Fig 1: Loss of excitatory synapses in hippocampal cell culture is CRH/BDNF/NF-?B-dependent.a IHC for Shank2 (green), Vglut1 (red), MAP2 (magenta) (scale bar = 5 µm), western blot analysis for all different treatment regimens (CRH; the CRHR1 blocker NBI30775; the corticosterone receptor blocker RU38486; and BDNF) and the relative quantification. b IHC for Shank2 (green), Vglut1 (red), MAP2 (magenta) and CRHR1 (white) with colocalization and trilocalization analysis (scale bar = 5 µm). c IHC for phospho-NF-?B p65 (green, scale bar = 5 µm) and the relative quantification of the signal within the nuclear compartment. d IHC for phospho-IKB-a and the relative quantification of the signal within the cytosolic compartment. e IHC for and compartment analysis of p65. f IHC (left panel) for Shank2 (green), Vglut1 (red), MAP2 (magenta) (scale bar = 5 µm), WB of BDNF (right panel) for all different treatment regimens blocking the NF-?B pathway (JSH (the translocation blocker JSH- 23); SC (the IKK-ß inhibitor SC-514); and LAC (the NF-?B activation blocker lactacystin)) and the relative quantification. g IHC for ATG5, immunoblot analysis of h BCN-1, i LC3, and j Lamp-2 and the relative quantification normalized to ß-actin. k TEM of acquired synapses after 15 min of CRH treatment (arrows indicate membrane-associated vesicles (scale bar = 0.5 µm)) and the relative quantification of docked vesicles and multivesicular bodies (MVBs)/synapses (a two-tailed unpaired T-test was used). l Quantification of the number of excitatory synapses (left) by IHC and WB analysis of BDNF expression (right) after treatment with 100 nM CRH for 30 min and Leupeptin hemisulfate + 5 µM E64 (CRH++) compared with control treatment. N = 3–5; the error bars represent the SEMs; one-way ANOVA and Bonferroni’s post hoc comparison test were performed (*P = 0.05, **P = 0.005, ***P = 0.0005, ****P = 0.0001).