PMID: 28430606 PMCID: PMC5464800 DOI: 10.18632/oncotarget.15405
MATERIALS AND METHODS
Animals
Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China). Prior to the experiment, rats were quarantined for two weeks at the Northeast Agricultural University (Harbin, China). All experiments were performed in accordance with the guidelines outlined by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China).

Mating and drug administration
Thirty-six Wistar rats were divided into 12 cages (one male and two females per cage) with an iron mesh at the bottom. On the next morning the vaginal suppository was investigated through the iron mesh. When sperm was detected, female rats were annotated as pregnant at day 0 (P0). The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55]. The total volume of ketamine stayed below 2 mL/100 mg. Ketamine-treated offspring were recorded as K group, while individuals within the control group were recorded as C group. The first day after birth was recorded as B0. During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure ​Figure11).
Sample collections
Rat pups were sacrificed at B30 via cervical dislocation, and were recovered to collect brain tissue for Nissl staining (n = 24, 1/dam), Golgi staining (n = 24, 1/dam), and western blotting (n = 72, 3/dam). A subset of their hippocampuses were quickly dispensed on ice, put into a freezing tube, and frozen in liquid nitrogen, while other tissues were preserved in 10% formalin.