Y-maze test is used to assesses short-term spatial memory in rodents. The maze was made of wood and 3 arms were positioned at equal angles. The serial arm entries were recorded for 5 min. Passing by half of the arm was recorded as entry. Alternation was considered as non-overlapping triplet set entries. The test was executed on the 39th day of birth. Alteration rates were calculated with followed formula, ‘Percentage of alternation (%) = (alternation / total number of arm entries- 2) × 100’. Closer to 100% alteration percentage of rat it means spatial memory function is better than lower alteration rat.

In rodent, passive avoidance test is a popular and easily used behavior test measuring long-term memory related to hippocampus function [20]. The apparatus consisted of light and dark acryl compartment. These two compartments were separated with a guillotine door. During the acquisition trial, rats were placed in the light compartment for 10s of habituation and guillotine door was opened. Immediately after rat entered the dark compartment, door was closed meanwhile electrical shock delivered (0.75mA, 50Hz) for 2 s through stainless steel floor. 24h later, the retention test was proceeded. The latency time until entering the dark compartment was measured without electrical shock. The maximum latency time was 600s. The test was executed on the 39th day of birth after Y-maze test. The latency time positively correlates with long-term memory [20].

Using immunohistochemical techniques, we evaluated c-fos and CREB expression in hippocampus. c-fos and CREB are transcription factors that bind to DNA and regulate BDNF expression. Animals were sacrificed under anesthesia. Then, animals were perfused and immersed with ice-cold PBS followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). Fixed brain tissues were storage in the sucrose solution for 3days on 4°C. Serial coronal sections of brains were cut into 40um thick with a microtome (CM3050S, Leica, Nussloch, Germany) and were immersed in storing solution. The brain sections were incubated in the primary antibody c-fos (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-CREB (1:1000; Millipore, Temecula, CA, USA) for 24h. Then sections were washed with PBS and incubated in a biotinylated anti-rabbit secondary antibody (1:400; Vector Laboratories, Brulingame, CA, USA) for 1hr. Next, the sections were incubated in avidin-biotin-peroxidase complex (Vector Laboratories, Brulingame, CA, USA) for 1hr. For the developing of color, 0.4% diaminobenzidine (Vector Laboratories, Brulingame, CA, USA) was used as the chromogen. The number of c-fos and phospho-CREB positive cells in the hippocampus was measured with image analysis system (ImagePro Plus, Media Cyberbetics Inc., Silver Spring, MD, USA).

The BDNF and trkB protein levels in hippocampus were evaluated by western blot. Increased expression of BDNF and trkB indicate the hippocampal synaptic plasticity and LTP. Hippocampal tissues were homogenized in a lysis buffer (iNtRON Biosystems, Seongnam, Korea). Proteins were resolved by 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and electro-transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 (TBST). Membranes were then incubated with anti-BDNF (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-trkB (1:1000; Cell Signaling Technology, Beverly, MA, USA) for a day. Membranes were incubated for 2h with horseradish peroxidase conjugated secondary antibody (anti-rabbit antibody, 1:5000, Molecular Probes, Eugene, OR, USA) in 5% skim milk in TBST and developed using enhanced chemiluminescence western blotting detection system (SuperSignal West Pico, Thermo Scientific, Rockford, IL, USA). The relative optical densities of the bands visible on X-ray film were scanned and digitized by Image J 1.42q software (NIH, Bethesda, MD, USA). All relative intensities were normalized to β-actin expression in the same sample.