Figure 1:

MSUS paradigm. (a) For MSUS, naïve males and primiparous females are mated, and starting 1 day after birth until PND14 (PND1–14), pups (F1) are separated from their mother (F0) for 3 h daily at an unpredictable time. (b) During separation, dams are unpredictably exposed to either an acute (5 min) swim in 18°C water or a 20-min physical restraint in a tube anytime (unpredictably) during the 3 h. Between PND15 and weaning at PND21, pups are left undisturbed with their mother and are then raised normally until adulthood. Control animals are produced and raised normally (left). (c) When adult (>3 months), F1 males are mated with naïve primiparous control females to produce F2 offspring, and breeding is repeated with males of each generation to produce F3, F4, F5, and F6 offspring. Male breeders are removed from the mating cage after 1 week and are never in contact with their offspring. MSUS is applied only to F1 pups but not to any of the following offspring. When adult, animals from each generation undergo behavioural and metabolic testing. The present study used F5 and F6 animals. Created with BioRender.com