Fig. 3: Reconstruction of CA3 PN local input field with coCATS.
From: Imaging brain tissue architecture across millimeter to nanometer scales
a, Orthogonal views of a coCATS imaging volume recorded with z-STED at near-isotropic resolution in neuropil of an organotypic hippocampal brain slice (N2V, raw data: Supplementary Fig. 4). Yellow lines indicate position of displayed planes. Label: ATTO643-NHS. b, Magnified view of the boxed region in a. Asterisks: pSCRs. Imaging data are representative of coCATS in n = 10 organotypic slices. c, Left: CA3 PNs in an organotypic hippocampal slice whole-cell patch-clamp recorded and filled with fluorescent dye (Lucifer yellow). Right: magnified view of a piece of proximal dendrite in the boxed region. MIP, maximum intensity projection. d, Action potential response of the middle PN elicited by current injection (inset). e,f, Spontaneous post-synaptic potentials (PSPs) and post-synaptic currents (PSCs) recorded from middle PN. g, coCATS (gray, z-STED, STAR RED-NHS, N2V, single z-section of volumetric dataset) overlaid with the intracellular label (yellow, confocal) of the middle PN provides super-resolved information on its local microenvironment. h, 3D rendering of the same proximal dendrite (gold) and 57 structures synaptically connected to it, reconstructed from the volumetric coCATS data. Connectivity was inferred by the presence of pSCRs between the positively labeled dendrite and the respective adjacent structures. i, 3D rendering of two MFBs (violet and gray) forming complex connections with one thorny excrescence of the proximal dendrite. pSCRs are indicated in white (identified by deep learning model from Fig. 2j,k). j, Violin plots with median (line) and quartiles (dashed lines) of the volumes of MFBs (nMFB = 40) contacting the recorded PN and its spines (nspine = 68). k,l, Quantification of connectivity pattern of individual MFBs and PN spines for that dendrite. Data in c–g are representative of coCATS labeling in combination with functional recordings and dye filling of various cell types in n = 6 organotypic slices. 3D reconstruction as in h and i was performed for n = 1 specimen, and analysis in j–l comprised nspine = 68 spine structures and nMFB = 40 MFBs. Three additional MFBs were only partially contained within the imaging volume and, thus, not included in quantifications.
