SUPPLEMENTARY INFORMATION

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doi: 10.1038/nature06994 A phosphatase cascade by which rewarding stimuli control nucleosomal response A. Stipanovich*, E. Valjent*, M. Matamales*, A. Nishi, J.H. Ahn, M. Maroteaux, J.

1 doi: /nature06994 A phosphatase cascade by which rewarding stimuli control nucleosomal response A. Stipanovich*, E. Valjent*, M. Matamales*, A. Nishi, J.H. Ahn, M. Maroteaux, J. Bertran-Gonzalez, K. Brami-Cherrier, H. Enslen, A.G. Corbillé, O. Filhol, A.C. Nairn, P. Greengard, D. Hervé, and J. A. Girault *equal contribution Content: 12 pages, 13 supplementary figures, 1 supplementary table. Supplementary Figure 1: Cocaine and morphine trigger nuclear accumulation of DARPP-32 in vivo. (a) Double immunolabeling of DARPP-32 (green) and a nuclear marker (TO-PRO-3, red) in mouse dorsal striatum, 15 min. after i.p. injection of saline or cocaine (20 mg/kg), single confocal sections. Intranuclear localization of DARPP-32 immunoreactivity is shown on xz and yz orthogonal sections along the yellow lines from the merged image. Bar: 2 µm. (b) Wild type mice were treated with morphine 5 mg/kg (s.c.) and DARPP-32 immunoreactivity studied in NAc shell 15 min later (see quantification in Fig.1b). Bar: 10 µm. 1

Supplementary Figure 2: Role of D1R in cocaine-induced DARPP-32 nuclear accumulation. (a) Nuclear accumulation of DARPP-32 occurs in D1R-expressing neurons 8 min after cocaine injection.

2 Supplementary Figure 2: Role of D1R in cocaine-induced DARPP-32 nuclear accumulation. (a) Nuclear accumulation of DARPP-32 occurs in D1R-expressing neurons 8 min after cocaine injection. Double fluorescence of DARPP-32 immunoreactivity (red) and EGFP (green) in the dorsal striatum of drd1a-egfp, which express EGFP under the control of D1R promoter 17, treated for 8 minutes with saline (S) or 20 mg/kg cocaine (C). In cocaine-treated mice DARPP-32 immunoreactivity is higher in nuclei of GFP-positive (D1Rexpressing neurons, arrowheads) than in GFP-negative neurons (arrows). Bar: 10 µm. (b) The % of neurons with nuclear DARPP-32 was counted in the GFP-positive (D1R+) or negative (D1R-) neuronal populations (right panel): two-way ANOVA: interaction between genotype and cocaine F (1,12) =4.33, NS ; effect of genotype, F (1,12) =10.07, p<0.01; effect of cocaine, F (1,12) =14.47, p<0.01; Bonferroni test: **p<0.01 Sal vs Coc. (c) Cocaineinduced nuclear accumulation of DARPP-32 is absent in D1R knockout mice. Wild type (WT) or D1R knockout mutant mice (D1R KO) were treated with saline (Sal) or cocaine (Coc) 20 mg/kg (i.p.) and DARPP-32 immunoreactivity studied in dorsal striatum 10 min later. Two-way ANOVA: interaction between genotype and cocaine F (1,8) =21.9, p<0.01; effect of genotype, F (1,8) =12.7, p<0.01; effect of cocaine, F (1,8) =5.71, p<0.05; Bonferroni test: **p<0.01 Sal vs. Coc; oo p<0.01 D1R KO Coc vs wild type Coc. 2

Supplementary Figure 3: Stimulation of D1R induces nuclear accumulation of endogenous DARPP-32 through a camp-dependent mechanism in striatal neurons in culture.

3 Supplementary Figure 3: Stimulation of D1R induces nuclear accumulation of endogenous DARPP-32 through a camp-dependent mechanism in striatal neurons in culture. (a) Neurons were treated for 15 min with vehicle or a D1R agonist, SKF81297 (10 µm). Vehicle, D1R antagonist SCH23390 (1 µm) or camp antagonist Rp-cAMPS (100 µm) were added 15 min before SKF DARPP-32 immunofluorescence and Sytox nuclear staining are shown as indicated. Bar 5 µm. (b) Quantification of results in (a). One-way ANOVA F (5,37) =20.4, p<0.001, Bonferroni test ***p<0.001 basal vs. SKF; ooo p<0.001 antagonist vs. vehicle. (c) Timecourse of nuclear accumulation of DARPP-32 in striatal neurons in response to a PKA activator Sp-5,6-DClcBIMPS (cbimps, 200 µm). Two-way ANOVA: interaction between time and treatment, F (3, 17) =3.22, p<0.05; effect of treatment F (1,17) =26.43, p<0.0001; effect of time, F (3,17) =4.63, p<0.05; Bonferroni test: *,p<0.05 and **p<0.01 Sp vs. vehicle. Data are means ± SEM. 3

4 Supplementary Figure 4: D1R stimulation triggers nuclear accumulation of DARPP-32-GFP in striatal neurons in culture. (a) Striatal neurons were co-transfected with D32-GFP (direct fluorescence, green) and D1R (immunostaining, red). Treatment with a D1R agonist, SKF81297 (10 µm) for 15 min, promotes nuclear accumulation of D32-GFP, as well as D1R internalization. (b) Quantification of results in (a). Data are means ± SEM. Unpaired t-test: ***p< (c) Confocal microscopy demonstrates co-localization of D32-GFP (direct fluorescence, green) with a nuclear marker (Sytox, red). D32-GFP transfected neurons were treated with SKF81297 for 15 min. The third panels show xz and yz orthogonal sections along the indicated lines from the merged image. Bars: 5 µm. 4

Supplementary Figure 5: Leptomycin B triggers nuclear accumulation of D32-GFP. (a) Time-lapse videomicroscopy of D32-GFP in a transfected striatal neuron in response to leptomycin B (LMB, 10 ng/ml).

5 Supplementary Figure 5: Leptomycin B triggers nuclear accumulation of D32-GFP. (a) Time-lapse videomicroscopy of D32-GFP in a transfected striatal neuron in response to leptomycin B (LMB, 10 ng/ml). Still frames at the indicated times are shown. See full video as Supplementary file Supp-Fig5_LMB_video.AVI. The nucleus/cytoplasm fluorescence ratio was plotted as a function of time. (b) CHO-K1 cells were transfected with D32-GFP and treated for two hours with vehicle (Veh) or leptomycin B (LMB, 10 ng/ml). xy (upper panel) and xz (lower panel) of single confocal sections are shown. (c) CHO-K1 cells were transfected with D32-GFP wild type (WT) or in which Leu-109 was mutated to alanine (L109A). Bars: 5 µm. In (b) and (c) the proportion of cells with nuclear GFP was: WT, 16%; LMB, 100%; L109A, 100%. 5

6 Supplementary Figure 6: Thr-34, Thr-75, and Ser-130 are not involved in the regulation of the cytonuclear shuttling of DARPP-32. (a) Striatal neurons were transfected with D1R and D32-GFP wild type (WT) or bearing mutations of either of three major phosphorylation sites (T34A, T75A, and S130A) and treated for 15 min with vehicle or 10 µm SKF81297 as indicated. The D1R agonist induced a similar nuclear accumulation of wild type and mutated D32-GFP. One way ANOVA: F (7,31) =33.8, p<0.001, Bonferroni test ***p<0.001 SKF vs. basal. (b) The effects of cocaine on nuclear localization of DARPP-32 were similar in wild type and T34A- DARPP-32 mutant mice. Two-way ANOVA: interaction between genotype and cocaine F (1,17) =1.16, NS; effect of genotype, F (1,17) =0.14, NS; effect of cocaine, F (1,17) =35.6, p<0.01; Bonferroni test: **p<0.01, ***p<0.001 Sal vs. Coc. (c) The effects of cocaine on nuclear localization of DARPP-32 were similar in wild type and T75A- DARPP-32 mutant mice. Two-way ANOVA: interaction between genotype and cocaine F (1,17) =0.33, NS; effect of genotype, F (1,17) =0.08, NS; effect of cocaine, F (1,17) =24.2, p<0.01; Bonferroni test: **p<0.01 Sal vs. Coc. Data are means ± SEM. 6

Supplementary Figure 7: Phosphorylation of Ser-97 controls intracellular localization of DARPP-32. (a) S97 mutation alters localization of DARPP-32.

7 Supplementary Figure 7: Phosphorylation of Ser-97 controls intracellular localization of DARPP-32. (a) S97 mutation alters localization of DARPP-32. Striatal neurons were transfected with D1R and D32-GFP wild type (WT) or in which Ser-97 was mutated to alanine (S97A), or glutamate (S97E), and treated for 15 min with vehicle or 10 µm SKF81297 as indicated. (b) Inhibition of CK2 by TBB promotes DARPP-32 nuclear accumulation. Striatal neurons were transfected with D1R and wild type D32-GFP, and treated with vehicle or 10 µm SKF81297 in the absence (upper row) or presence (lower row) of TBB, 45 min before SKF). (c) Mutation of Ser-97 to glutamate (S97E) prevents the effects of TBB (50 µm). (d) Okadaic acid prevents D1Rinduced nuclear accumulation of DARPP-32. Striatal neurons transfected with D32-GFP, were treated with vehicle or SKF81297 as in (a), in the absence or presence of okadaic acid (OA, 500 nm, 45 min before SKF81297). Quantifications of these results are shown in Fig. 3. Scale bars: 5 µm. 7

8 Supplementary Figure 8: Dose response curves of the effects of forskolin on the phosphorylation of DARPP-32 Thr-34 and Ser-97 in striatal slices. Mouse striatal slices were incubated with the indicated concentrations of forskolin for 20 min, and analyzed by immunoblotting with antibodies specific for P-Thr-34- DARPP-32 (a) or P-Ser-97-DARPP-32 (b), as in Fig.3f. Data are means ± SEM for 4-7 experiments. One-way ANOVA (P-Thr-34, F (5,36) =32.72, p<0.0001; P-Ser-97,F (5, 32) =9.549, p<0.0001) followed by Bonferroni test: ***p< Supplementary Figure 9: Role of the B56δ subunit of PP2A in the regulation of DARPP-32 Ser-97 phosphorylation. (a) The B56δ subunit of PP2A is highly expressed in the striatum and cortex as compared to other tissues. The indicated amounts of protein from various tissues were immunoblotted with antibodies against B56δ. (b) The presence of B56δ promotes the dephosphorylation of DARPP-32 Ser-97 in response to forskolin. HEK293 cells were transfected with Myc-tagged DARPP-32 and vector, or B56δ or Bα subunit of PP2A, and incubated with vehicle or 10 µm forskolin for 10 min. DARPP-32 phosphorylation was assessed by immunoblotting with phosphospecific antibody against phospho-ser-97 and anti-myc antibody for total DARPP-32. Quantification of these results is shown in Fig. 3h. 8

9 Supplementary Figure 10: Spontaneous locomotor activity of S97A mutant mice. (a) Basal locomotor activity was recorded for 1 hour in a circular maze. (b) The same experiment was repeated on 3 consecutive days. Basal locomotor activity and habituation were virtually identical in wild type and S97A mutant mice. (c and d) Detailed time-course of the responses to cocaine (c) and morphine (d) in wild type and Ser-97-Ala DARPP-32 mutant mice. Twoway ANOVA: Cocaine: interaction between genotype and time F (14,480) =1.05, NS; effect of genotype, F (1, 480)=5.28, p<0.05; effect of time, F (14,480) =25.2, p<0.01. Morphine: interaction between genotype and time F (29,390) =1.86, p<0.04; effect of genotype, F (1,390) =158.41, p<0.01; effect of time, F (29,390) =9.20, p<0.01. Bonferroni test: *p<0.01 wt vs. S97A. (e and f) Acute and sensitized locomotor responses to cocaine and morphine in wild type and S97A mutant mice. Mice received an injection of cocaine (20 mg/kg, e, 16 mice per group) or morphine (5 mg/kg, f, 8 mice per group) on day 1 and a second injection on day 7. Locomotor activity in response to the first and second injection is shown for each mouse. Arrows indicate the position of the mean in each group. Statistical analyses were done with paired t-test; p values are indicated. (g) Learning of an operant paradigm for food selfadministration was similar in wild type and S97A mutant mice. Data are expressed as a percentage of correct responses under FR1, FR5 and reversal FR5 reinforcement schedule, during five consecutive days each. 9

Supplementary Figure 11: Mutation of Ser-97 to alanine does not alter the phosphorylation of Thr-34 in response to camp stimulation in transfected cells.

10 Supplementary Figure 11: Mutation of Ser-97 to alanine does not alter the phosphorylation of Thr-34 in response to camp stimulation in transfected cells. COS-7 cells were transfected with wild type or S97A-D32- GFP. In these transfected COS-7 cells S97A-D32-GFP was abundant in the cytoplasm (data not shown). Transfected cells were incubated for 15 min in the presence of vehicle or a PKA activator Sp-5,6-DCl-cBIMPS (cbimps, 200 µm). (a) The phosphorylation of Thr-34 and the total amount of D32-GFP were determined by immunoblotting with phosphospecific and total DARPP-32 antibodies, respectively. (b) Immunoreactivity for P- T34 was quantified and normalized to the total amount of D32-GFP. Data are means + SEM, two-way ANOVA interaction between construct and treatment F (1,16) =0.26, NS, effect of construct F (1,16) =0.26, NS, effect of treatment F (1,16) =90.8, p< Bonferroni test: ***p< Supplementary Figure 12: D1R stimulation in response to SKF81297 increases phosphorylation of H3 on Ser-10 in DARPP-32-transfected neurons. Striatal neurons transfected with D32-GFP and D1R, were treated for 15 min with vehicle or SKF81297 (10 µm) as indicated. Quantification of results shown in Fig. 5b: Arrows indicate median values for the various groups. SKF81297 increased the immunoreactivity of phospho-ser-10-h3 (P-H3, Mann Whitney test, p<0.001) and phospho-ser-10/acetyl-lys-14-h3 (P/Ac-H3, Mann Whitney test, p<0.001), but did not alter the acetylation of Lys-14-H3 (Ac-H3, Mann Whitney test, NS). 10

Supplementary Figure 13: Localization of CK2 in the striatum. Sections from mouse striatum were immunostained for DARPP-32 (green) and CK2 alpha subunit (red).

11 Supplementary Figure 13: Localization of CK2 in the striatum. Sections from mouse striatum were immunostained for DARPP-32 (green) and CK2 alpha subunit (red). Mice had received 20 mg/kg of cocaine 10 min prior to sacrifice to trigger nuclear accumulation of DARPP-32. CK2 immunoreactivity was detected in the cytoplasm and, usually with a slightly lower intensity, in the nucleus of most DARPP-32-positive neurons. The intracellular localization of CK2 appeared similar in saline-treated and cocaine-treated mice (data not shown). Bar: 20 µm. 11

12 Supplementary Table 1: ANOVA analysis of results presented in Figures 1-4 Set of data Type of ANOVA F p Figure 1 1b Cocaine time-course One-way F (6,42) =26.6 <0.01 1c DStr One-way F (2,14) =15.38 < c NAc core One-way F (2,14) =14.45 < c NAc shell One-way F (2,14) =17.93 <0.001 Figure 2 2c- Mutagenesis of hydrophobic residues in the NES One-way F (4,21) =35.0 < d- Effects of SKF81297 One-way F (5,22) =35.2 <0.001 Figure 3 3a- S97 mutation One-way F (7,42) =16.6 < b- Inhibition of CK2 by TBB One-way F (3,16) =14.9 < c- Mutation of Ser-97 to glutamate One-way F (3,12) =14.6 < d- Okadaic acid effects One-way F (3, 21) =38.9 < e- SKF81297 effects on Thr-34 One-way F (4, 28) =39.8 < e- SKF81297 effects on Ser-97 One-way F (4, 33) =10.7 < f- Forskolin effects on Thr-34 One-way F (4, 19) =13.0 < f- Forskolin effects on Ser-97 One-way F (4, 18) =11.5 < g- P-Thr-34-DARPP-32 One-way F (3,24) =16.3 < g- P-Ser-97-DARPP-32 One-way F (3,26) =24.7 <0.001 Figure 4 4a- Dorsal striatum: genotype-cocaine interaction Two-way F (1,19) =11.5 <0.01 4a- Dorsal striatum: genotype effect Two-way F (1,19) =5.58 <0.05 4a- Dorsal striatum: cocaine effect Two-way F (1,19) =3.81 >0.05 4a- NAc core: genotype-cocaine interaction Two-way F (1,8) =24.0 <0.01 4a- NAc core: genotype effect Two-way F (1,8) =6.5 <0.05 4a- NAc core: cocaine effect Two-way F (1,8) =1.9 >0.05 4a- NAc shell: genotype-cocaine interaction Two-way F (1,10) =12.0 <0.01 4a- NAc shell: genotype effect Two-way F (1, 10) =1.49 >0.05 4a- NAc shell: cocaine effect Two-way F (1,10) =5.00 <0.05 4c- Cocaine CPP: genotype-treatment interaction Two-way F (1,26) =3.16 >0.05 4c- Cocaine CPP: genotype effect Two-way F (1,26) =3.76 >0.05 4c- Cocaine CPP: treatment effect Two-way F (1, 26) =14.21 <0.01 CPP: Conditioned place preference 12

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figure 1. Behavioural effects of ketamine in non-stressed and stressed mice. Naive C57BL/6 adult male mice (n=10/group) were given a single dose of saline vehicle or ketamine (3.0 mg/kg,