Adult Sprague Dawley females are bred in our animal facility, litters are culled to 8-10 pups with an even sex ratio, and maternal care and aggression testing is done in the rats home cage. The experimental subjects remain in the same room through the study. One inch high cage separators made of clear Plexiglas are placed in the cage on day 20 or 21 of gestation. These divide the cage into 4 quadrants to prevent pups from crawling back to the dam when placed in the cage. Typical maternal behavior is confirmed for all dams on the day of birth (day 1 of lactation) through direct observation of pup retrieval, grooming, nursing, and self grooming. Intruder males are smaller or similarly sized Sprague Dawley males, and females are never presented with the same male twice. Maternal care and maternal aggression are video recorded on the morning of days 2, 9, and 16 of lactation as described below:

1. Remove the litter from the dam, weigh males and females separately, and place entire litter in a clean holding cage for 60 min.
2. At the end of the 60 min separation, weigh males and female pups separately prior to replacing litter in the home cage of the dam, dividing the pups as evenly as possible between the 3 non-nest quadrants of the cage. Start video recording prior to the reintroduction to ensure all pup retrieval is recorded.
3. Video record maternal care for 30 min. Allow dams to interact with their litters for another 90 min. 120 min (30 min recording + additional 90 min) after placing pups in the dam’s cage, weigh them again to determine milk intake over the two hour period. One pup is euthanized from each litter on behavioral testing days following the 120 min nursing period to extract a milk sample from the stomach. The purpose of collecting the milk from the pups is to assess the levels of hormones and other factors that may mediate the behavioral and/or physiology effects of chronic social stress on offspring.
4. Return pups to dam after weighing, and introduce a smaller or similarly sized novel male intruder to the cage for 30 min to test for maternal aggression. Start video recording during the introduction to ensure that all aggression is recorded. For control subjects, the male is removed after the 30 min recording. For Chronic Social Stress subjects, the males are left in for another 30 min. On non-behavioral testing days during the day 2-16 period, novel intruder males are placed in the cages of CSS subjects for one hour without video recording. The social stress exposures are stopped if injury occurs, and injured animals are treated and removed from the study.
5. A bottle with 0.02% saccharin is added to the dam home cage at 3 PM on days 2, 9, and 16 alternating between the right and left side. Both plain water and saccharin bottles are weighed at 3 PM, then again at 8 AM the next morning to determine saccharin preference.
6. Pups are weaned on day 23, and dams are euthanized at this point to obtain basal plasma samples and extract the brain for gene expression analysis of physiologically and behaviorally relevant target nuclei.

Maternal Behavior Scoring

The maternal care aggression behaviors described in Tables 1 and 2 are scored from the videos using Odlog behavioral analysis software. Keys are assigned to each behavior, and the key is held down while each behavior is being performed to record the duration of that behavior. Frequencies are the sum of the key strokes for each behavior. This program creates both a complete data file of time stamped five second bins and a summary table with the total frequencies and durations for the defined behaviors over the 30 min recording. Both maternal aggression and maternal care are scored for the maternal aggression videos, as the dams do respond maternally to the pups while the male intruder is in the cage.

Soon after placing the pups in the cage, the dam will retrieve each pup individually back to the nest. Full retrieval is scored once when all pups are in the nest. While full retrieval is typically only scored once, it may sometimes be scored twice if the dam relocates the entire litter to a new site after an initial full retrieval. Nesting behavior is scored whenever the nesting material is manipulated by the dam with her mouth or paws. Pup grooming includes licking or manipulation of the pup’s fur by the dam with her paws. Nursing scoring is initiated after the dam has been in a stationary crouching or prone nursing posture for 10 seconds. This is to allow time for pups to start suckling. Total maternal care is the combined duration of nesting, pup grooming, and nursing, but may be restricted to grooming and nursing to focus on pup-directed behavior. Self grooming is defined as manipulation of the dam’s own fur with her mouth or paws. Activity is scored during any locomotor activity not associated with the previously defined behaviors.

Maternal aggression consists of attacking, biting, kicking, and pinning. Attacking is defined as the boxing or tackling of the intruder male. Biting is scored when the dam uses her teeth on the intruder, and pinning is recorded when the dam holds the male down with her limbs or body. Total aggression is the sum of attacking, biting, kicking, and pinning.

Estatística

Maternal behaviors, milk intake, and saccharin preference of the control and CSS dams were compared on each lactation day with one-tailed t-tests to assess the effects of CSS on individual days. Growth data on days 2, 9, and 16 of lactation, were compared with one-tailed t-tests on weight (in grams) on day 2 or the percentage growth relative to day 2 (day 9 or 16 weight/day 2 weight x 100). Expression levels for OXT mRNA were normalized against three combined housekeeping genes, b-actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Relative OXT mRNA expression levels were compared with individual t-tests on each peptide or receptor for each brain region. All graphical results are presented as group means + SEM, and the level of statistical significance was p ≤ 0.05.