From P7 to 14, pups were placed onto separate containers individually without heating. From P2 to 7, pups were placed together on a heating pad maintained at 37℃ during the separation period. In the maternal separation group (MS), pups were removed from their home cages from P2 to P14 for 3 hours daily at randomized time points during the light cycle. At the end of the separation period, both pups and dams were returned to the home cage.
Additionally, cages containing the dams were placed in a separate room to prevent any potential communication among the dams and pups. For both maternal separation procedures, dams were moved from home cages to a clean cage with ad libitum food and water during separation. In the maternal control (MC) group, with the exception of routine bi-weekly cage changes, pups were left untouched with the dams until weaning at P21. Other exclusions are listed in the figure legends. During the course of testing, one cage in the MC group in the MSEW cohort exhibited excessive fighting and three mice progressively sustained severe injuries and were excluded from some tests. The MS cohort originally consisted of 9 MS and 8 MC mice while the MSEW cohort was comprised of 9 MSEW mice and 12 MC mice. All tests were conducted on the same cohorts of animals. All experimental procedures were conducted in accordance with guidelines for the care and use of laboratory animals for scientific purposes with approved protocols from the Institutional Animal Care and Use Committees of Duke-NUS Medical School (Protocol number: 2014/SHS/999). Upon weaning, littermates were group-housed (up to 4 per cage). All the pups from one dam (a litter) were randomly assigned to one of the groups. If birth occurred, age was designated as P0. Breeding pairs were checked daily for litters. Food and water were provided ad libitum on a 12-hr light/dark cycle (lights on at 0700 h). Intriguingly, we could not observe the predicted behavioral anomalies upon either MS or MSEW paradigm.Ĭ57BL/6J breeding pairs were housed in a specific pathogen-free environment and maintained at 22℃ with 55% humidity. We hypothesized that the MSEW paradigm would result in increased severity in anxiety- and depressive-like behavior as well as enhanced contextual and cue fear learning as reported. Following the two different paradigms, animals underwent multiple behavioral tests relevant to depression-like and anxiety-like behaviors and cognitive functions at P60. Here, we employed two different, early-life stress paradigms – a conventional protocol with 3-hour separation from P2 to P14 (MS) and a relatively recent protocol with longer time for maternal separation together with early weaning at P17 (MSEW) in C57BL/6J mice.
While maternal separation paradigms in rat models are relatively well-established, behavioral effects upon MS paradigms in mice are often inconsistent ( Table 1), which may be due to subtle differences among separation paradigms, animal handling, or housing environments.
In typical maternal separation paradigms, pups undergo 3 hours or more episodes of separation from the dam daily, which has been linked to increased levels of stress hormones and behavioral changes in adult animals. To identify underlying mechanisms of early-life, stress-induced brain dysfunction, maternal separation (MS) paradigms were developed in rodents. Early-life aversive experiences including childhood abuse, trauma, and neglect have been associated with increased risk for neuropsychiatric disorders, accompanied by potential neurophysiological changes such as the reduced volume and abnormal activation in the hippocampus, amygdala, and prefrontal cortex.
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