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. 2013 Jan 9;33(2):824–839. doi: 10.1523/JNEUROSCI.2229-12.2013

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Copyright © 2013 the authors 0270-6474/13/330824-16$15.00/0

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Figure 8. — RIM3γ and RIM4γ knock-down affects early axonal outgrowth. A, Hippocampal neurons transfected DIV 1 expressing GFP (Control), shRNAs against RIM3γ (shRIM3) and RIM4γ (shRIM4), shRNAs with few nucleotide exchanges (mutated shRIM3 and mutated shRIM4) and coexpressing the shRNAs with the respective resistant cDNAs (Rescue RIM3 and Rescue RIM4) were fixed and analyzed by confocal microscopy at DIV 5. B, C, Quantitative analysis of total axonal length (B) and the number of axonal branches (C) showed that axonal outgrowth and branching are strongly reduced after knock-down of RIM3γ (shRIM3) and RIM4γ (shRIM4) as compared with controls. Both parameters are unaffected after cotransfection of shRIM3 or shRIM4 and a resistant version of the respective RIM variant (Rescue RIM3 and RIM4) or using mutated shRNAs against RIM3γ (mutated shRIM3) and RIM4γ (mutated shRIM4). D, Measurements of axonal growth of hippocampal neurons at DIV 8–DIV 9 from the time-lapse experiment of Figure 7B revealed that neurons transfected with shRNAs grow with reduced velocity 24–48 h after transfection. Significance: one-way ANOVA with Tukey's multiple-comparison test, ***p < 0.001, **p < 0.01.