Supplementary Figure S1. Effect of stress during withdrawal on expression of sensitization to repeated cocaine exposure in WT and D2R / mice.

Transcription

1 Supplementary Figure S1. Effect of stress during withdrawal on expression of sensitization to repeated cocaine exposure in WT and D2R / mice. The time course of locomotor activity for WT (a, b) or D2R / (c, d) mice during expression of sensitization to cocaine (5 or 10 mg/kg, i.p.) after drug withdrawal without (a, c) or with (b, d) chronic stress was determined. Arrows denote time of injection with saline or cocaine. Data are means ± s.e.m. (n = 6 to 11 mice per group). *P < 0.05, **P < 0.01, ***P < versus the corresponding value for mice injected with saline during the initiation protocol (Student s t test). 1

2 Supplementary Figure S2. Effect of D-AP5 on the induction of LTD. A summary of LTD induction (a) and the mean percentage LTD at 50 to 60 min (b) in the NAc during incubation in the absence (control, n = 6) or presence (n = 5) of 50 μm D-AP5 are shown for WT mice. **P < 0.01 versus control. 2

3 Supplementary Figure S3. Behavioral sensitization to repeated cocaine exposure in WT and D2R / mice studied in Figure 4d g. a, Initiation of behavioral sensitization to cocaine (15 mg/kg, i.p.) as in Figure 2b; n = 14 to 21 mice per group. *P < 0.05, **P < 0.01, ***P < (Student s t test). b, Locomotor activity after the first (day 1, C1) and last (day 5, C5) injections of cocaine (cocaine day effect F1,50 = 38.13). c, C5/C1 ratio of locomotor counts for WT and D2R / mice (cocaine genotype interaction F1,49 = 6.67, P = ). d, e, Total locomotor activity counts for WT (d) and D2R / (e) mice during the 30-min session on each of the five test days in initiation of sensitization to cocaine (15 mg/kg) and for the 30 min after challenge injection of cocaine (10 mg/kg) in expression of sensitization as in Figure 2k and 2l; n = 6 to 9 mice per group. For expression of sensitization in d and e: cocaine stress interaction F1,32 = 4.07, P = (d); cocaine stress interaction F1,25 = 3.02, P = (e). Twoway ANOVA post hoc test for b and c as well as the expression of sensitization in d and e: ***P<0.001 versus the value for C1; P<0.01versus WT mice; &P<0.05 versus nonstressed value. All data are means ± s.e.m. 3

4 Supplementary Figure S4. Effect of chronic stress during withdrawal on behavioral sensitization to repeated cocaine exposure in WT mice injected with raclopride into the NAc core. a, Initiation of behavioral sensitization to cocaine (15 mg/kg, i.p.) as in Figure 2b. {Mice (n = 8 to 17 per group) were injected with vehicle (control) or raclopride (Rac, 5 nmol) 40 min before each injection of cocaine or saline in the test sessions. ***P < (Student s t test). b, Locomotor counts averaged for the five test sessions (cocaine effect F1,46 = 62.03, P < ). c, Locomotor activity after the first (day 1, C1) and last (day 5, C5) injections of cocaine (cocaine day effect F1,54 = 50.89, P < 0001). d, e, Total locomotor activity counts for mice (n = 5 to 9 per group) injected with vehicle (d) or raclopride (e) were measured during the 30-min session on each of the five test days in initiation of sensitization to cocaine (15 mg/kg) and for the 30 min after challenge injection of cocaine (10 mg/kg) in expression of sensitization as in Figure 2k and 2l. (d: cocaine effect F1,19 = 21.52, P = ; stress effect F1,19 = 0.57, P = ; interaction F1,19 = 0.12, P = e: cocaine effect F1,13 = 3.44, P = ; stress effect F1,13 = 4.27, P = ; interaction F1,13 = 3.81, P = ) Two way ANOVA post hoc test for b, c, and e: ###P < versus saline; ***P < versus C1; &P < 0.05 versus nonstressed. All data are means ± s.e.m. 4

5 Supplementay Figure S5. Effect of chronic stress on LTD induction in the NAc of saline pretreated mice injected with Lenti-shD2R or Lenti-control into the NAc core. a, SP-LFS induced LTD formation induction in the indicated groups (n = 7 to 12 mice per group) measured as in Figure 4c). b, Mean percentage LTD at 50 to 60 min (virus stress interaction F1,32 = 0.16, P = ). 5

6 Supplementary Methods Behavioral analysis Elevated plus-maze test. The elevated plus-maze was made of acrylic and consisted of two open arms and two closed arms, each with a length of 67 cm, width of 7 cm, and height of 55 cm. Mice were placed on the central platform facing an open arm. The time spent in the open arms with all four paws and the number of entries into the open arms from closed arms are thought to be inversely related to anxiety level and were measured over 5 min. Head dipping in open arms was considered to occur when the head of a mouse protruded over the edge of an open arm. Open-field test. Locomotor activity was evaluated with the use of an Activity Monitor (Med Associates, St. Albans, VT), consisting of an open-field chamber (43.2 by 43.2 by 30.5 cm) fitted with a 16 by 16 array of photocells for measurement of horizontal movements. The open field was divided into a central zone (20.3 by 20.3 cm) and a peripheral zone. The total distance traveled in the central zone, which is thought to be inversely related to anxiety level, was determined over 60 min. Forced swim test. The forced swim test was performed by dropping a mouse in water and recording its behavior. The apparatus consisted of a plastic cylinder (internal diameter, 10 cm; height, 25 cm) containing a 19-cm column of water at 23 C. The mouse was thus not able to support itself by touching the bottom of the apparatus with its paws or tail. An animal was considered to be immobile when it floated in an upright position and made only small movements to maintain its head above water. The total duration of immobility, which is thought to reflect a depressive state, was determined during a 6-min test session. Corticosterone assay Blood samples were collected with the use of the tail clip technique 56 between 1400 and 1500 hours, and plasma was then isolated and stored at 80 C until assayed for corticosterone (final dilution, 1:200) with a competitive immunoassay (Correlate-EIA Corticosterone kit; Assay Designs, Ann Arbor, MI). Virus construction An shrna targeting mouse D2R mrna was designed on the basis of an shrna previously used to target rat D2R mrna 57. The shrna was delivered and constitutively expressed with the use of the plentilox 3.7 (pll3.7) RNAi vector system. For preparation of viral particles, HEK 293T cells were transfected with a vector encoding the D2R 6

7 shrna (5 -TGCGTAGCAGCCGAGCTTTCTTCAAGAGAGAAAGCTCGGCTGCTACGC TTTTTTC-3 ) or with the empty vector, together with VSV-G and Δ8.9 packaging plasmids (medium was replaced after 6 h). The culture supernatants were collected 24, 48, and 72 h after transfection, pooled, mixed with 40% polyethylene glycol (final concentration, 10%), and incubated overnight at 4 C. The mixture was centrifuged for 30 min at 3000 rpm to isolate the precipitated viral particles, and the pellet was resuspended in ice-cold phosphate-buffered saline (PBS) before concentration of the particles by centrifugation at 29,000 rpm for 2 h at 4 C. The viral titer of the resulting pellet was determined by flow cytometry. Virus was divided into portions and stored in light-protected boxes at 80 C until use. Stereotaxic surgery, cannula placement, and virus and raclopride injection Stereotaxic surgery was performed on mice at 21 days of age. Animals were anesthetized by i.p. injection of 1.6 μl of Zoletil and 0.05 μl of xylazine (Rompun, Bayer) per gram of body weight and were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). For injection of lentiviruses, a 31-gauge syringe needle was used to bilaterally infuse 2 μl (3 105 to Bilateral tissue punches were obtained from the NAc core, and total RNA was isolated with the use of Trizol and RQ1 RNase-free DNase (Invitrogen) Reverse transcription of the RNA was performed with SuperScript II reverse transcriptase (Invitrogen), and the resulting cdna was transducing units) of virus into the NAc core at an angle of 0 (AP +1.4; ML +1.3; DV 4.5) at a rate of 0.2 μl/min. For cannula surgery, a 26-gauge stainless steel guide cannula was implanted into the NAc core (AP +1.4; ML +1.1; DV 4.4). Microinfusion of S( )-raclopride (+)-tartrate salt (Sigma-Aldrich, St. Louis, MO) at 5 nmol per side was performed 40 min before measurement of locomotor activity and every other day during the withdrawal and recovery periods. Raclopride or saline was administered into the NAc core in a volume of 1 μl at a rate of 0.2 μl/min with a 22-gauge 25-μl Hamilton microsyringe connected to a 33-gauge internal cannula and an automated syringe pump (KD Scientific, USA). Mice were allowed to recover for 5 days after surgery (virus injection or cannula placement) before being subjected to cocaine sensitization experiments. Reverse transcription and real-time PCR analysis Bilateral tissue punches were obtained from the NAc core, and total RNA was isolated with the use of Trizol and RQ1 RNase-free DNase (Invitrogen) Reverse transcription of the RNA was performed with SuperScript II reverse transcriptase (Invitrogen), and the resulting cdna was 7

8 subjected to real-time PCR analysis in triplicate with the use of a LightCycler 480 system (Roche, Mannheim, Germany) and with primers (forward and reverse, respectively) specific for mouse D2R (5ʹ-TCTTCTGGTGGCCACACTGGTTAT-3ʹ and 5ʹ-ACAGGTTCAAGATGCTTGCTGTGC-3ʹ) and mouse β-actin (5ʹ-ACTATTGGCAACGAGCGGTT-3ʹ and 5ʹ-TGTCAGCAATGCCTGGGTACAT-3'). Quantitation was performed with the method 58. ΔΔCt Slice preparation and surface receptor biotinylation Surface AMPAR expression in brain slices was measured as described previously 59. In brief, coronal brain slices (thickness, 300 μm) containing the NAc were prepared with a vibratome (Oxford Instruments, Abingdon, UK) in an ice-cold dissection buffer (87 mm NaCl, 2.5 mm KCl, 1.25 mm NaH 2 PO 4, 25 mm NaHCO 3, 1 mm CaCl 2, 3 mm MgCl 2, 10 mm dextrose, 212 mm sucrose) saturated with 5% CO 2 and 95% O 2. The slices were then incubated for 90 min in an ice-cold artificial cerebrospinal fluid (ACSF: 124 mm NaCl, 5 mm KCl, 1.23 mm NaH 2 PO 4, 26 mm NaHCO 3, 10 mm dextrose, 1 mm MgCl 2, 2 mm CaCl 2 ) that was also saturated with 5% CO 2 and 95% O 2 before their transfer to ice-cold PBS containing sulfo-nhs-lc-biotin (Pierce, Rockford, IL) at 1 mg/ml and incubation for an additional 20 min. The slices were washed three times for 5 min with ice-cold Tris-buffered saline and then transferred to ice-cold PBS for microdissection. NAc slices were dissected bilaterally on an ice-cold platform (Harris Micropunch, hole of 1.00 mm diameter). The microdissected tissue was homogenized in RIPA buffer (150 mm NaCl, 10 mm NaH 2 PO 4, 2 mm EDTA, 50 mm NaF, 10 mm sodium pyrophosphate, 10 mm iodoacetamide, 1 mm sodium orthovanadate, 1% Triton X-100, 0.5% SDS, 0.5% sodium deoxycholate) supplemented with 100 nm phenylmethylsulfonyl fluoride, and the total protein concentration of the homogenate was determined with the Bradford assay. Total protein (40 to 60 μg) was subjected to precipitation with avidin-agarose (NeutrAvidinAgarose; Thermo Scientific, Rockford, IL) for isolation of biotinylated proteins. The resulting precipitate as well as the total homogenate were subjected to immunoblot analysis with antibodies to GluR1 (AB 1504; Millipore, Billerica, MA) for determination of surface and total GluR1 expression, respectively; the blot was also probed with antibodies to β-actin (1:25,000 dilution of MAB1501;Chemicon, 8

9 Temecula, CA) as a loading control. The detailed immunoblot protocol was described previously 19. Blots were subjected to densitometric quantification of total GluR1 expression normalized by β- actin and of surface GluR1 expression normalized by total GluR1. Supplementary References 56. Abatan, O. I., Welch, K. B. & Nemzek, J. A. Evaluation of saphenous venipuncture and modified tail-clip blood collection in mice. J. Am. Assoc. Lab. Anim. Sci. 47, 8 15 (2008). 57. Johnson, P. M. & Kenny, P. J. Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats. Nat. Neurosci. 13, (2010). 58. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, (2008). 59. Heynen, A. J. et al. Molecular mechanism for loss of visual cortical responsiveness following brief monocular deprivation. Nat. Neurosci. 6, (2003). 9