Transmission electron microscope analysis

The hippocampal tissues were fixed with 4% glutaraldehyde (Beyotime Biotech. Shanghai, China) for 2 hours and with 1% osmium tetroxide (OsO4) for 25 to 30 minutes, and washed with phosphate-buffered solution (PBS, ZSGB Bio. Tech., Beijing, China) for 2 times, 10 minutes per time. Then, the hippocampal tissues were dehydrated in graded ethanol (Sigma-Aldrich, St. Louis, MO, USA), including 50% and 70% ethanol for 1 time, 90% ethanol for 2 times and 100% ethanol for 3 time, for 12 minutes per time. The hippocampal tissues were continuously embedded in acetone and embedding regent (Cat. No. EPON812, Sigma-Aldrich, St. Louis, MO, USA). The hippocampal tissues were sliced into sections with ultramicrotome (Mode: OMU3, Leica Reichert, Germany) and stained with uranyl acetate (Beyotime Biotech. Shanghai, China) and lead citrated (ZSGB Bio. Tech., Beijing, China). Eventually, the tissue sections were observed with Philips TECNAI-10 transmission electron microscope (Mode: TECNAI-10, Philips, Holland, Switzerland) as the previous report described [21].