Fig 2: Fenofibrate administration rescued the NAcS dopaminergic response to rewarding social stimulus in male rats blunted by VPA exposure. The levels of Thr34 DARPP-32 phosphorylation at baseline and following the interaction with an unknown conspecific or after sucrose consumption were measured in the NAcS by immunoblotting in male (a, b) and female rats (d, e). a Three-way ANOVA, social stimulus: F1,40 = 72.13, p < 0.0001; VPA exposure: F1,40 = 11.59, p = 0.0015; FBR administration: F1,40 = 10.70, p = 0.022; interaction of VPA exposure × social stimulus: F1,40 = 11.82, p = 0.0014; interaction of FBR administration × social stimulus: F1,40 = 16.02, p = 0.0003; interaction VPA exposure × FBR administration × social stimulus: F1,40 = 4.109, p = 0.0494; post hoc comparison: ***p < 0.001 vs. the respective baseline group. Values are expressed as mean ± SEM and calculated as percentage of the baseline values of the saline-SD group; n = 6. b Three-way ANOVA, sucrose consumption: F1,40 = 11.86, p = 0.0014; VPA exposure: F1,40 = 11.83, p = 0.0014; FBR administration: F1,40 = 0.36, n.s.; interaction of VPA exposure × FBR administration: F1,40 = 2.19, n.s.; interaction of VPA exposure × sucrose consumption: F1,40 = 12.68, p = 0.0010; interaction of VPA exposure × FBR treatment × sucrose consumption: F1,40 = 2.57, n.s; post hoc comparison: **p < 0.01, *p < 0.05 vs. the respective baseline group. Values are expressed as mean ± SEM and calculated as percentage of the baseline values of the saline-SD group; n = 6. c Representative immunoblotting in males. d Three-way ANOVA, social stimulus: F1,40 = 48.48, p < 0.0001; VPA exposure: F1,40 = 7.59, p = 0.0088; FBR administration: F1,40 = 0.19, n.s.; interaction of VPA exposure × social stimulus: F1,40 = 14.12, p = 0.0005; interaction of VPA exposure × FBR administration: F1,40 = 2.51, n.s.; interaction of VPA exposure × FBR treatment × social stimulus: F1,40 = 2.52, n.s.; post hoc comparison: ***p < 0.001, **p < 0.01 vs. the respective baseline group. Values are expressed as mean ± SEM and calculated as percentage of the baseline values of the saline-SD group; n = 6. e Three-way ANOVA, sucrose consumption: F1,32 = 35.08, p < 0.0001; VPA exposure: F1,32 = 0.15, n.s; FBR administration: F1,32 = 0.007, n.s.; interaction of VPA exposure x FBR administration: F1,40 = 2.19, n.s.; interaction of VPA exposure × sucrose consumption: F1,32 = 0.45, n.s.; interaction of VPA exposure × FBR treatment × sucrose consumption: F1,32 = 0.69, n.s. Values are expressed as mean ± SEM and calculated as percentage of the baseline values of the saline-SD group; n = 5. f Representative immunoblotting in females. Values are expressed as mean ± SEM and calculated as percentage of the baseline values of the saline-SD group. The levels of PPARa were measured by immunoblotting in the VTA of male (g) and female rats from the subgroups not exposed to the social or sucrose stimulus (h). g Two-way ANOVA, VPA exposure: F1,24 = 1.75, n.s.; FBR administration: F1,24 = 4.97, p = 0.0354; interaction: F1,24 = 0.226, n.s. Values are expressed as mean ± SEM and calculated as percentage of the saline-SD group values; n = 7. h Two-way ANOVA, VPA exposure: F1,24 = 0.176, n.s.; FBR administration: F1,24 = 4.39, p < 0.049; interaction: F1,24 = 0.00024, n.s. i Representative immunoblotting in males (upper panel) and females (lower panel). Values are expressed as mean ± SEM and calculated as percentage of the saline-SD group values; n = 7

Fig 3: Outline of experimental protocols. ASD-like symptoms were induced in Sprague-Dawley male and female rat offspring of dams that had received an intraperitoneal injection of valproic acid (VPA, 500 mg/kg) at gestational day 12.5 (G 12.5). Offspring were first checked for negative geotaxis and olfactory discrimination (tests of developmental milestones) and from postnatal day (PND) 21 to the end of the experimental procedures were fed with a standard diet (SD) or fenofibrate-enriched diet (FBR). After 4 weeks of treatment, animals were subjected to behavioral and/or neurochemical analysis. a From PND 48–53, the four experimental groups (each group n = 12) were behaviorally tested to evaluate the level of anxiety (elevated plus maze test), social interaction (three-chamber test), locomotor activity, and stereotypies. One week after the end of behavioral tests, animals were sacrificed and brain regions were dissected out for immunoblotting assays. b The four experimental groups (n = 12) of second cohort underwent behavioral screening to evaluate social transmission of food preference and perseverative behavior (marble burying test). In addition, animals were tested for the two-bottle sucrose preference as an index of hedonic response. One week later, animals were sacrificed and brain regions were dissected out for immunoblotting assays. c The third cohort was used to determine by immunoblotting the Thr34 phosphorylation levels of DARPP-32 in response to social interaction or nonsocial stimulus (sucrose consumption) in the shell of NAc (NAcS). At PND 48–53, half animals in each group were sacrificed at baseline and half 30 min after a 10 min-interaction with a novel conspecific (social stimulus) or 30 min after consumption of 10 sucrose pellets. For each experimental group in this cohort, the rats not exposed to the social or sucrose stimulus were also used to assay the PPARa levels in the VTA

Fig 4: Caffeine stimulates adducin phosphorylation through DARPP-32.(a) Striatal samples were prepared from C57BL/6 mice 40 min after intraperitoneal injection of saline or 7.5 mg kg-1 caffeine. pThr75 and total DARPP-32, and actin as loading control, were analysed by immunoblotting. Lower panel, independent data points are plotted and means±s.e.m. are shown; n=9 per group from 2 experiments; Student's t-test, t16=2.53, *P<0.05. (b) Samples obtained as in a were immunoblotted for pSer713 and total ß-adducin and actin. Lower panel as in a, n=7 per group from 2 experiments; Student's t-test, t14=3.66, **P<0.01. (c) T75A knock-in mutant mice or WT littermates were injected with saline or caffeine as in a and killed 40 min later. Striatal samples were analysed as in b. Lower panel as in a, n=12–15 per group from >3 experiments; two-way ANOVA: caffeine factor, F(1,60)=6.28, P=0.015; genotype factor, F(1,60)=0.001, not significant (NS); no interaction, F(1,60)=2.48. Šidák post hoc test, caffeine versus saline: *P=0.01.

Fig 5: ß-adducin alters DARPP-32 mobility and interacts with it in neurons.(a) Mouse striatal neurons in culture were transfected with WT, S97A or S97D-DARPP-32-GFP and fluorescence monitored before and after photobleaching (photobleached area, dotted line). Scale bar, 10 µm. (b–e) GFP fluorescence was recorded in cells co-transfected with myc vector (open bars or symbols) or myc-ß-adducin (blue bars or symbols). (b) Quantification of average recovery (30–60 s). Independent data points are plotted and means±s.e.m. are shown, n=14–18 per group from 3 experiments; two-way ANOVA: ß-adducin factor, F(1,95)=65.47, P<10-4; Ser97 mutation factor, F(2,95)=19.82, P<10-4; interaction, F(2,95)=4.01, P=0.02. Šidák's test, presence versus absence of ß-adducin, *P<0.05, ****P<10-4; effects of Ser97 mutation in the presence of ß-adducin, °P<0.05; °°°°P<10-4. (c) Time course of recovery after photobleaching of WT DARPP-32-GFP (WT) fluorescence, in the absence or presence of co-transfected ß-adducin. Data are means ± s.e.m. Two-way ANOVA: effect of ß-adducin, F(1,1947)=198.6, P<10-4; time factor, F(58,1947)=177.2, P<10-4; no interaction, F(58,1947)=1.15. (d) Same as in c for S97A-DARPP-32-GFP. Two-way ANOVA: effect of ß-adducin, F(1,1740)=91.56, P<10-4; time factor, F(59,1740)=76.94, P<10-4; no interaction, F(58,1947)=0.45. (e) Same as in c for S97D-DARPP-32-GFP. Two-way ANOVA: effect of ß-adducin, F(1,1800)=135.1, P<10-4; time factor, F(59,1800)=135.0, P<10-4; no interaction, F(58,1947)=1.31. (f) For acceptor photobleaching, striatal neurons in culture were co-transfected as indicated with mCherry (mCh) or ß-adducin–mCherry (ßAdmCh), and GFP or GFP fused to either WT DARPP-32 (D32GFP) or S97A-DARPP-32 (S97AGFP) or S97D-DARPP-32 (S97DGFP). Top row, low-magnification single confocal sections indicating the area blown up in bottom rows (rectangles). Bottom rows, pre-bleach, post-bleach and calculated FRET images, respectively. Photobleached areas are indicated by dotted lines. FRET efficiency was calculated by measuring GFP fluorescence before and after mCherry photobleaching (pseudocolour scale indicated at bottom left). Scale bars, 10 µm. (g) Quantification of FRET efficiency as in f. Independent data points are plotted and means±s.e.m. are shown. Statistical analysis, one-way ANOVA: F(4,223)=26.97; P<0.0001. Tukey's test: *P<0.05; ****P<10-4.