The overall goal of the following experiment is to create an ecologically relevant rodent model to study postpartum mood disorders. First, the pups undergo a period of maternal separation. The pups are then returned to the home cage, and maternal care behaviors are recorded to induce social stress.
A novel male intruder is introduced. Maternal aggression is recorded for 30 minutes or longer for the induction of chronic social stress. Our results indicate that chronic social stress is an effective and reliable paradigm for depressing maternal care.
Another advantage of this technique over existing stress-induced depression models like repeated restraint, social defeat, or chronic mild stress paradigms, which use a randomized variety of stressors, is that social stress does not introduce major logistical and or physiological confounds. The use of chronic social stress paradigms may lead to the identification of improved preventative measures and treatments for mood disorders in mothers and in their offspring. Adults bragged dolly females are bred in-house and testing is done in the home cage, located in the same room throughout the study.
At day 20 or 21 of pregnancy, one inch high cage separators made of clear plexiglass are placed in the home cage. These divide the cage into four quadrants to prevent pups from crawling back to the dam during testing. Typical maternal behavior is confirmed for all dams on the day of birth.
Through direct observation of pup retrieval, grooming, nursing, and self grooming. The intruder males used in this study are smaller or similarly sized. Sprague Dolly, males and females are never presented with the same male twice.
Using males that are similar in size to the dams helps prevent and infanticide. A video camera is mounted near the cage so that behaviors can be recorded during testing. To begin, remove the litter from the dam, weigh the male and female pup separately, and then place them into a clean holding cage for 60 minutes.
At the end of this 60 minute separation, weigh the pups again. Before introducing the pups back to their home cage, start the video recording to make sure that all pup retrieval behavior is captured. Divide the pup as evenly as possible between the three non-test quadrants of the cage.
After this reintroduction record maternal care behavior for 30 minutes. Maternal care behaviors typically include the retrieval of pups back to the nest nesting behaviors, pup or self grooming, or nursing. When the dam has interacted with the litter for a total of 120 minutes, weigh the pups once more to determine milk intake.
Over the two hour period, one pup is euthanized from each litter on behavioral testing days following this nursing period. To extract a milk sample from the stomach, we turn the pups to the dam. After weighing, after starting the video recording, introduce a smaller or similarly sized novel male intruder to the cage for 30 minutes to test for maternal aggression.
Maternal aggression behaviors typically include attacking, biting, kicking, or pinning of the intruder male by the maternal female. For control subjects, the male is removed after the 30 minute maternal aggression video recording on days two, nine and 16 of lactation for chronic social stress subjects, the males are left with the dams for 60 minutes each day. Over the 15 day period between days two and 16 of lactation on days two, nine and 16, the first 30 minutes are recorded similar to the control subjects on days two, nine and 16 of testing a bottle with 0.02%Saccharin is added to the dam home cage at 3:00 PM alternating between the right and left side.
The bottles are later weighed to determine saccharin preference. The pups are weaned on day 23 and the dams are euthanized to obtain samples for further analysis. The adult female offspring of chronic social stress dams later produce their own pups.
For further study, odd log behavioral analysis software is used to score the maternal behavior recorded during the study. Both maternal aggression and maternal care are scored for all maternal aggression videos. This program creates both a complete data file of timestamped five second bins and a summary table with the total frequencies and durations for the selected behaviors over the 30 minute recording.
Chronic social stress caused by exposing a lactating dam to a male intruder for one hour a day for 15 days resulted in a decrease in the duration of pup grooming and total maternal care. By day nine of lactation by day 16 of lactation, the dam showed an increase in self grooming, a measure of anxiety and aggressive behaviors. Correspondingly milk intake by the pups decreased compared to controls on days nine and 16 of lactation pup growth was significantly attenuated on both of these days.
Social stress also decreased saccharin preference and indicator of an hedonia by taste 16 of testing milk intake by the pups of adult female. Offspring of social stress dams was decreased on lactation days two and 16 saccharin preference of the early life chronic social stress F1 dams was also lower. On lactation days two and 16, oxytocin mRNA expression was attenuated in the supra optic nucleus of early life stressed F1 dams and similar effects were found in the paraventricular nucleus.
When establishing a chronic social stress protocol, it's important to minimize exposure to additional stressors such as transportation of dams in their litters, excessive human disturbance, soiled cages, and other aversive acoustic oral factory stimuli. A stable housing facility and consistent animal husbandry will produce the most consistent data. Following this procedure, a wide variety of tissues can be sampled at multiple time points to investigate the effects of chronic social stress on other organ systems.
In addition, social stressors can be applied at multiple life history stages to investigate both susceptibility and resilience to stress induced disorders.
