Transmission electron microscopy imaging

Immediately after the animals were sacrificed, the hippocampal tissues were harvested, fixed for 24 hours, then placed in 2.5% glutaraldehyde solution for 2 hours and then 1% reagent enzyme for 2 hours. After fixation, the tissues were rinsed with distilled water and placed in a gradient of ethanol and acetone solutions for dehydration; after dehydration, the tissues were placed in a mixture of epoxy resin and acetone overnight (16 hours) at room temperature, embedded in epoxy resin, and dried for storage using a thermostat. The tissue blocks were trimmed, cut into 100-nm ultrathin sections using a Leica EM UC7 ultramicrotome (Leica, Weztlar, Germany), and stained with uranyl acetate and lead citrate (Zhongjingkeyi Technology Co., Ltd., Beijing, China) for 10 and 5 minutes, respectively. The sections were allowed to dry and observed by transmission electron microscopy (TEM) (JEM-1400Flash, JEOL Japan Electronics Co., Ltd., Tokyo, Japan). For each group of animals, four thin-layer sections, each obtained from a different animal, were placed in a copper mesh for TEM acquisition. First, five fields were acquired at ×2000. Next, for each field, we took two random photographs at ×8000 and ×10,000. For each group of animals, > 50 synapses were included in the assessment. Image capture and TEM assessment were performed by two pathologists blinded to group information, and disagreements were resolved by negotiated consensus.