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. 2018 Jul 6;11:223. doi: 10.3389/fnmol.2018.00223

Figure 3.

Figure 3

The ID-related mutation C18Y prevents synaptic potentiation by a constitutively active form of Rac1, Rac1b. (A,B) Scatterplots show eEPSC amplitudes for single pairs of CA1 pyramidal neurons transfected with Rac1b (green) or Rac1b C18Y (red) and their corresponding control neurons (open circles). Filled circles show mean ± SEM (insets). Current traces from control (black) and transfected (green for Rac1b, red for Rac1b C18Y) neurons are shown (Scale bars: 20 ms for AMPA, 50 ms for NMDA, 20 pA). Bar graphs show the average eEPSC amplitudes (± SEM) of neurons expressing Rac1b or Rac1b C18Y normalized to their respective average control eEPSC amplitudes. Wilcoxon Rank Sum Tests were used to compare across independent conditions (i.e., Rac1b and Rac1b C18Y in (A,B), *P < 0.05). (A) Rac1b expression increased AMPAR-eEPSC amplitude (n = 12 pairs, *P < 0.05, Wilcoxon Signed Rank Test). Rac1b C18Y expression reduced AMPAR-eEPSC amplitude (n = 9 pairs, *P < 0.05, Wilcoxon Signed Rank Test). (B) Rac1b expression increased NMDAR-eEPSC amplitude (n = 10 pairs, *P < 0.05, Wilcoxon Signed Rank Test). Rac1b C18Y expression did not affect NMDAR-eEPSC amplitude (n = 8 pairs, P > 0.05, Wilcoxon Signed Rank Test). (C) Rac1b and Rac1b C18Y expression did not affect PPF ratios at interstimulus intervals of 20, 40, 70 and 100 ms (Upper plot: 20 ms: n = 6 pairs, 40 ms: n = 8 pairs, 70 ms: n = 4 pairs, 100 ms: n = 6 pairs, P > 0.05, Student’s t-test; Lower plot: 20 ms: n = 6 pairs, 40 ms: n = 8 pairs, 70 ms: n = 5 pairs, 100 ms: n = 6 pairs, P > 0.05, Student’s t-test). Peak 1-scaled current traces from control (black) and transfected (green for Rac1b, red for Rac1b C18Y) neurons are shown. (Scale bar: 20 ms). n.s., not significant. (D) CV analysis of AMPAR-eEPSC from pairs of control and Rac1b/Rac1b C18Y neurons and of NMDAR-eEPSCs from pairs of control and Rac1b neurons. CV−2 ratios are graphed against the mean amplitude ratio for each pair (open circles) (green for Rac1b AMPAR-eEPSC: n = 12 pairs; black for Rac1b NMDAR-eEPSC: n = 10 pairs; red for Rac1b C18Y AMPAR-eEPSC: n = 9 pairs). Filled circles show mean ± SEM. Dashed lines show linear regression and 95% confidence intervals. (E) Rac1b expression but not Rac1b C18Y expression increased dendritic spine density. Representative dendritic spine images from neurons transfected with GFP (control), Rac1b, or Rac1b C18Y are shown on the left (Scale bars: 5 μm). The bar graph shows average spine density (mean ± SEM) of neurons expressing Rac1b (control: 0.17 ± 0.015 spines/μM, n = 11; Rac1b: 0.31 ± 0.031 spines/μM, n = 10 pairs, *P < 0.05, Student’s t-test) or Rac1b C18Y (control: 0.24 ± 0.075 spines/μM, n = 7; Rac1b C18Y: 0.19 ± 0.033 spines/μM, n = 13, P > 0.05, Student’s t-test) normalized and compared to GFP expressing control neurons. Wilcoxon Rank Sum Test was used to compare across independent conditions (i.e., Rac1b and Rac1b C18Y in E, *P < 0.05).