Figure 2: Shrm4 is key mediator of dendritic spine formation and morphology. | Nature Communications

Figure 2: Shrm4 is key mediator of dendritic spine formation and morphology.

From: Epilepsy and intellectual disability linked protein Shrm4 interaction with GABABRs shapes inhibitory neurotransmission

Figure 2

(a) Sholl analyses were performed on 18DIV rat hippocampal neurons transfected at 8DIV with GFP-coexpressing knockdown shRNA#1 or scrambled#1. No difference in dendritic arborization and diameter was observed between the two conditions. Scale bars, 10 μm. (b) Images of rat hippocampal neurons transfected at 8DIV with Shrm4 scrambled#1, shRNA#1 or rescue constructs and immunostained for PSD-95, Bassoon, GluA2 and Synapsin post-synaptogenesis at 18DIV. Scale bar, 10 μm. (c) Numbers of fluorescence puncta/15 μm dendrite (left panel) and mean fluorescence intensity of synaptic markers (right panel). The effect of Shrm4 silencing could be rescued by coexpressing wild-type Shrm4 cDNA resistant to knockdown by shRNA#1 (Data normalized to scrambled#1 controls) Histograms show mean ±s.e.m.; see Supplementary Table 2 for statistical tests. (d) Normalized spine density per 20 μm of hippocampal neurons transfected at 8DIV for Shrm4 knockdown and rescue. See Supplementary Table 2 for statistical tests. (e) Spine head width and (f) length for scrambled, knockdown and rescue cells. See Supplementary Table 2 for statistical tests. (g) Percentages of mushroom, stubby and thin spines for scrambled, knockdown and rescue cells. Histograms show mean ±s.e.m.; see Supplementary Table 2 for statistical tests. (h) (Left) Images of rat hippocampal neurons transfected at 8DIV with scrambled, GABAB1 knockdown shRNA or rescue constructs and analysed at 18DIV. (Right) Histogram showing normalized spine density per 20 μm of hippocampal neurons transfected at 8DIV for scrambled, GABAB1 knockdown and rescue; Histograms show mean±s.e.m.; n=6–8 cells; Scrambled versus shRNA **P=0.0056; shRNA versus rescue *P=0.0108, one-way ANOVA-Mann–Whitney. Scale bars, 10 μm. (i) (Left) Images of 17DIV rat hippocampal neurons transfected at 5DIV with pSuper and treated from 8DIV to 13DIV with a Tat control or Tat-859–870 peptide. (Right) Histogram showing spine density per 40 μm dendrite of 17DIV-treated hippocampal neurons; n=10; ***P=0.0001; t-test. Scale bar, 10 μm. (j) Representative Ca2+signals from hippocampal neurons expressing dsRed-coexpressing knockdown shRNA#1 or scrambled#1 along with GCaMP6 fast under basal conditions in modified Krebs solution (in mM: 140 NaCl, 2.5 CaCl2, 4.7 KCl, 11 glucose, and 5 HEPES, pH 7.4. Scale bar, 5 μm. (k) Representative Ca2+ signals imaged from single dendrites (grey) and averaged traces (black) using GCaMP6f. (l) Maximum and averaged ΔF/F0 values of Ca2+ signals in scrambled#1 and shRNA#1 condition. (m) Time course of averaged ΔF/F0 values and single exponential fits (red) of Ca2+ signals in scrambled#1 and shRNA#1 condition. (n) Mean rise times and (o) decay τ of Ca2+ signals in scrambled#1 and shRNA#1 condition n=10–15; histograms are means±s.e.m. *P<0.05, **P<0.01, ***P<0.001; t-test.

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