Our research aims to identify circuit level abnormalities resulting from trauma exposure during various stages of neuro development, we have developed a model of early adolescent physical trauma to better understand the impact of childhood trauma on adult brain functioning. Behavioral health professionals are seeing an increase in patients suffering from the lasting impacts of childhood abuse. However, most current rodent research focuses on trauma exposure in adulthood or the impact of infant neglect.
Neither of these directly addresses the unique challenges seen in adults who have a history of childhood abuse. Our protocol produces a consistent avoidant phenotype in over two thirds of defeated animals in adulthood. Additionally, by using younger males without breeding experience housed with females, our protocol is able to produce robustly aggressive CFW males without the use of additional adult B6 mice to instigate aggression, increasing the ease and thorough put of the protocol.
Our research will help contribute to the growing understanding of how trauma exposure at different stages of development has a unique biological, behavioral and anatomical impact on the brain, and hopefully assist in the new development of treatment technologies for individuals who've experienced childhood abuse. To begin, pair house CFW males with CFW OVX females for one week to allow for sexual experience and increase territorial aggression in the male. After one week, remove the CFW OVX female from the cage and place it in a clean, empty cage.
Then introduce the stimulus C57 male into the cage with the CFW male for one minute and record the latency of the attack. Afterward, remove the stimulus C 57 male and place the CFW OVX female back in the cage with the CFW male before returning them to the housing room Group house CFW males that either do not attack or show an attack latency of more than 30 seconds on two or more days. Use these non-aggressive CFW males as stimulus animals during open field social interaction testing.
When adolescent C57 males are four weeks old, weigh each animal and record the weight. Separate the animals into two equal cohorts, control and defeated while ensuring that the weights between the two cohorts are not significantly different. House one control adolescent C57 on one side of a plexiglass partition with another control adolescent C57 on the other side of the partition.
On the 11th day house, each control adolescent beside a different control adolescent with a plexiglass partition between the two for the remainder of the experiment. Weigh and record the weight of the adolescent C57 that is to be defeated. Wounds larger than four millimeters in diameter shown here to scale, require immediate euthanasia of the test animal.
Then remove the CFW OVX female from the cage with the resident CFW male and house it with other OVX females. Afterward, introduce the adolescent C 57 to the cage of the resident CFW male and start the stopwatch. Remove the adolescent from the cage after 30 bites or five minutes, whichever occurs first and record the attack data.
Then place the plexiglass partition in the middle of the CFW male resident's cage, ensuring that the CFW resident is on one side and the defeated adolescent is on the other. On the 11th day, house the defeated adolescents beside other defeated adolescents with a plexiglass partition between them for the duration of the experiment. Habituate the control and defeated animals to the testing room for one hour.
Record all sessions with animal tracking software. Place the wire cup against the left wall of the 44 by 44 by 44 centimeter acrylic arena at the midpoint of the wall. Draw a two centimeter circular interaction zone around the cup.
Now place the C 57 in the corner of the arena with an empty wire cup for 150 seconds. Then remove the C 57 from the arena and place it in a clean, empty cage. Place a non-aggressive unfamiliar CFW adult male inside the wire cup and place the wire cup inside the arena.
Reintroduce the C 57 to the arena with the CFW containing wire cup for 150 seconds. After returning both animals to their respective cages, clean the arena with water and 70%ethanol to clean any areas with urine or feces before testing the next C 57. Calculate the length of time any body point is within the interaction zone for both the session with the empty cup and the session with the CFW containing cup.
Divide the time spent in the interaction zone with the CFW containing cup by the time spent in the interaction zone with the empty cup to produce the social interaction ratio. Perform the open field social interaction at any time after the 10 day defeat period, depending on the developmental window of interest. Defeated animals showed a significant reduction in both social interaction ratio and raw social interaction time compared to controls at all tested time points.
The most pronounced reduction in social interaction ratio and time occurred two weeks after defeat with defeated animals showing increased time spent with an empty cup. Heat maps confirmed that defeated mice spent more time in the corners and less time in the interaction zone across all time points.
