Supplementary Figure 1

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1 Supplementary Figure 1 PDE4 phosphorylation state-specific antibody generation, distribution of PDE4 in brain, and cgmp levels in striatal slices. (a) Identification of the dependent phosphorylation site on 1 by LC-LC MS analysis. The presence of a single charged peptide (SDSDYDLpSPK) indicates the site of phosphorylation at amino acid residue Ser145 of the rat 1 sequence, with ps denoting the position of the phospho-serine. The spectrum depicts the y-ion series as a vertical line in the peptide sequence and corresponding site of y-ion Nature Neuroscience: doi:1.138/nn.466

2 generation (i.e. y 1, y 2, etc.). (b) Time-course of in vitro phosphorylation of recombinant 1 by PKA. Time-dependent 32 P incorporation, Coomassie-stained (CBB) 1, and quantified reaction stoichiometry are shown. (c) PDE4 family member and Cdk5 brain tissue distribution. Immunoblots with pan-specific PDE4A, 4B, and 4D antibodies are shown with individual isoforms indicated. Blots for Cdk5 and GAPDH are also shown. ob, olfactory bulb; ctx, cortex; str, striatum; nac, nucleus accumbens; hip, hippocampus; cer, cerebellum. (d) Validation of phosphorylation state-specific antisera to phospho-ser133 1 [ ] phosphorylated by PKA. Dot blot analysis to confirm phosphorylation statespecificity of antibody. Selective detection of phospho- versus dephospho-peptide with corresponding Pyronin Y staining of total peptide is shown. (e) Immunoblot of lysates from striatal slices incubated in the absence (Control) or presence of forskolin (1 µm, 1 min) probed with antibody. (f) Validation of phosphorylation state-specific antisera to phospho-ser145 1 [ (Cdk5)] phosphorylated by Cdk5. Immunoblot analysis of recombinant 1 phosphorylated (+) or mock phosphorylated ( ) in vitro with Cdk5 using (Cdk5) versus total PDE4 antibodies (top panel). Blots of PC12 cells transfected with 1 treated with control buffer or Indo A (1 µm, 6 min) are shown in the bottom panel. (g) Cdk5 inhibition with indolinone A (IndoA, 1 µm) does not affect cgmp levels in the absence or presence of sodium nitroprusside (SNP), an activator of guanylate cyclases, in striatal slices (n = 4 8. All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

3 Supplementary Figure 2 Reciprocal relationship of Cdk5 and PKA activity in striatum. (a) Evaluation of PKA-mediated phosphorylation in response to treatment of striatal slices with 5 µm roscovitine. Representative blots for PKA-mediated phospho-ser133 (ps133) CREB, phospho-ser9 (ps9) synapsin, phospho-thr34 (P-T34) DARPP-32, phospho-ser845 (ps845) GluR1, and respective total protein are shown with quantification (n = 3 6). (b) The dose-dependent, reciprocal relationship between PKA- and dependent phosphorylation of DARPP-32. Immunoblots (left) of lysates from mouse striatal slices treated with the Cdk5 inhibitor roscovitine ( 5 µm, 6 min) for phospho-thr75 (P-T75), phospho-thr34 (P- T34), and total DARPP-32 (D-32) are shown with quantification (right). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

4 a Cdk5 activity level 32 P CBB c Ros b (Cdk5) (Cdk5)/ c c / c d cgmp (pmol per mg tissue) 4 2 Striatum ns c Supplementary Figure 3 Decreased Cdk5 activity and PDE4 phosphorylation in Cdk5 c. (a) Immunoprecipitation Cdk5 kinase activity assay using striatal lysate from versus Cdk5 c. The effect of treatment of immunoprecipitate from control samples with Cdk5 inhibitor roscovitine (Ros, 1 μm) is also shown. Radiographic and Coomassie stained gels with quantification are shown (n = 6 9). (b, c) PDE4 phosphorylation levels at the Cdk5 (b) and PKA sites (c) in lysates of ventral striatum from Cdk5 c and control mice (n = 4). (d) Levels of cgmp are unaltered in striatum from Cdk5 c and control mice (n = 4-5). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

5 a b c Normalized NMDA current 11 control PKI wash Cdk5 c Time (min) NMDA PKI control.1 na Cdk5 c NMDA PKI control.5 s Reduction of I NMDA by PKI (%) c d e f I NMDA (pa) ns Cm (pf) Cdk5 c Cdk5 c Cdk5 c I NMDA density (pa/pf) Supplementary Figure 4 PKA regulation of NMDA receptor currents is altered in Cdk5 c mice. (a) Plot of normalized peak NMDA receptor currents showing the effect of PKI (.2 µm, myristoylated) in striatal neurons isolated from and Cdk5 c mice. (b) Representative NMDA receptor current traces from data of panel a). (c) Cumulative data showing the percentage reduction of NMDA receptor currents by PKI in neurons from and Cdk5 c mice (n = 9 1). (d-f) Dot plots showing the NMDA receptor current amplitudes (d), capacitance (e), and current densities (f) in neurons from and Cdk5 c mice (n = 12). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

6 a Spine density (spines per μm) 1..5 c c b Spine subtype (%) Thin Mushroom Stubby c c Time (s) Interaction Zone Social Defeat Time (s) Corner Zone No Target Target No Target Target c d Ambulatory counts (au) Locomotion c e Time in zone (s) Interaction Zone Social Interaction Time in zone (s) Corner Zone c No Target Target No Target Target Nature Neuroscience: doi:1.138/nn.466

7 Supplementary Figure 5 Cdk5 c exhibit reduced spine density in medium spiny neurons and altered behavioral responses to stress. (a) Dendritic spine density of medium spiny neurons in the ventral striatum is decreased in Cdk5 mice as compared to controls (n = cells from four mice per genotype). (b) No change in spine types, such as thin, mushroom, stubby was observed between genotypes. (c) The effect of Cdk5 c on social defeat. Social avoidance was assessed by time spent interacting with the social interaction target. Graphs depict time spent in interaction zone (left) and corner zones (right) in present (Target) or absence (No Target) of interactor mouse (n = 7 13). (d) Assessment of cage activity in Cdk5 c and control mice (n = 13 14). (e) The effect of Cdk5 c on social interaction. Graphs depict time spent in interaction zone (left) and corner zones (right) in presence (Target) or absence (No Target) of interactor mouse (n = 17 24). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

8 a b (Cdk5) / h Control post- FST (Cdk5)/ Control 1h post- FST c e / / Control Control 1h post- SI 1h post- CUS d f (Cdk5) (Cdk5)/ (Cdk5) (Cdk5)/ Control 1h Control post- SI 1h post- CUS Supplementary Figure 6 Forced swim test, social defeat and chronic unpredictable stress induce changes in PDE4 phosphorylation (a,b) Quantitative immunoblot analysis of ventral striatum lysates from mice 1 h after FST (n = 4) compared to non-swim controls (n = 4) for and (Cdk5). (c,d) Analysis of ventral striatum lysates from chronic socially defeated mice (n = 1) and non-defeated controls (n = 6) 1 h after social interaction testing for and (Cdk5). (e,f) Analysis of ventral striatum lysates mice undergoing chronic unpredictable stress (n = 6) and non-stressed controls (n = 5) for and (Cdk5). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

9 Supplementary Figure 7 Behavioral characterization of virus-mediated Cdk5 knockout in ventral striatum. (a) Immunostain showing Cdk5 knockout in the ventral striatum induced by stereotactic bilateral infusion of AAV2-Cre into the NAc of homozygous floxed Cdk5 (fl/fl AAV) and mice ( AAV). Arrowheads at the border of viral transduction fields indicate locations of inserts (right). (b) Assessment of cage activity in fl/fl AAV and AAV (n = 7 8). (c,d) Elevated plus maze performance of fl/fl AAV and AAV (n = 7 8) indicating time in zones (c) and locomotor activity (d). (e) Social interaction (SI) in fl/fl AAV and AAV (n = 1). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

10 Supplementary Figure 8 Reduced PDE4 phosphorylation in striatum of. (a) Immunostain showing D1 dopamine receptor promoter-driven Cre expression via a GFP reporter (top) and Cdk5 loss in medium spiny neurons of ventral striatum (encircled; bottom). (b) Reduced Cdk5 expression in striatal lysates from (n = 6). (c,d) Quantitative immunoblot analysis of ventral striatum lysates from mice and controls (n = 6) for (c) and (Cdk5) (d). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

11 a c Immobility (s) Time (s) Forced Swim Test Social Defeat Interaction Zone 6 No Target Target Latency (s) 2 1 Time (s) 4 2 Corner Zone No Target Target b d Normalized time struggling Distance traveled (m) 1..5 Locomotion during SD Tail Suspension No Target ns Target e h Social interaction ratio Ambulatory counts (au) Social Interaction Locomotion Ambulatory counts (au) f Time in zone (s) Center Elevated Plus Maze ns Bins (1 min) Closed arms Open arms i 1 5 Distance traveled (m)15 Open Field g Distance traveled (m) Time in center (s) EPM locomotion ns Supplementary Figure 9 Cdk5 knockout in D1 dopamine receptor neurons alters stress-induced behavioral responses. (a,b) exhibit reduced immobility and increased latency to initiation of floating in the FST (n = 9 16) (a) and increased time struggling in the TST (n = 12 14) (b). (c) Effect of on social defeat. Social avoidance was assessed by time spent interacting with the social target. Graphs depict time spent in interaction zone (left) and corner zones (right) in presence (Target) or absence (No Target) of Nature Neuroscience: doi:1.138/nn.466

12 interactor mouse (n = 8 1). (d) Locomotor activity during social interaction testing (n = 8 1). (e) Social interaction (SI) in (n = 1). (f,g) Elevated plus maze performance of and control mice (n = 7) indicating time in zones (f) and locomotor activity (g). (h) Assessment of cage activity in and control mice (n = 12 13). (i) Evaluation of locomotor activity and center times in the open field assay for and control mice (n = 9 1). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

13 Supplementary Figure 1 Increased water intake in mice. (a) Effect of Cdk5 knockout in the mouse lines on sucrose preference test (SPT), a test of anhedonia that does not rely on locomotor activity. Graph depicts sucrose preference for (n = 1) and control mice (n = 15). (b) Total liquid consumption per day measured during water, sucrose and choice (water and sucrose) phase of the SPT. (c) Normalized intake volumes for each bottle during water and sucrose phase show no bias. (d) mice exhibit normal water intake during the night cycle, but have increased water intake during the day cycle (n = 5 6). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

14 a Two-trial Forced Swim Test Immobility (s) Latency (s) Vehicle PKA inhibitor Vehicle PKA inhibitor b Two-trial Forced Swim Test Immobility (s) Latency (s) 4 2 c Vehicle PKA inhibitor Vehicle PKA inhibitor Supplementary Figure 11 Reversal of forced swim test phenotype by PKA inhibition in and Cdk5 c. (a,b) Effect of infusion of the PKA inhibitor Rp-cAMPs into ventral striatum of (a) and Cdk5 c (b) as assessed by the two-trial FST (n = 6 1). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

15 a c Normalized pt75 D-32 level _ _ SKF PDE4 sip b Two-trial Forced Swim Test Ambulatory counts (au) Pre-FST Post-FST scrambled PDE4 sip Immobility (s) Latency (s) 2 1 d 2 scrambled R7-PDE4 sip scrambled R7-PDE4 sip Two-trial Forced Swim Test 45 Immobility (s) Latency (s) 3 15 e S133A (Cdk5) S145A S133A S145A PDE4 sip scrambled S133A S145A S133A PDE4 scrambled S145A sip (Cdk5)/ 1..5 S133A PDE4 S145A sip / 1..5 S133A PDE4 S145A sip Supplementary Figure 12 In vitro and in vivo assessment of PDE4-siP specificity and its effect on PDE4 phosphorylation. (a) Effect of the D1 dopamine receptor agonist SKF81297 (SKF), PDE4-siP, or both on the defined Cdk5 site, phospho-thr75 DARPP-32 (pt75 D-32). P <.5 compared to SKF81297 only; one-way ANOVA and Nature Neuroscience: doi:1.138/nn.466

16 Newman-Keuls test, (n = 4 8). (b) Evaluation of cage activity in mice infused with PDE4-siP (n = 5) and scrambled peptide (n = 9) before (4 min) and after (3 min) FST. (c) Effect of infusion of the PDE4 peptide with an N-terminal poly-arginine (R7) cell membrane permeablizing tag into NAc on the two-trial FST (n = 5 7). (d) Effect of infusion of peptides, in which the PKA (S133A; n = 8), Cdk5 (S145A; n = 8), or both serine sites (S133A/S145A; n = 8) were mutated to alanine, into NAc on the two-trial FST (n = 5 1) and compared to infusions with PDE4-siP (n = 9) and scrambled peptide (n = 7). (e) Infusion of the PDE4-siP into the ventral striatum has no impact on PDE4 phosphorylation levels at the Cdk5 and PKA sites within the dorsal striatum (n = 5 6). All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

17 Fig 1f Fig 1g PDE4 D Flag Fig 2b Fig 2e ps845 GluR1 Cdk5 5 GluR1 β-actin (Cdk5) Fig 2f pt34 D β-actin Fig 3b 1 75 Cdk Supplementary Figure 13 Whole blot images of the immunoblots presented in the main figures Nature Neuroscience: doi:1.138/nn (Cdk5) Fig 4g PDE4 (Cdk5) β-actin

18 Supplementary Tables Supplementary Table 1. Monoamine levels in striatum, hippocampus and frontal cortex of Cdk5 c and control mice. High-performance liquid chromatography (HPLC) analysis of striatal, hippocampal and frontal cortex tissue from Cdk5 c and control mice ( n = 4; c n = 6). DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanilic acid; 3-MT, 3-methoxytyramine; 5-HT, serotonin. All values are indicated in nm per gram of tissue. All data shown are means ± s.e.m., P <.5, P <.1, P <.1. Nature Neuroscience: doi:1.138/nn.466

19 Target/Ab Name Company/Source Catalogue number /Reference Remark Flag tag Cell Signaling Technology #8146 Mab phospho-creb (Ser133) Cell Signaling Technology #9196 Mab total CREB Cell Signaling Technology #9197 Rab phospho-darpp-32 (Thr34) Cell Signaling Technology #12438 Rab phospho-darpp-32 (Thr75) Cell Signaling Technology #231 Rab total DARPP-32 Hugh C. Hemmings Hemmings & Greengard, J Neurosci Mab 6, (1986) Cdk5 PhosphoSolutions 38-CDK5 Mab phospho-glur1 (Ser845) PhosphoSolutions p Rab total GluR1 PhosphoSolutions 895-GluR1 Mab phospho-tyrosine PhosphoSolutions p158-4 Rab hydroxylase (Thr4) total tyrosine hydroxylase PhosphoSolutions 225-THRAB Rab Cdk5 (C-8) Santa Cruz sc-173 Rab p35 (C-19) Santa Cruz sc-82 Rab FITC Abcam ab19224 Goat ab NeuN Millipore MAB377 Mab PDE4A Miles D. Housley Huston et al, J Biol Chem 271, Rab Miles D. Housley (1996); Huston et al, Biochem J 328 (Pt2), (1997); Sheep ab PDE4D Miles D. Housley Bolger et al, Biochem J 328 (Pt2), Sheep ab (1997). phospho-pde4 (Cdk5) James A. Bibb Rab phospho-pde4 James A. Bibb Rab phospho-synapsin (Ser9) Sigma S867 Rab Synapsin Sigma S193 Rab -actin Sigma A2228 Mab GAPDH Sigma G9545 Rab Supplementary Table 2. Primary antibodies used for immunoblotting and/or immunochemistry. Nature Neuroscience: doi:1.138/nn.466