Double Immunofluorescence Staining
In order to obtain histological results, mice were anesthetized with 1% barbital sodium (100 mg/kg) (Aladdin, China) before being transcranially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA). After collecting the brains, they were fixed in 4% PFA at 4 °C for 24 hours and cryopreserved in 30% sucrose until sectioning. A sliding microtome (Leica, Germany) was used to section the brain coronally into 35 m sections containing the hippocampal area which is about 2 mm from the Bregma. Then the brain sections from hippocampus were blocked for 10 min in PBS containing 10% bovine serum albumin (BSA) and then incubated overnight at 4 °C with the primary antibodies NeuN (Mouse monoclonal) (1:100, Abcam, USA) and synaptophysin (Rabbit monoclonal) (1:100, CST, USA) or PSD95 (Rabbit monoclonal) (1:100, CST, USA). Subsequently, the sections were incubated with the secondary antibodies [Alexa Fluor 555-conjugated anti-mouse (1:1000 dilution, Thermo Fisher Scientific, USA), Alexa Fluor 488-conjugated anti-rabbit (1:1000 dilution, Thermo Fisher Scientific, USA)] for 2h at room temperature.  Each of the steps above was followed by three rinses in PBS lasting 10 minutes each. At the end the sections were mounted by fluorescent mounting medium (Sigma, USA). Laser-scanning confocal microscopes (Leica TCS SP2, Germany) were used to capture the confocal images.