Mice were deeply anesthetized with an overdose of isoflurane and transcardially perfused with PBS followed by ice-cold 2% PFA in PBS. The brains were quickly removed and cut into coronal sections with 300 μm thickness using a vibratome in ice-cold 0.1 M sodium phosphate buffer (pH 7.4), supplemented with 2 mM CaCl2. The hippocampus area was dissected from the coronal sections, and further fixed with 2% GA and 2% PFA in 0.1 M sodium phosphate buffer, supplemented with 2 mM CaCl2 at 4 °C for 3 h. The samples were stored in 2% PFA in 0.1 M NaCac prior further processing. The samples were then washed with 0.1 M NaCac (pH 7.4) and osmicated with 2% osmium tetroxide in 0.1 M NaCac for 1.5 h, followed by 2.5% ferrocyanide in 0.1 M NaCac for another 1.5 h. After washes with water, the samples were immersed with 1% thiocarbohydrazide at 40 °C for 30 min followed by treatments with 1% unbuffered osmium for 1 h, and 1% UA in water at 4 °C overnight. Between the treatments, the samples were washed with ion-exchanged water 5 times for 5 min. For embedding, samples were dehydrated through a graded ethanol series into pure acetone followed by gradual infiltration into Durcupan™ according to the manufacturer’s instructions. After polymerization at 60 °C for 48 h, the embedded samples were trimmed and mounted on aluminum pins with conductive glue, prior to coating the exposed hippocampal surface with a thin layer of platinum. The target volumes in the hippocampus were imaged by a Zeiss FIB-SEM using a 2.5 nm pixel size with 5 nm milling depth. The SEM micrographs were acquired with 0.31 nA electron beam, 1.3 or 1.4 keV, dwell time 2 − 3 µs with 1 − 2 times line averaging. Both backscattered and secondary electron signals were collected using Inlens detectors. The new surface for serial FIB-SEM imaging was generated by FIB milling with a 0.7 nA beam current at an acceleration voltage of 30 kV. The datasets were collected and aligned using Zeiss Atlas 5 software with a 3D tomography module. The representative synapses in the electron microscopy data sets generated were manually segmented using MIB software, and the vesicles and endosomal structures which were located mostly in the volume covering the depth of 300 µm were included in the models. Visualization of the models was done by Amira software. Also dataset from WT mouse with 10 nm pixel, slicing 10 nm.

2024