Characterization of TrkC KD used in the current study. A, Design of lentiviral shRNA vectors for KD of TrkC. H1, Human H1 promoter; IRES, internal ribosome entry sequence; Ub, ubiquitin promoter. B, Levels of TrkC mRNA were measured at DIV12–13 by quantitative RT-PCR in cultured cortical neurons infected at DIV3 with lentiviruses expressing the indicated candidate TrkC shRNAs (T3–T9). Dashed line indicates 70% KD cutoff level for tests of biological effects. C, Rat hippocampal neurons were transfected with lentiviral vectors expressing shRNAs targeting TrkC (T4, T7, or T9) or control vectors lacking shRNA expression (sh-Control) at DIV8 and analyzed at DIV14 by double immunofluorescence with antibodies to EGFP (green) and synapsin (red). Scale bar, 10 μm (applies to all images). D, Summary graphs of the effects of lentiviral vectors expressing shRNAs targeting TrkC (T4, T7, or T9) in neurons on puncta density (left), puncta size (middle), and puncta intensity (right) measured using synapsin as a synaptic marker. All data are shown as means ± SEM (*p < 0.05, 3*p < 0.001; ANOVA with post hoc Tukey's test). E, F, Representative images (E) and quantification results (F) demonstrating no alteration in dendritic branch number in NT-3- or TrkC-deficient cultured neurons. Hippocampal neurons were infected with lentiviruses expressing sh-Control (Control), sh-NT-3, or sh-TrkC at DIV3 and analyzed at DIV11 using Sholl analysis. All data are shown as means ± SEM. “n” denotes the number of neurons as follows: sh-Control, n = 21; sh-NT-3, n = 22; and sh-TrkC, n = 17. Scale bar, 60 μm (applies to all images).
