Fig. 3. Downregulation of Shank3 in D1R MSNs induces alterations in inflammatory mediators and Trpv4 expression.

a Experimental design. Drd1a-dTomato mice were injected neonatally in the NAc with scr or shShank3 virus. At P30 the NAc was dissected and FACsorted in 4 different cell populations (D1R-tom+, D1R-tom-, D1R-tom+::AAV and D1R-tom−::AAV). For each cell population we carried out bulk RNA sequencing. b Worst-case scenario selected altered genes in scr vs sh testing clearly discriminated infected cells, both D1R+ and D1R− in PCA analysis. c While non-infected samples do not share common genes significantly altered in scr vs sh testing, infected D1R+ and D1R- share a core set of 68 altered genes. d Overall GO:Term analysis of infected D1R+ significantly altered genes highlights the relevance of inflammatory mechanisms, as well as cell adhesion-, localization- and movement-related functions. e D1R-tom+ altered genes include genes expressing proteins directly involved in electrophysiological properties, including the Transient receptor potential vanilloid 4 (Trpv4). f Real-time PCR analysis of NAc dissected from P6- or P90-injected mice confirm the upregulation of Trpv4 in P6 sh-infected mice (unpaired t-tests: P6 – Trpv4 t₍₄₎ = 2.980, p = 0.041; P6 – Trpv1 t₍₄₎ = 0.367, p = 0.732; P6 – HCN1 t₍₄₎ = 0.318, p = 0.766; P90 – Trpv4 t₍₅₎ = 0.4203, p = 0.69). g Experimental design. Drd1a-dTomato mice were injected neonatally in the NAc with scr or shShank3 virus and whole-cell patch clamp recordings were performed during early adulthood. h Right: example traces from 300 pA depolarizing current injection in D1R+ MSNs infected with scrShank3 treated with vehicle (left), D1R+ MSNs infected with shShank3 treated with vehicle (middle) and D1R+ MSNs infected with shShank3 treated with HC-067047 (right). Left: number of action potentials (nAPs) across increasing depolarizing current steps (0−500 pA) for D1R-tom+::scrShank3 and shShank3 MSNs in the presence of Trpv4 antagonist (HC-067047) (repeated measures ANOVA, drug main effect F_{(2, 31)} = 5.883, p = 0.007, current steps main effect F_{(10, 310)} = 24.15, p < 0.001, drug by current steps interaction F_{(20, 310)} = 1.685, p = 0.035, n = 12 cells, 4 mice (shShank3-Veh), n = 10 cells, 3 mice (shShank3-Trpv4), n = 12 cells, 4 mice (scrShank3-Veh)). i Experimental design. C57BL6/j mice were injected neonatally in the NAc with scr or shShank3 virus and at P50-60 were bilaterally cannulated above the NAc. After 7 days, mice underwent the three-chamber social interaction assay. ScrShank3 were infused with vehicle (aCSF/DMSO 0.3%). On the other hand, shShank3 mice were infused with either vehicle (aCSF/DMSO 0.3%) or HC-067047 (2 µg in aCSF/DMSO 0.3%) 10 min before to start the test. i′ Representative image of the injection site and cannula placement above the NAc (scale bar: 250 µm). Left: time around the target during the social preference test for mice infected with scrShank3 and infused with vehicle (paired-samples t-test for object- vs. social: j t₍₅₎ = 6.304, p = 0.002), mice infected with shShank3 and infused with vehicle (paired-samples t-test for object- vs. social: k t₍₇₎ = 0.869, p = 0.414) or with HC-067047 (paired-samples t-test for object- vs. social: l t₍₇₎ = 4.324, p = 0.004). Right: juvenile preference index for mice infused either with vehicle or with HC-067047 (one-sample t-tests against chance level = 0.5: j t₍₅₎ = 6.459, p = 0.001; k t₍₇₎ = 1.02, p = 0.342; l t₍₇₎ = 6.078, p = 0.001). m Juvenile preference index comparison between shShank3-vehicle and shShank3-HC-067047 (paired-samples t-test for object- vs. social: t₍₇₎ = 2.6, p = 0.035). Error bars report SEM.