EM was performed as previously described (61). Briefly, mouse brain tissues were blocked in the fixative reagent (G1102, Servicebio) at 4°C for 2 hours and were postfixed with 1% osmium tetroxide for 2 hours. Tissues were dehydrated in graded ethanol solutions (50 to 100% and two times of acetone, 15 min each) and were embedded in EMbed 812 (90529-77-4, Structure Probe Inc., West Chester, USA) at 60°C for 48 hours. Brain tissues were prepared in ultrathin slices (60 to 80 nm) with an ultramicrotome (Leica EM UC7, Leica). Slices were stained with uranyl acetate in pure ethanol for 15 min, in lead citrate for 15 min and were air-dried. Images were captured with a transmission electron microscope (Hitachi, Japan). Both synapse density and axonal myelination were counted under the 13,500× magnification, postsynaptic (PSD) length and thickness were calculated under 46,000× magnification by ImageJ.

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