Journal: Frontiers in Molecular Neuroscience

Article Title: Autism Spectrum Disorder Risk Factor Met Regulates the Organization of Inhibitory Synapses

doi: 10.3389/fnmol.2021.659856

Figure Lengend Snippet: Hepatocyte growth factor (HGF) increases gephyrin clustering in vitro . Hippocampal neurons were left untreated or were treated with DMSO, HGF, or PHA-665752. Representative images of gephyrin- or MAP2-stained hippocampal neurons are shown in the absence (A–A”) or presence (B–B”) of HGF. Overview images are shown in ( A,B ; scale bar 20 μm) while insets are enlarged in ( A’,A”,B’,B” ; scale bar 10 μm). Panels (A’,B’) depict confocal images, (A”,B”) processed images for the quantification of gephyrin cluster (red) densities within masks of MAP2-positive dendritic segments (25 μm). ( C) Quantification of gephyrin cluster densities. One-way ANOVA and Tukey’s multiple comparison test F (4,364) = 0.7245. CTR: n = 75, HGF: n = 71, DMSO n = 78, PHA n = 73, PHA/HGF n = 72. *** p < 0.0001, error bars SEM. (D,E) Untreated hippocampal neurons (D) or treated with HGF (E) were stained for phosphorylated p70S6K (pS6K, scale bar 20 μm). Insets are enlarged in ( D’,E’ ; scale bar 10 μm) for determination of mean fluorescence intensities of phosphorylated p70S6K signals within a mask of MAP2-positive cell bodies. Dashed outlines mark the area of the cell bodies. (F) Quantification of phosphorylated p70S6K intensities. CTR n = 57, DMSO n = 83, HGF n = 83, PHA n = 68, PHA/HGF n = 77. One-way ANOVA and Tukey’s multiple comparison test F (4,363) = 3.369. *** p < 0.0001, error bars: SEM.

Article Snippet: Inhibitors were dissolved in dimethylsulfoxide (DMSO), HGF (Merck KGaA, Darmstadt, Germany) in PBS and added to hippocampal neurons (DIV 9) to final concentrations of 50 ng/ml for HGF, 200 nm for mTOR inhibitor rapamycin (Merck), and 1 μM PHA-665752 (Merck KGaA, Darmstadt, Germany).

Techniques: In Vitro, Staining, Fluorescence