Fig 2: 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.

Fig 3: DARPP-32 interacts with the MARCKS domain of ß-adducin.(a) Rat striatal lysates were incubated with Sepharose coupled to purified recombinant DARPP-32 or to casein as control. After washing, proteins were eluted with 150 mM NaCl, analysed by SDS–polyacrylamide gel electrophoresis and immunoblotted for ß-adducin. (b) Striatal lysates from DARPP-32-GFP transgenic mice and WT littermates were incubated with Sepharose-bound GFP antibody. Immune precipitates were analysed by immunoblotting for ß-adducin (upper panel) or GFP (lower panel). (c) The amounts of ß-adducin co-immunoprecipitated with GFP from WT or DARPP-32-GFP transgenic mice as in c were quantified. Independent data points are plotted and means±s.e.m. are shown, n=4 per group, Student's t-test, t6=2.68, *P<0.05. (d) Domain organization of ß-adducin and its fragments used in the current study (all fused to an N-terminal 13-residue myc peptide, black). MARCKS, myristoylated alanine-rich C-kinase substrate homology domain. The position of the phosphorylated Ser713 in the MARCKS domain is indicated. (e) COS-7 cells were transfected with empty myc vector (-) or myc-tagged full-length ß-adducin or its fragments (+). Myc-tagged proteins were immunoprecipitated with agarose-coupled myc antibody. Beads were then incubated with lysates of DARPP-32-GFP-transfected cells and bound DARPP-32 detected by immunoblotting. (f) Quantification of DARPP-32-GFP association with ß-adducin fragments. The background level is indicated by the amount of DARPP-32 precipitated with beads coated with lysates of myc vector-transfected COS-7 cells (-, dotted line). Independent data points are plotted and means±s.e.m. are shown; n=5–13 per group from >3 experiments; one-way ANOVA: F(5,40)=5.82, P<0.001. Tukey‘s test versus no ß-adducin (-): *P<0.05; **P<0.01. Independent data points are plotted and means±s.e.m. are shown.

Fig 4: 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 5: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ?FosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. *p < 0.05 and **p < 0.01 represent significant differences from control. (C) Representative confocal images of pERK assessed in the three types of MSNs: D1R-only (red arrow), Enk-only (D2R, green arrow) or D1R and ENK (D1-D2 heteromer)-expressing neurons (yellow arrow) in MSNs from NAc of saline- or SKF 83959-treated rats. (D) Quantification of pERK fluorescence in MSNs in the NAc. Results are the mean ± SEM of data after removing the non-specific background (n = number of MSNs from N = at least 3 rats/condition). (**p < 0.001; ***p < 0.0001). (E) Representative immunohistochemistry images and their quantification obtained using an antibody against ?FosB and a secondary antibody conjugated to Alexa-488. Nuclei are stained by DAPI. Rats were treated for 7 days with cocaine (10 mg/kg, i.p.) without or with co-injection of SKF 83959 (1 mg/kg, s.c.). Disrupting the heteromer by repeated injections of TAT-D1 had the same effect as repeated injections of cocaine. Results are means ± SD obtained by the analysis of n = 1,500–1,700 neurons from the NAc of N = 3 rats/condition.