Fig. 2: In vitro and in vivo synaptic defects in morphology and function.

A Impaired spine maturation in cultured neurons derived from rat hippocampi affected by mutant Shank1. Neurons were transfected at DIV8 and examined at DIV18. A, a Examples of spines from neurons transfected with GFP constructs (in green) and Shank1 constructs (in red). The scale bar is 2 µm. A, b, d Quantitation of spine length (µm) and head width (µm) (8–10 neurons and >650 spines examined from three to five independent experiments for each construct) (one-way ANOVA). A, c, e Comparisons of the percentages of spines with length ≤ 1 µm or head width ≤ 1 µm between mutant and WT Shank1 neurons (chi-square test with Bonferroni correction). B Abnormal morphology of dendritic spines of CA1 neurons observed in Shank1 R882H-KI mice with Golgi staining (35–40 neurons and >1500 spines from 3 mice per genotype). B, a Representative images of secondary dendrites. The scale bar is 5 µm. B, b, c Reductions in spine length and head width in R882H-KI mice, presented in cumulative frequency plots (Kruskal-Wallis test followed by Kolmogorov-Smirnov test) and bar graphs (one-way ANOVA). B, d Reduced spine density in R882H-KI mice (one-way ANOVA). C Ultrastructure of excitatory synapses in the hippocampus observed by TEM (>120 PSDs from 3 mice for each genotype). C, a Representative TEM images of CA1 synapses. Arrowheads, synaptic vesicles; arrows, PSDs; and stars, dendritic spines. The scale bar is 100 nm. C, b, c Elongated length and thinner thickness of PSDs, presented in cumulative frequency plots (Kolmogorov-Smirnov test) and scatterplots (unpaired Student’s t-test). D Perturbed glutamatergic synaptic function in Shank1 R882H-KI mice. D, a Long-term potentiation was significantly decreased during the last 50 min of recordings in HOM mice, as is evident from the average time plot (repeated-measures ANOVA, genotype, P = 0.008, time, P < 0.0001, interaction, P < 0.0001; 7–8 slices from 3–4 mice for each genotype). D, b The frequency of mEPSCs was reduced, while the amplitude was not affected, in HOM mice (unpaired Student’s t-test; 8–10 cells from 4 mice for each genotype). All data are presented as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for groups compared with WT.