Specific pathogen-free Wistar rats (No. 2015-0018, certification
number: 42000600014526, licence number: SCXK (Hubei), with weights of 200 ± 20 g for females and 280 ± 20 g for males) were obtained from the Experimental Center of the Hubei Medical Scientific Academy (Wuhan, China). Animal experiments were performed at the Center for Animal Experiments of Wuhan University (Wuhan, China),which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The Committee on the Ethics of Animal Experiments of the Wuhan University School of Medicine approved the protocol (permit number: 201719). All animal experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee.Animals were housed in cages with padding in an air-conditioned room under standard conditions (room temperature: 18–22 °C; humidity: 40%–60%; light cycle: 12 h light-dark cycle; 10–15 air changes per hour) and allowed free access to rat chow and tap water. All rats were acclimated one week before experimentation, and two female rats were then placed together with one male rat overnight in a cage for mating. The appearance of sperm in the vaginal smears confirmed mating, and the day of mating was set as gestational day (GD) 0. As shown in Fig. 1, a group of pregnant rats received 0.2 mg/kg examethasone (No. H42020019, Shuanghe Pharmaceutical Company,Wuhan, China) subcutaneously every day starting from GD9. On GD20,the pregnant rats were anaesthetized using diethyl ether (No.one0009318, Hengyuan Biotech Co., Ltd., Shanghai, China) and sacrificed. Pregnant rats with litters sizes of 12–14 (the male/female ratio was approximately 1:1) were considered qualified (n = 14 pregnant rats per group), and male fetal rats were collected. Four whole brains were randomly collected for each group and fixed in 10% formalin,paraffin embedded and sectioned. The hippocampal samples that were dissected and collected from each littermate were pooled together and immediately frozen in liquid nitrogen for subsequent analyses. Another group of pregnant rats were allowed to deliver spontaneously at term.The number of pups per litter was randomly selected to 8 to ensure adequate and equal nutrition until weaning. At postnatal week 4 (PW4),
10 male pups from 10 different mothers were selected randomly for each group. At PW26, all pups were subjected to behavioural tests.After the tests, all rats were anaesthetized and sacrificed to collect the whole brain or hippocampal tissues.
