Fig. 1. siRNA-mediated suppression of Shank3, Nlgn3, Fmr1, Mecp2 or Tsc1 in the dorsal striatum produced mild to severe autistic-like phenotypes. (A) Experimental design of siRNA injection into the dorsal striatum and following behavioral tests. A diagram showing siRNA injection sites in the dorsal striatum (AP, +1.0; ML, ±1.5; DV, −3.6 mm). Representative photomicrograph showing the dorsal striatum injected with fluorescence-tagged siRNA-control. Brain sections were prepared 2 days after siRNA injection. dST: dorsal striatum; cc: corpus callosum. (B) Real-time PCR data showing the level of siRNA-induced knockdown of Shank3, Nlgn3, Fmr1, Mecp2 and Tsc1 in HT22 cells. Cells were harvested 24 h after siRNA transfection. (C, D) Grooming (C) and digging (D) behaviors of mice with the knockdown of Shank3, Nlgn3, Fmr1, Mecp2 or Tsc1 in the dorsal striatum. (E, F) Experimental design of the social novelty preference test using U-field two-choice field (E). The exploration time of mice with the knockdown of Shank3, Nlgn3, Fmr1, Mecp2 or Tsc1 in the dorsal striatum (F). (G, H) Experimental design of novel object preference (G), and sniffing time against the object of mice with the knockdown of Shank3, Nlgn3, Fmr1, Mecp2 or Tsc1 in the dorsal striatum (H). n=6–8 animals. (I) Real-time PCR data showing the siRNA-induced knockdown of Mecp2 and Tsc1 in the dorsal striatum of WT mice (I). n=3 animals, and 3 PCR repeats for each siRNA. (J, K) Experimental design of the three chamber test (J). Social interaction time between stranger vs. empty cage for mice with the knockdown of Mecp2 and Tsc1 in the dorsal striatum (K). (L, M) Locomotion in the open field test (L), and marble burying (M) behaviors of mice with the knockdown of Mecp2 and Tsc1 in the dorsal striatum. ** in Figure 1M denotes the difference between Con-siRNA and Tsc1-siRNA at p<0.01 at time points marked by arrowheads. n=6–9 animals. Data are presented as mean±SEM. * and ** denote the differences between the indicated groups at p<0.05 and p<0.01, respectively (Student's t-test, two-way ANOVA, two-way repeated measures ANOVA and Holm-Sidak post hoc test).
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