After recordings, slices were placed in a fixative containing 4% FA and 0.2% picric acid in 0.1 M PB (pH = 7.4) for 12 hr at 4°C. They then were embedded in agarose (2%) and re-sectioned at ~150 µm thickness. The biocytin-filled cells were visualized with Cy3-conjugated streptavidin (1:1000, Jackson ImmunoResearch, Bar Harbor, ME) in TBS containing 0.2% Triton X-100. Sections were then treated with uranyl acetate, dehydrated in a graded series of ethanol, incubated in acetonitrile, and flat-embedded in epoxy resin (Durcupan) as described in Holderith et al., 2020 (link). Putative contacts between the recorded neurons were identified with visual inspection at high magnification (60×, 1.35 NA objective, Olympus FV1000 microscope, Tokyo, Japan). Tissue blocks containing the biocytin-filled processes were re-embedded, and ultrathin (70 or 200 nm) serial sections were cut and mounted on adhesive Superfrost Ultra plus slides. Potential contact sites between the presynaptic PC boutons, and the postsynaptic dendrites were identified on the ultrathin sections, imaged using a confocal microscope (Olympus FV1000), and reconstructed with a custom-made ImageJ plugin (HyperStackStitcher, 3DHistech, available on the website: http://www.nusserlab.hu/software.html ).
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