Western blotting
Immediately after the sucrose consumption test, all rats were decapitated and the brains were rapidly removed on ice. The hippocampus were dissected bilaterally according to the atlas of Paxinos and Watson.[22] Tissues were homogenized in 20 volumes of buffer (pH 7.4, containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA Ethylenediamine tetraacetic acid disodium, 1 mM EGTA Ethyleneglycol bis (2-aminoethyl) tetraacetic acid, 1% Triton X-100, 2.5 mM sodium orthovanadate, 1 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). Protein content of lysates was determined using bicinchoninic acid (Pierce Biotechnology, Rockford, IL, USA). Lysates were mixed with 5 × sodium dodecyl sulfate (SDS) to prepare for certain concentration of sample solutions. Thereafter proteins were separated by 10% SDS-polyacrylamide gels and were blotted onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA) by electrophoretic transfer. Blots were incubated in blocking buffer (5% nonfat dry milk powder in tris-buffered saline containing 0.1% Tween-20, [TBST]) for 1 h at room temperature and washed 3 times with TBST for 10 min each. Blots were then incubated overnight at 4°C with 1:1000 diluted primary antibodies including mouse anti-β-actin, rabbit anti-ERK1/2 and mouse anti-P-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then washed 3 times for 10 min each in TBST. Blots were incubated with appropriate Horseradish peroxidase -labeled goat anti-mouse or goat anti-rabbit secondary antibody IgG (Santa Cruz) for 2 h at room temperature, washed 3 times for 10 min each in TBST, treated with ECL reagents (Amersham) and exposed to film. Band intensity was quantified by Glyko BandScan software (Glyko Inc., Novato, CA, USA). The relative level of each signal protein was calculated as the ratio of total gray of each signal protein/β-actin.