We investigate the impact of adolescent social adversity on neurodevelopment and behavioral outcomes, specifically focusing on male and female C57BL/6 mice. We model social distress using an adapted version of the chronic social defeat paradigm. Our model, called accelerated social defeat stress, is tailored for its use in adolescent C57BL/6 mice.
The social defeat stress protocol requires aggressive behavior from the resilient mouse. Existing studies are often conducted in the mid-adolescent period or lack a specificity in terms of postnatal day of exposure. Some studies extend into early adulthood or employ strains of mice that are not commonly used.
Implementing the social defeat 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 adolescence is a short period of life that encompasses discrete temporal windows of vulnerability. The AcSD model successfully induces social avoidance 24 hours later in both males and females. It is short enough to allow exposure during discrete periods within adolescence.
It allows the segregation of resilient and susceptible mice and is the first model available to study social defeat stress in adolescent C57BL/6 female mice. Our model was generated to be used in adolescent male and female C57BL/6 mice. This four day stress paradigm, with two physical attack sessions per day, uses an adult C57BL/6 male mouse to prime the CD-1 mouse for aggressiveness, ensuring that the CD-1 mouse readily attacks the experimental male or female mouse.
To begin, introduce an adolescent C57BL/6 mouse into the same cage as primed CD-1 mouse. When the CD-1 mouse bites or places its teeth on any part of the adolescent mouse's body, register it as an attack. When the C57BL/6 mouse moves both of its hind paws away from the position they were in initially, it is considered moving away.
Separate the animals with a plastic ruler if the CD-1 mouse bites more than once in succession. Similarly, separate the animals if the CD-1 mouse bites and does not let go, or if the C57BL/6 mouse is trapped in a corner or pinned down by the CD-1 mouse and cannot move away. To screen and prime the CD-1 mice, put on a pair of nitrile examination gloves.
On the first day, introduce an adult male C57BL/6 postnatal day 65 or older mouse as an intruder into the cage of the CD-1 mice for three minutes. Register the latency to the first aggression and how many aggressions are performed. From the second to the last day of screening and priming, introduce an adult male C57BL/6 postnatal day 65 or older for one minute, and then swap it with an adolescent C57BL/6 postnatal day 25 mouse for five minutes.
If the priming is for aggressiveness towards female C57BL/6 mice, perform the screening and priming with adolescent female mice. Register the latency to the first aggression, the number of attacks, and the wounds received by all the C57BL/6 mice. If the CD-1 mouse does not approach the adolescent C57BL/6 mice for two days during the screening or priming, do not include the CD-1 mouse.
To begin, use acrylic glass dividers to separate rack cages into two compartments. Add hard wood chip bedding to both sides of the cage, then place a cotton square on each side. Secure the wire top with binder clips.
Then place the cover lid without the protective cotton over the cage. Place the CD-1 mouse on one side of the divider. Label the cage with a tag placed outside it for easy identification of each mouse.
For the defeat sessions, make a schedule of two sessions per day for four days with a different aggressor for each session. Weigh a C57BL/6 adolescent mouse. Next, introduce a male C57BL/6 postnatal day 65 or older mouse for 30 seconds into the CD-1 side of the defeat cage.
Swap it with an adolescent C57BL/6 postnatal day 24 to 28 mouse for a 10 minute long defeat episode or until 10 attacks occur. Register the latency to the first aggression, the number of attacks, and the wounds received by all the C57BL/6 mice. Separate the animals after each attack with a plastic ruler.
After every session of defeat, check for injuries and treat them with pain relief cream. Register the number of injuries. Place the defeated adolescent C57BL/6 mouse in the free compartment next to its aggressor and leave it there until the next session.
Start a new session with the experimental and aggressor mice that were not paired in the previous session. Monitor the injuries of the adult C57BL/6 male mice used for priming. For the control mice, place two control mice per cage, one on each side of the cage.
House them in the social defeat apparatus in a separate room from the social defeat mice. For every session occurring in the defeat group, rotate the mice in the control group such that each experimental mouse is introduced in the cage of an unfamiliar C57BL/6 mouse. The CD-1 mice attacked the C57BL/6 mice in a consistent manner across multiple cohorts.
Adolescent males received more attacks with primed CD-1 mice during accelerated social defeat than adolescent males exposed to a chronic social defeat protocol. The primed CD-1 mice presented aggressive attacks towards the adolescent C57BL/6 male and female mice. No interaction was found between day and cohort across experiments.
When comparing the number of attacks received by resilient and susceptible mice undergoing accelerated social defeat, no difference is found if experiments are run in males or females.