescent C57BL/6 mice. Equipment required modifications for adolescent C57BL/6 mice The first limitation was that the equipment used for the social defeat apparatus was designed for adult mice. Because of their size, adolescent C57BL/6 mice at PND 21 were able…

Discussion

Consistent, aggressive behavior in CD-1 mice
During the screening phase, it is very important to take note of all the behaviors displayed by the CD-1 (chasing, mounting behaviors, sniffing, grooming, or biting) and to closely follow these records when selecting the CD-1 mice for the AcSD. It is likely that a CD-1 mouse which interacts with the adolescent mouse without attacking it will develop aggressiveness towards adolescent mice during priming. In contrast, a CD-1 mouse which attacks an adu…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by the Canadian Institutes for Health Research (CF Grant Numbers: MOP-74709; PJT 190045), the National Institute on Drug Abuse (CF Grant number: R01DA037911), the Natural Sciences and Engineering Research Council of Canada (CF Grant Number: 2982226). Andrea Pantoja-Urban was supported by The National Council for Humanities, Science and Technologies/Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) from México and FRQNT – Merit scholarship program for foreign students (PBEEE). Samuel Richer was supported by a scholarship from the Integrated Program of Neuroscience at McGill University. Figure illustrations were created using templates from BioRender.com.

Materials

C57BL/6 adolescent mice	In house breeding		Mice were breeded at the Neurophenotyping Centre of the Douglas Mental Health University Institute.
C57BL/6 adult mice	Charles River Laboratories	Strain Code: 027	Mice are ordered so as to arrive at PND>65 and are group housed (four mice per cage) in standard mice cages.
C57BL/6J adolescent mice	Jackson Labs	Strain Code: 000664; RRID:IMSR_JAX:000664	Mice are ordered so as to arrive at PND 24 and are group housed (four mice per cage) in standard mice cages.
CD-1 mice	Charles River Laboratories	Strain Code: 022	Mice retired breeders more than three months of age and singled housed throughout.
Cleaning solution	Virox Animal Health	DIN 02537222	Prevail: Accelerated Hydrogen Peroxide. Desinfectant cleaner and deodorizer.
Clear perforated acrylic glass divider	Manufactured by Douglas Hospital, custom order		0.6 cm (w) × 45.7 cm (d) × 22.23 cm (h); perforations of 0.6 cm diameter. The dividers are perforated allowing sensory but no physical contact between the pair of mice.
Clear rectangular rat cages	Allentown		24 cm (w) × 48.3 cm (d) × 22.23 (h).
Cotton squares for bedding	Inotiv Envigo	T.6060 iso-BLOX	2.5 cm x 2.5 cm. Added to the social defeat apparatus.
Hard woodchip bedding	Inotiv Envigo	Teklad 7090, 7115	Sani-chip bedding.
Large binder clips to secure the steel-wire tops	STAPLES	Item #: 132429, Model #: 24178-CA	51 mm
Medium binder clips to secure the steel-wire tops		Item #: 132367, Model #: 24172-CA	32 mm, in case the cover lids of the rat cages do not close with the large clips
Pain relief cream	Polysporin		Plus Pain Relief Cream (red format, NOT ointment), 2 Antibiotics plus lidocaine hydrochloride
Paired Steel-wire tops			24 cm (w) × 48 cm (d) with 0.6 cm (w) of separation between the grill
Removable wire-mesh enclosure	Johnston industrial plastics		11 cm (w) × 6.8 cm (d) × 42 cm (h) custom order; two per social interaction test arena secured in precut clear polycarbonate
Social interaction open-field arena	PEXIGLAS		45 cm (w) × 45 cm (d) × 49 cm (h), custom-crafted from opaque acrylic glass (Plexiglas)
Stopwatch			For timing defeat sessions
Video camera with infrared lights	Swann	SRDVR-44580V	Swann Camera – 4 Channel 1080p Digital Video Recorder & 2 x PRO-T853
Video tracking software	Topscan		2.0 Clever Systems Inc.

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