Fig 2: Increased striatal DARPP-32 mRNA expression during the early, but not late, phase of motor skilled learning.(A) Regional analysis of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) mRNA expression within sub-regions of the striatum of (top panel) early phase (EP) active controls (white bars) vs. EP trained rats (red bars), and of (bottom panel) late phase (LP) active controls (white bars) vs. LP trained rats (blue bars). Data in bar graphs show mean optical (OD) density values for DARPP-32 mRNA in the DMS, DLS, VLS, AcbC, and AcbSh. The results are presented as means ± SEM; n = 5 per group. Asterisks indicate where trained rats differ significantly (*P < 0.05 and **P < 0.01) from active control rats. (B) Representative autoradiographs showing the mRNA expression levels of Drd1 at the level of the striatum of active controls and trained rats during the early and late phases of motor skill learning. For more details, see Figs 1 and 2.

Fig 3: 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 4: DARPP-32 Ser97 phosphorylation enhances ß-adducin interaction.(a) COS-7 cells were co-transfected with DARPP-32-GFP and full-length myc-ß-adducin or its fragments. PhosphoSer97-DARPP-32 (pSer97), total DARPP-32 and actin as loading control were analysed by immunoblotting. (b) Quantification of results as in a. n=13–28 per group from >3 experiments; one-way ANOVA: F(5,114)=5.1, P<0.001; Tukey's test versus myc vector: *P<0.05; **P<0.01; ***P<0.001. (c) Phosphorylation of DARPP-32 Thr34, Thr75 and Ser97 (pThr34, pThr75 and pSer97, respectively), total DARPP-32 and actin were analysed by immunoblotting striatal lysates from WT or ß-adducin KO mice. (d) Quantification of results as in c. n=5–14 mice per group from two experiments; Student's t-test, pThr34, t8=1.68, P=0.13; pThr75, t8=1.70, P=0.13; pSer97, t22=2.71, *P=0.013; NS, not significant. (e) Purified DARPP-32 and CK2a2ß2 were incubated with 100 µM ATP at 37 °C for 30 min in the absence (-) or presence (+) of purified ß-adducin. pSer97 and total DARPP-32 were analysed by immunoblotting. (f) Quantification of results as in e. n=10 per group from >3 experiments; Student's t-test, t18=4.04, ***P<0.001. (g) Beads coated or not with ß-adducin were incubated with lysates of COS-7 cells transfected with WT, S97A or S97D-DARPP-32-GFP. DARPP-32 was detected in ß-adducin precipitates by immunoblotting. (h) Quantification of results as in g as ratios of DARPP-32 precipitated in the presence (+)/absence (-) of ß-adducin. n=6 per group; one-way ANOVA: F(2,15)=14.26; P<0.001. Tukey's test versus S97A: *P<0.05; ***P<0.001. (i) Beads coated (+) or not (-) with ß-adducin were incubated for 2 h at 4 °C with purified recombinant DARPP-32 phosphorylated or not by CK2, as indicated. After washing, DARPP-32 bound to the beads, and DARPP-32 and pSer97 in the input were analysed by immunoblotting. (j) Quantification of data as in i. n=9–12 per group from >3 experiments; two-way ANOVA: ß-adducin factor, F(1,38)=22.18, P<10-4; CK2 phosphorylation factor, F(1,38)=10.41, P=0.003; no interaction, F(1,38)=2.14; Šidák's test versus no ß-adducin, *P<0.05, ***P<0.001; phospho versus non-phospho, °°P<0.01. In b,d,f,h,j, independent data points are plotted and means±s.e.m. are shown.

Fig 5: NEE alters dendritic spines in nucleus accumbens.WT and mutant mice were killed in their home cage or after 24 h in a NEE. Spines were analysed in the nucleus accumbens using the Golgi-Cox method (see Supplementary Fig. 6). (a) Spine length was measured in WT (left panel) and ß-adducin KO (right panel) littermate mice (196–252 spines per group) and plotted as cumulative frequency. NEE increased spine length in WT but not in KO mice; Gehan–Breslow–Wilcoxon test: WT, ?2=32.92, degrees of freedom (DF)=1, P<10-4; KO, ?2=9.133, DF=1, P=0.0025. (b) Quantification of spine width in the same samples as in a, plotted as cumulative frequency. NEE increased spine width in WT but not in KO mice; Gehan–Breslow–Wilcoxon test: WT, ?2=5.992, DF=1, P=0.014; KO, ?2=0.04, DF=1, not significant (NS). (c) Analysis of spine length in WT (left panel) and Thr75Ala (T75A) DARPP-32 mutant littermate mice. NEE increased spine length in WT but not in T75A littermates; Gehan–Breslow–Wilcoxon test: WT, ?2=9.836, DF=1, P=0.0017; T75A, ?2=1.958, DF=1, NS. (d) Quantification of spine width in the same samples as in c, plotted as cumulative frequency. NEE increased spine width in WT but not in KO mice; Gehan–Breslow–Wilcoxon test: WT, ?2=13.78, DF=1, P=0.0002; KO, ?2=0.03, DF=1, NS; T75A, ?2=0.049, DF=1, NS.