Fig 2: Effects of Shank3b mutation on the dendrite and synapse development in ACC. (A) Sholl analysis of Golgi images of pyramidal neurons in WT and Shank3b−/− ACC 4 weeks post-birth. Notice the fewer branches in the basal dendrites of Shank3b−/− mice of 4 weeks old. (B) Imaris analysis of spines in basal dendrites of WT and Shank3b−/− neurons. Notice the decrease of total spine density and stubby spines. (C) Double-immunostaining of vesicular glutamate transporter 2 (vGlut2)/MAP2 and Western-blotting of vGluT2. Notice the weaker expression of vGluT2 in Shank3b−/− ACC. (D) Electron microscopic study of synaptic ultrastructure in WT and Shank3b−/− ACC at 4 weeks post-birth. The average length and thickness of post-synaptic density (PSD) was reduced. (E) Patch-clamp recording of miniature excitatory post-synaptic current (mEPSC) in WT and Shank3b−/− ACC. Notice the reduction of frequency of mEPSC in Shank3b−/− ACC. Bars = 2 μm (A,B), 150 μm (C) and 135 nm (D). Values represent mean ± SEM. *P < 0.05. **P < 0.01. N = 6 mice per group in (A,B), 30 neurons per group in (C), five mice per group in (D) and six neurons per group (E). Two-way ANOVA (A), One-way ANOVA (B) and Student’s t-test (C–E).

Fig 3: T. gondii infection decreased VGLUT2 expression in the mouse olfactory bulb. a Brains from mock-infected control mice and mice orally infected with 10 ME49 cysts for 30 and 60 days were collected after perfusion with ice-cold 4% PFA. Brain sections were fixed and stained with anti-VGLUT1 (green), anti-VGLUT2 (red), and mounted with DAPI (blue). Whole brain images were scanned using Leica Biosystems Scanscope and processed with image scope. Representative images were taken from the olfactory bulb at × 20 magnification. n = 4–5 mice per group, the experiment was repeated three times. Scale bar = 50 μm. b, c At least five different fields of the olfactory bulb and pre-frontal cortex were randomly selected from 2 to 4 mice per group. The intensity of VGLUT2 (b) and VGLUT1 (c) signal at different time points post infection was quantified. d Representative images of the olfactory bulb of mock-infected and T. gondii-infected mice stained with anti-GAD67 (green) and VGLUT2 (red). Scale bar = 50 μm, n = 4 mice per group. e The mean fluorescent intensity for GAD67 (green) was measured using Axiovision software from five different fields (n = 4 mice per group). f Representative image from the olfactory bulb of infected mice stained with anti-GAD67 (green) and anti-T. gondii (red). Scale bar = 50 μm. g Western blot analysis of VGLUT1/2 (55 and 65 kDa, respectively) and GAD67 (67 kDa) protein levels using whole brain samples from mock-infected controls and T. gondii-infected mice at days 30 and 60 post infection. GAPDH (37 kDa) was used as loading control. Blot shown is representative of three independent experiments. h–j Densitometric analysis of Western blot data from g. The results are expressed as the ratio of densities of GAD67 or VGLUT1/2-specific bands to GAPDH bands. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001

Fig 4: Effects of VPA-treatment on the expression of excitatory and inhibitory neuronal markers of human NPCs. (A) Experimental design. (B,C) Immunostaining and Western-blotting of Tuj-1 in control (Con) and VPA-treated human NPCs. (D,E) Immunostaining and Western-blotting of CaMKII in control (Con) and VPA-treated human NPCs. (F,G) Immunostaining and Western-blotting of VGLUT2 in control (Con) and VPA-treated human NPCs. Notice the increase of Tuj-1, CaMKII and VGLUT2 in VPA-treated cells. (H,I) Immunostaining and Western-blotting of DLX2 in control (Con) and VPA-treated human NPCs. (J,K) Immunostaining and Western-blotting of NKX2.1 in control (Con) and VPA-treated human NPCs. Bars = 50 μm. N = 3 batches of cells. Student’s t-test. **P < 0.01. ***P < 0.001. ns means no significance. NIM, neural induction medium. EB, embryonic body. NB, neurobasal. VPA, valproic acid. Con, control.

Fig 5: Enrichment of siRNA chemistries in different retinal cell types(A) Cross-section showing efficient distribution of monomer siRNA labeled with Cy3 across entire retinal cross-section. Image shown in grayscale to better visualize the Cy3 signal. (B) Western blot quantifications with cell type–specific antibodies from protein extracts of FACS cells that were Cy3+. The y axis shows relative enrichment in percentage when compared to total protein extracts from an uninjected retina. Antibodies: rhodopsin (rod PR cells), CA (cone PR cells), GS (Müller glia cells), Lim1 (mainly horizontal cells), PKCα (mainly rod bipolar cells), VGAT (amacrine cells, horizontal cells), and VGLUT2 (ganglion cells). A multiple t test was performed to determine significance with uninjected total retinal extracts. N = 3 samples; error bars: SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.