Fig. 6

TSP-1 prevents spine alterations in Fmr1 KO neurons. Analysis of the effects of TSP-1 administration on dendritic spine morphology in Fmr1 KO hippocampal neurons at 17 DIV. Continuous treatment with recombinant TSP-1 (250 ng/ml) averted the spine alterations in Fmr1 KO neuronal cultures. a Cumulative frequency distribution of spine lengths. b rTSP-1 induced a significant reduction in spine length and (c) the density of filopodia-like spines in the Fmr1 KO neurons. d A marked increase in the density of stubby spines was observed following the application of rTSP-1. Heat-inactivated human recombinant TSP-1 (HI-TSP1) or gabapentin (GBP; 32 ug) in combination with rTSP-1 did not exert a neuroprotective effect on spines. One-way ANOVA with Bonferroni correction was used to analyze the data. All data are expressed as mean ± SEM. *p < 0.05; n = 75 neurons per group, N = 3 independent experiments. e Dendritic segments labeled with fluorescent DiI to illustrate typical spine morphologies found in Fmr1 KO neurons and following treatment with exogenous TSP-1, GBP/TSP-1 or HI-TSP-1, respectively. Scale bars: 10 μm. f Excitatory synapse formation is normalized in Fmr1 KO following rTSP-1 treatment. Data was analyzed by Student’s t-test. Data are presented as the mean ± SEM, ***p < 0.001; n = 75 neurons per group, N = 3 independent experiments. g Double immunofluorescence of dendritic segments with PSD-95 (red) and synaptophysin (green) to identify excitatory synapses. Scale bars: 10 μm