GoKawiilHuman participants
Brain tissues used in this study were provided by the Douglas Brain Bank (RRID:SCR_025991) with ethical approval from the Research Ethics Board (REB) of the Centre Intégré Universitaire de Santé et de Services Sociaux (CIUSSS) de l’Ouest-de-l'Île-de-Montréal. Analyses were conducted in accordance with all relevant guidelines and regulations. Informed consent from next of kin was obtained for each individual included in this study. Psychological autopsies, considered the gold standard for obtaining information on deceased individuals51,52, were conducted. In brief, these consist of a series of proxy-based, structured interviews assessing psychopathology with next of kin and complemented by reviews of medical records, as previously described51. Groups were matched for depression diagnosis, and were otherwise neurotypical individuals who died suddenly without prolonged agonal periods and did not have evidence of axis I disorders. Groups were matched for postmortem interval, tissue pH and RNA Integrity number. Frozen histological grade samples of grey and white matter were dissected from the subiculum by expert neuroanatomists and stored at –80 °C. Dissections were performed on 0.5-cm-thick coronal sections with the guidance of a human brain atlas53 (http://www.thehumanbrain.info/brain/bn_brain_atlas/brain.html). Subiculum samples were obtained from sections equivalent to plate 43 of the atlas (level of lateral geniculate nucleus), by dissecting through the hippocampal fissure with a slight upward angle, up to the beginning of the CA1 region. All procedures complied with ethical regulations for research involving human tissue. Demographic information can be found in Supplementary Table 11.
Animals
Wild-type C57BL6/J mice were purchased from Jackson Laboratories at 8 weeks old, and maintained on a 12 h:12 h light:dark cycle throughout the entirety of the experiments. Mice were housed in temperature- and humidity-controlled conditions (approximately 22 ± 2 °C and 50 ± 10% humidity) with ad libitum access to food and water throughout the entirety of the experiments. All behavioural testing occurred during the light cycle. Experimenters were blind to experimental group, and the order of testing was counterbalanced during behavioural experiments. All animal procedures were performed in accordance with NIH guidelines and with the approval of the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai.
Breeding
Adult virgin female mice were pair bred in house with age-matched males. Males were removed after a maximum of 5 days, and pregnant females were singly housed at least 2 days before parturition. Pups were counted on the day of birth (0 dpp) and weaned at 21 dpp. Only dams with litters between 4–10 pups were used for all experiments. On the day of weaning, dams were group-housed into cages of 3–5 mice with animals of the same experimental condition. For timed breeding, copulation plugs were checked every morning within 1 h after lights on, where confirmation of a plug was designated as embryonic day 0.5 (E0.5), signalling the immediate removal of the female to her own cage with a nestlet. Virgin NP females were age-matched for each experimental cohort. Mating + no pregnancy females were confirmed for the presence of a copulation plug. For mating + pregnancy + birth dams, pups were removed at 0 dpp within 3 h after lights-on to minimize maternal-offspring interactions. For comparison of RE dams to pup sensitized virgin females, litters were culled to 4 pups to equate litter size with the number of pups used for each sensitized female. In all other experiments, litters were not culled.
Postpartum stress paradigm
Stress RE females were subjected to limited nesting and maternal separation from 10–20 dpp (late stress RE) or 2–12 dpp (early stress RE), as previously described54,55,56. On each day of separation, the entire litter was removed to a clean cage with Sani-Chip bedding for 3–4 h. Separations occurred during the light cycle, and the timing varied each day to minimize predictability and acclimatization. EnviroDri nesting material was depleted to one-third of the amount in control cages during the days of separation. Following pup weaning on 21 dpp, the nesting material was restored to normal levels, and dams were group-housed into cages of 3–5 with mice of the same experimental condition.
Pup sensitization
Pup sensitization was conducted as previously described16,57. On the first day of pup sensitization, virgin NP females were presented with 4 pups (postnatal day 5) from a cage consisting of a lactating donor dam and her litter. Donor pups were exchanged for satiated pups from the same litter every 8–12 h for 21 days. Donor pups were weighed daily to ensure continual weight-gain throughout the experiment, and only pups that exhibited consistent weight gain were used. Behavioural observations were conducted during the first four days of sensitization. Each dam was observed for 30 min during the light phase for the following maternal behaviours: licking/grooming, crouching and nestbuilding. During these observation periods, females were closely monitored to ensure that they did not display aggressive behaviour toward the donor pups. On the last day of sensitization, donor pups were weaned from the cage, and sensitized females were group-housed into cages of 3–5 mice with animals of the same experimental condition.
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