Table 1.

Statistical analysis.

 Figure Data structure Type of test Sample size Statistical data 2C (left + right)Open field habituation following VEH or CNO treatment (hM4Di, rM3Ds and control mice*) Normal distribution Mixed model analysis with two between-subject factors (group and treatment) and one repeated measure (time), followed by Pairwise Comparisons using the Planned Comparisons procedure (InVivoStat) 15 CNO hM4Di,12 CNO rM3Ds,13 CNO control;12 VEH hM4Di,17 VEH rM3Ds,6 VEH control FTIME(17,1173) = 47.95, p < 0.0001FGROUP(2,69) = 12.18, p < 0.001FTREATMENT(1,69) = 0.50, p = 0.48FGENOTYPE×TREATMENT(2,69) = 7.36, p = 0.0013 Planned Comparisons, relative to control (WT), df = 1242: VEH treated: No significant difference CNO treated: 25: hM4Di, *, p = 0.0064, t = 2.731 & rM3Ds,  p = 0.324, t = 0.99 30: hM4Di, *, p = 0.042, t = 2.03 & rM3Ds,  p = 0.14, t = 1.47 35: hM4Di, *, p = 0.016, t = 2.41 & rM3Ds,  *, p = 0.021, t = 2.31 40: hM4Di, ***, p = 0.0003, t = 3.59 & rM3Ds,  p = 0.2827, t = 1.07 45: hM4Di, ****, p < 0.0001, t = 3.95 & rM3Ds,  p = 0.07, t = 1.8 50: hM4Di, ****, p < 0.0001, t3.93 & rM3Ds,  p = 0.068, t = 1.82 55: hM4Di, ****, p < 0.0001, t = 5.1 & rM3Ds,  p = 0.15, t = 1.42 60: hM4Di, ****, p < 0.0001, t = 4.49 & rM3Ds,  *, p = 0.0171, t = 2.38 65: hM4Di, ****, p < 0.0001, t = 4.55 & rM3Ds,  p = 0.13, t = 1.49 70: hM4Di, **, p = 0.0054, t = 2.79 & rM3Ds,  **, p = 0.0073, t = 2.68 75: hM4Di, **, p = 0.007, t = 2.70 & rM3Ds,  p = 0.51, t = 0.66 80: hM4Di, ***, p = 0.0032, t = 2.96 & rM3Ds,  p = 0.34, t = 0.95 85: hM4Di, *, p = 0.016, t = 2.42 & rM3Ds,  p = 0.2, t = 1.28 2DTotal distance traveled during open field habituation with VEH or CNO pretreatment (hM4Di, rM3Ds and control mice) Normal distribution Two-way ANOVA followed by Pairwise Comparisons using the ‘Planned Comparisons’ procedure (InVivoStat) 15 CNO hM4Di,12 CNO rM3Ds,13 CNO control12 VEH hM4Di,17 VEH rM3Ds,6 VEH control FTREATMENT(1,69) = 0.02, p = 0.89 FGROUP(2,69) = 14.93, p < 0.0001 FINTERACTION(2,69) = 5.37, p = 0.0068Planned Comparison,Within-treatment, relative to control, df = 69; CNO - hM4Di: **, p = 0.0012, t = 3.39CNO - rM3Ds: *, p = 0.010, t = 2.66VEH - hM4Di: p = 0.908, t = 0.12VEH - rM3Ds: p = 0.255, t = 1.15Within-group, df = 69; Control: p = 0.680, t = 0.41 hM4Di: *, p = 0.0103, t = 2.63 rM3Ds: p = 0.0605, t = 1.9 3AHabituation phase prior to treatment (0–90 min) (hM4Di and control mice). Normal distribution Mixed-model analysis with two between subject factors (group and treatment) and one repeated measure (time) (InVivoStat) 9 saline control,12 cocaine control,12 saline hM4Di,21 cocaine hM4Di FTIME(8,400) = 86.89, p < 0.0001FGROUP(1,50) = 2.46, p = 0.123 FTREATMENT(1,50) = 2.06, p = 0.157 3AAcute cocaine response after CNO and habituation (120–180 min) (hM4Di and control mice). Normal distribution Mixed-model analysis with two between-subject factors (group and treatment) and one repeated measure (time), followed by Pairwise Comparisons using the ‘Planned Comparisons’ procedure. (InVivoStat) 9 saline control,12 cocaine control,12 saline hM4Di,21 cocaine hM4Di FTIME(6,300) = 11.57, p < 0.0001FGROUP(1,50) = 7.68, p = 0.0078FTREATMENT(1,50) = 36.17, p < 0.0001FGROUP × TREATMENT(1,50) = 3.52, p = 0.0667Planned Comparison, relative to control, df = 350?:Cocaine hM4Di vs cocaine control:120: p = 0.1362, t = 1.49130: *, p = 0.0432, t = 2.03 140: **, p = 0.001, t = 3.33 150: ***, p = 0.0008, t = 3.39 160: **, p = 0.0027, t = 3.01 170: *, p = 0.0304, t = 2.17180: p = 0.0635, t = 1.86 3B Total distance traveled after cocaine (120–180 min) (hM4Di and control mice). Normal distribution Two-way ANOVA followed by pairwise comparisons using the ‘Planned Comparisons’ procedure. (InVivoStat) 9 saline control,12 cocaine control,12 saline hM4Di,21 cocaine hM4Di FGROUP(1,50) = 9.71, p = 0.003FTREATMENT(1,50) = 25, p < 0.0001FINTERACTION(1,50) = 2.52, p = 0.119Planned Comparison, df = 50? Within-treatment, hM4Di vs control; Cocaine: **, p = 0.0005, t = 3.70Saline: p = 0.3276, t = 0.99Within-group, saline vs cocaine;Control: ****, p < 0.0001, t = 4.25hM4Di: **, p = 0.0097, t = 2.69 3EAMPAR/NMDAR ratio (hM4Di mice) Normal distribution Unpaired t test 6 saline-saline:4 CNO-cocaine p = 0.02, t = 2.89, df = 8 4DAcute cocaine response after CNO including habituation (hM4DiVTA→NAc and control mice). Normal distribution Two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test 6 control,5 hM4DiVTA→NAc FTIME(17,153) = 11.55, p < 0.0001, FGROUP(1,9) = 6.754, p = 0.0288 and FINTERACTION(17,153) = 3.455, p < 0.0001Multiple comparison, Group, df = 162130: p = 0.0120, t = 3.471140: p < 0.0001, t = 5.643150: p = 0.0002, t = 4.587 4E, left Total distance traveled during habituation (0–90 min) (hM4DiVTA→NAc and control mice). Normal distribution Unpaired t test 6 control,5 hM4DiVTA→NAc p = 0.0681, t = 2.072, df = 9 4E, rightTotal distance traveled after cocaine (120–180 min) (hM4DiVTA→NAc and control mice). Normal distribution Unpaired t test 6 control,5 hM4DiVTA→NAc p = 0.037, t = 2.439, df = 9 5B Cocaine sensitization of hM4Di and control mice, day 1 and day 2 of induction (saline control mice) Normal distribution Two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test on induction day 1 7 CNO control saline,4 CNO hM4Di saline,4 VEH hM4Di saline Induction day 1 (left);FTIME(5,60) = 27.52, p < 0.0001, FGROUP(2,12) = 4.688, p = 0.0313FINTERACTION(10,60) = 1.338, p = 0.2316Multiple comparison, relative to CNO hM4Di saline, df = 72VEH hM4Di saline10: p = 0.016, t = 2.72820: p = 0.0341, t = 2.442CNO control saline10: p = 0.0024, t = 3.36720: p = 0.0075, t = 2.998Induction day 2 (right);FTIME(5,60) = 16.46, p < 0.0001, FGROUP(2,12) = 3.249, p = 0.0745, FINTERACTION(10,60) = 1.344, p = 0.2288 5CTotal distances traveled during cocaine sensitization of hM4Di and control mice, induction days and challenges Normal distribution Two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test 15 saline controls (7 CNO controls, 4 CNO hM4Di, 4 VEH hM4Di) 10 CNO control cocaine, 10 CNO hM4Di cocaine, 7 VEH hM4Di cocaine FTIME(8,304) = 38.71, p < 0.0001, FGROUP(3,38) = 35.84, p < 0.0001 and FINTERACTION(24,304) = 5.546, p < 0.0001Multiple comparison, Group, df = 342CNO hM4Di cocaine vs VEH hM4Di cocaine: Day1: p = 0.171, t = 2.20 Day2: p = 0.0003, t = 4.095 Day3: p = 0.121, t = 2.335 Day4: p = 0.0096, t = 3.182 Day5: p = 0.0439, t = 2.698 Day6: p < 0.0001, t = 4.513 Challenge I: p > 0.999, t = 0.9513 5ECocaine sensitization of hM4Di and control mice, total distance traveled during the initial 30 min on day 6 of induction Normal distribution One-way ANOVA followed by Bonferroni’s post- hoc test 15 saline controls,10 CNO control cocaine,10 CNO hM4Di cocaine, 7 VEH hM4Di cocaine F(3,38) = 36.06, p < 0.0001Multiple comparison, df = 38CNO hM4Di vs CNO control:p = 0.0004, t = 4.518CNO hM4Di vs VEH hM4Di:p = 0.0002, t = 4.710CNO hM4Di vs saline control: p = 0.0041, t = 3.697VEH hM4Di vs saline control:p < 0.0001, t = 8.368CNO control vs saline control: p < 0.0001, t = 8.646 5GCocaine sensitization of hM4Di and control mice, total distance traveled during the initial 30 min of Challenge I Normal distribution One-way ANOVA followed by Bonferroni’s post- hoc test 15 saline controls,10 CNO control cocaine,10 CNO hM4Di cocaine, 7 VEH hM4Di cocaine F(3,38) = 6.385, p = 0.0013Multiple comparison, df = 38CNO hM4Di vs saline control: p = 0.0132, t = 3.209VEH hM4Di vs saline control: p = 0.0119, t = 3.321CNO control vs saline control: p = 0.0081, t = 3.462 5ICocaine sensitization of hM4Di and control mice, total distance traveled during the initial 30 min of Challenge II Normal distribution One-way ANOVA 10 CNO control cocaine, 10 CNO hM4Di cocaine, 7 VEH hM4Di cocaine F(2,24) = 0.7356, p = 0.4897 5J Cocaine sensitization of hM4Di and control mice, total distance traveled during Challenge I and Challenge II Normal distribution Two-way repeated-measures ANOVA 10 CNO control cocaine, 10 CNO hM4Di cocaine, 7 VEH hM4Di cocaine FTIME(1,24) = 4.145, p = 0.0530, FGROUP(2,24) = 0.5531, p = 0.5823, FINTERACTION(2,24) = 0.2762, p = 0.7610 5K Effect of CNO on cocaine response in open field test after repeated cocaine administration in home-cage, no habituation (hM4Di and control mice). Normal distribution Two-way repeated measures ANOVA 5 hM4Di cocaine;5 control cocaine Day after last injectionFTIME(5,40) = 13.59, p < 0.0001,FGROUP(1,8) = 0.1655, p = 0.6948,FINTERACTION(5,40) = 0.3572, p = 0.8746 6BHabituation before and after chronic CNO/VEH administration (hM4Di mice) Normal distribution Two-way repeated measures ANOVA 4 hM4Di CNO,4 hM4Di VEH FTIME(5,30) = 8.266, p < 0.0001, FGROUP(1,6) = 0.1646, p = 0.699 FINTERACTION(5,30) = 0.3483, p = 0.8793 6C-D Protein levels of pTH and DAT after chronic CNO (hM4Di mice) Assuming normality Unpaired t-test 4 hM4Di CNO,4 hM4Di VEH C; pTH: p = 0.9225, t = 0.1014, df = 6; D; DAT: p = 0.713, t = 0.3857, df = 6 6EStriatal dopamine levels DAT after chronic CNO (hM4Di mice) Assuming normality Unpaired t-test 4 hM4Di CNO,4 hM4Di VEH NAc: p = 0.547, t = 0.63, df = 6 dStr: p = 0.45, t = 0.807, df = 6 7CCocaine conditioned place preference in hM4Di mice, time in paired (% of baseline) Normal distribution One-way ANOVA followed by Bonferroni’s post-hoc test 8 saline,6 cocaine CNO, 8 cocaine VEH F(2,19) = 6.549, p = 0.0069 Multiple comparison, relative to saline, df = 19 Cocaine VEH: p = 0.0060, t = 3.401Cocaine CNO: p = 0.0302, t = 2.672 7DCocaine conditioned place preference in hM4Di mice, time in paired Normal distribution Two-way repeated measures ANOVA followed by Bonferroni’s post-hoc test 8 saline,6 cocaine CNO, 8 cocaine VEH FTIME(8.152) = 8.656, p < 0.0001, FGROUP(2,19) = 2.174, p = 0.1412 FINTERACTION(16,152) = 1.833, p = 0.0315; Multiple comparison, relative to baseline, df = 152: Cocaine VEH: Post-test: p < 0.0001, t = 5.320Ext1: p = 0.0003, t = 4.267Ext2: p = 0.0084, t = 3.341Reinstatement: p = 0.0350, t = 2.893Cocaine CNO: Post-test: p = 0.0001, t = 4.451Ext1: p < 0.0001, t = 4.981Ext2: p = 0.0001, t = 4.421Ext3: p = 0.0002, t = 4.322Ext4: p = 0.0033, t = 3.608Ext5: p = 0.0117, t = 3.242 7ELocomotor activity during baseline-, post-, and reinstatement-test of the cocaine conditioned place preference Normal distribution Two-way repeated measures ANOVA followed by Bonferroni’s post-hoc test 8 saline,6 cocaine CNO, 8 cocaine VEH FTIME(2,38) = 38.60, p < 0.0001, FGROUP(2,19) = 9.012, p = 0.0018 FINTERACTION(4,38) = 5.036, p = 0.0023 Multiple comparison, relative to saline, df = 57: Cocaine VEH: Baseline: p > 0.999, t = 0.4795Post-test: p = 0.0009, t = 3.714 Reinstatement: p < 0.0001, t = 5.410 Cocaine CNO: Baseline: p > 0.999, t = 0.1025Post-test: p = 0.3743, t = 1.335Reinstatement: p = 0.1354, t = 1.863 7FLocomotor activity during conditioning sessions in paired compartment Normal distribution Two-way repeated measures ANOVA followed by Bonferroni’s post-hoc test 8 saline,6 cocaine CNO, 8 cocaine VEH FTIME(3,57) = 10.22, p < 0.0001, FGROUP(2,19) = 15.21, p = 0.0001 and FINTERACTION(6,57) = 7.515, p < 0.0001; Multiple comparison, relative to cocaine VEH, df = 76: Saline VEH: 1st: p = 0.0005, t = 3.8232nd: p = 0.0061, t = 3.0623rd: p < 0.0001, t = 6.1214th: p < 0.0001, t = 6.768Cocaine CNO: 3rd: p = 0.0002, t = 1.7874th: p = 0.0002, t = 4.124Multiple comparison, relative to 1st session, df = 57: Cocaine VEH: 3rd: p = 0.001, t = 4.4084th: p < 0.0001, t = 5.843 7H Conditioned place preference in hM4Di mice following CNO treatment, time in paired (% of baseline) Normal distribution Two-way repeated measures ANOVA followed by Bonferroni’s post-hoc test 7 saline (+CNO),8 saline (+CNO/VEH)8 saline (+VEH) from Fig. 7C,D FTIME(4,80) = 3.582, p = 0.0097, FGROUP(2,20) = 0.1793, p = 0.8372 FINTERACTION(8,80) = 0.8799, p = 0.5371 8C+DReward preference test in hM4Di mice, total volumes consumed Normal distribution Unpaired t-test 8 hM4Di CNO,8 hM4Di VEH p = 0.22, t = 1.27, df = 14 8EReward preference test in hM4Di mice, reward preference ratio (%) Normal distribution Unpaired t-test 8 hM4Di CNO,8 hM4Di VEH p = 0.30, t = 1.08, df = 14 8GTouchscreen-based motivational assay, number of touch trials Normal distribution Unpaired t-test 8 hM4Di CNO,8 hM4Di VEH p = 0.01, t = 2.83, df = 14 8HTouchscreen-based motivational assay, mean correct latency Normal distribution Unpaired t-test 8 hM4Di CNO,8 hM4Di VEH p = 0.04, t = 2.28, df = 14 8ITouchscreen-based motivational assay, mean correct latency Normal distribution Unpaired t-test 8 hM4Di CNO,8 hM4Di VEH p = 0.29, t = 1.11, df = 14
• Statistical analyses were performed in GraphPad Prism 6, except for behavioral data in Figs. 2 and 3, which were analyzed InVivoStat (Invivostat.co.uk).

• * Control mice are wild-type mice injected with the Cre-dependent AAV and do not express the DREADD transgenes.