All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (publication no. 85-23, revised 1985). The experiments were approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University (No: 15030254). Sprague-Dawley (SD) dams with pups were bred in our colony in a temperature-controlled (22-23°C) room on a 12 h light/dark cycle (lights on at 8:00 AM) with free access to food and water. Fourty PND-7 SD male rat pups (11-14 g) were used in our experiment and were randomly assigned to ketamine-treated and sham-treated groups. The grouping method was performed by using the methods described in our previous study . In the anesthesia group, ketamine was diluted in 0.9% normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg×4 injections). In the sham-treated group, rats received an equal volume of normal saline. Temperature probes were used to facilitate the control of temperature at 36.5±1°C by using computer-controlled heater/cooler plates that were integrated into the floor of the chamber. Between each injection, animals were returned to their individual chambers to help in maintaining body temperature and to reduce stress.
Morris Water Maze Test (MWM)
The apparatus and behavioral procedures of the MWM test have been previously described . Behavioral testing was conducted in a circular, black painted pool (180 cm diameter, 50 cm deep). The water temperature was maintained at 25±1℃. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and was placed in the center of the quadrant III, which was determined by using four starting locations (defined as Ⅰ, Ⅱ, Ⅲ and Ⅳ) at equal distances on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory setting, and all of the rats were trained four times per day, with the starting positions being randomized for each rat. When each of the rats found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be gently guided to the location and allowed to stay on the platform for 30 s, and the latency time in finding the hidden platform was recorded as 120 s. The average time from the 4 trials was represented as the daily result for each of the rats. Following the completion of the training, spatial memory was assessed in the probe tests, the hidden platform was removed and each of the rats was placed in the opposite quadrant. The rats were allowed to swim freely for 120 s. The number of times that each of the rats swam to cross the previous platform area and the number of times that each of the rats stayed in the target quadrant within 120 s were recorded. The paths of each of the animals were tracked by using a computerized video system. After every trial, each rat was placed in a heater plate for 1 to 2 min until they were dry, after which they were returned to their chambers. The data were analyzed by using software for the MWM (Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical university, Xuzhou, China.).
The experimental design that was used in the present study is outlined in Fig. 1. To label the dividing neurons in the early postnatal DG, we injected BrdU (5-bromo-2-deoxyuridine; Sigma), at a dosage of 100 mg/kg, into the rats. In experiments 1 and 2, animals received three consecutive BrdU injections on PND-7, 8 and 9 after exposure to the treatment.
Experiment 1 evaluated the survival rate of developmentally generated granule neurons in the adult DG. The animals were weaned at PND-35, after which they were housed in cages with free access to food and water for up to 2 months. Then, a portion of the animals were deeply anesthetized with 40 mg/kg ketamine and transcardially perfused with 0.9% normal saline, followed by a transfusion with 4% paraformaldehyde. The NeuN+/BrdU+ cells were examined by using double-immunofluorescence staining (n=5).
Experiment 2 evaluated the integration rate of developmentally generated granule neurons into the hippocampus-dependent memory networks in the DG. The animals were raised to an age of 3 months old, after which they were trained in the MWM task. Hippocampus-dependent memory was assessed following the training period (n=6). The expression of c-Fos was induced by neural activity, such as memory testing, all animals were deeply anesthetized with 40 mg/kg ketamine and sacrificed immediately after the completion of the MWM test. The experimental protocol is shown in Fig. 1. The c-Fos expression in NeuN+/BrdU+ cells was examined by triple-immunofluorescence staining (n=5). The integration rate of developmentally generated granule neurons into the hippocampal memory networks was estimated by calculating the proportion of c-Fos+/NeuN+/BrdU+ cells in the hippocampal DG.
Tissue Preparation and Immunofluorescence
The brains were postfixed in 4% paraformaldehyde and the coronal sections of the brains were cut consecutively at a thickness of 30 μm, at the point in which the hippocampus was initially exposed, the fifteenth section was taken and stored in PBS. The position of the hippocampus coronal sections selected in our study was approximately 2.80-2.85 mm posterior to the bregma for the 2 months old rats and approximately 2.90-2.95 mm posterior to the bregma for the 3 months old rats [15,16].
For the NeuN/BrdU double-immunofluorescence staining, the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37°C and by performing 3 washes with PBS for 5 min between each of these steps. The blocking of nonspecific epitopes with 10% donkey serum in PBS (which contained 0.3% Triton-X) for 2 h at room temperature preceded an overnight incubation at 4°C with the primary antibodies against NeuN (Mouse anti-NeuN monoclonal antibody; 1:200; Millipore, Massachusetts, USA) and BrdU (Rabbit anti-BrdU monoclonal antibody; 1:500; Abcam, San Francisco, USA). On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen Carlsbad, USA) for 2 h at room temperature.
For the Fos/NeuN/BrdU triple labeling, identical procedures were performed by using a primary rabbit anti-c-Fos polyclonal antibody (1:200; Abcam), a mouse anti-NeuN antibody (1:200; Millipore) and a rat anti-BrdU monoclonal antibody (1:500; Abcam). On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.
The photographs of the stained sections were taken by using a laser scanning confocal microscope (Fluoview 1000, Olympus, Japan), and a skilled pathologist, who was blinded to the study conditions, examined the labeled sections. The numbers of double-positive or triple-positive cells in the hippocampal DG were quantified by using Image-Pro Plus software (Media Cybernetics Inc., Bethesda, USA).
Brain Tissue Harvest and Western Blot Analysis
The rats selected in sham group and ketamine group were deeply anesthetized with ketamine and decapitated at 2 months or 3 months old respectively (n=3). The hippocampal DG tissue was dissected carefully with an anatomic microscope (leica EZ4HD). The harvested hippocampal DG tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15 min at 4°C and were resolved by 12% polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4°C with the primary antibodies rabbit anti-caspase-3 antibody (Cell Signaling Technology) and β-tubulin (Abcam). The membranes were then incubated with the appropriate secondary alkaline phosphatase-conjugated antibody (Abcam) for 1 h. The band intensity was quantified using Image J software.
The statistical analysis was conducted by using SPSS 13.0 (SPSS Inc., Chicago, USA), and the graphs were created by using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, USA). The data were analyzed by using the Mann-Whitney U test. The interaction between the time and group factors, which was determined by using a two-way ANOVA, was used to analyze the differences in escape latency between the rats in the control group and the rats that were treated with ketamine in the MWM. The data are presented as the mean±SD, and P<0.05 was considered to be statistically significant.