Journal: Molecular Psychiatry

Article Title: A CRHR1 antagonist prevents synaptic loss and memory deficits in a trauma-induced delirium-like syndrome

doi: 10.1038/s41380-020-0659-y

Figure Lengend Snippet: 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-κB p65 (green, scale bar = 5 μm) and the relative quantification of the signal within the nuclear compartment. d IHC for phospho-IKB-α 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-κB pathway (JSH (the translocation blocker JSH- 23); SC (the IKK-β inhibitor SC-514); and LAC (the NF-κB 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 β-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).

Article Snippet: The following primary antibodies were purchased from commercial suppliers: Vglut1 (1:500, Synaptic Systems GmbH, #135304), Gephyrin (1:500, Synaptic Systems GmbH, #147003), Vgat (1:500, Synaptic Systems GmbH, #131011), Iba1 (1:250, Wako Chemical GmbH, #NCNP24), GFAP (1:500, Sigma-Aldrich, #G3893 ) , NeuN (1:1000, Millipore, #MAB377), C1q (1:1000, Abcam, #182451), CRH (1:1000, Abcam, #8901), p62 (1:500, Abcam, #56416), Map2 (1:500, EnCor Biotechnology Inc., #CPCA-MAP2), Synaptotagmin-1 (1:500, Synaptic Systems, #105311C5), ATG5 (Abcam, #AB108327), NF-κB p65 (1:500, Santa Cruz, #SC8008), phospho-NF-κB (Thr435) (1:500, Thermo Fisher Scientific, #PA5–37724), phospho-IκBα (Ser32) (1:500, Cell Signaling, #2859), CRHR1 (1:500, Everest Biotech, #EB08035), and ChAT (1:250, Synaptic System, #297015).

Techniques: Western Blot, Blocking Assay, Translocation Assay, Activation Assay, Two Tailed Test, Expressing