(a) qPCR analysis of NAc of acute and chronic (7 days) cocaine treated mice (20 mg/kg/day IP) demonstrated transient increases in Dnmt3a mRNA levels 4 hr after the final injection (*P<0.05, n=14 acute, n=7 chronic) and a decrease after 24 hr (**P<0.005, n=6 acute, n=16 chronic). Dnmt1 (#P=0.08) and Dnmt3b transcripts were not altered significantly by chronic cocaine, however, acute cocaine significantly reduced Dnmt1 expression at 24 hr (*P<0.05, n=6). (b) Dnmt3a mRNA was significantly reduced in the NAc of chronic (13 days) self-administering rats examined 24 hr after the last drug dose (*P<0.05, #P=0.12, n=6 control, n=7 self-administration). (c) ChIP analysis revealed a significant cocaine-induced increase (4 hr) and decrease (24 hr) in me3K4H3 binding −500 bp upstream to the Dnmt3a promoter (*P<0.05, n=4, 5 mice pooled/n), with no regulation seen −2000 bp upstream from the promoter. Data are mean ± sem.

(a) Chronic (7 days) methionine (0.78 g/kg/2x/day SC) diminished the rewarding effects of cocaine in the CPP paradigm (*P<0.05, n=9). (b) Continuous intra-NAc infusion over 7 days of the DNMT inhibitor RG108 (100 µm) decreased global DNA methylation levels in NAc (*P<0.005, n=6 vehicle, n=7 RG108), (c) increased cocaine CPP at 10 mg/kg IP (**P<0.0005, n=9), (d) and enhanced the induction of locomotor sensitization to chronic cocaine (20 mg/kg IP) (*P<0.05, n=8). (e) Verification of anatomical placement and viral infection in NAc after HSV-Dnmt3a-GFP injection; immunostaining for GFP is shown. Cartoon adapted from MBSC atlas, Figure 18, 1.54 mm Bregma. (f) HSV-Dnmt3a increased global DNA methylation levels (*P<0.05, n=8 HSV-GFP, n=5 HSV-Dnmt3a). (g) Intra-NAc HSV-Dnmt3a significantly attenuated cocaine reward at 10 mg/kg cocaine (*P<0.05, n=10), with a trend seen at a lower dose (#P=0.13, n=10). (h) Intra-NAc AAV-Cre injected into floxed-Dnmt3a mice significantly increased cocaine CPP at 7.5 mg/kg (*P<0.05, n=14 AAV-CRE, n=18 AAV-GFP), with no effect seen in floxed Dnmt1 mice. All raw CPP data are provided in Supplemental Table 1. Data are mean ± sem.

(a) qPCR of NAc taken 1 or 10 days after chronic (10 days) social defeat shows a significant increase in Dnmt3a levels at both time points (*P<0.05). Inset displays social avoidance, a depressive-like behavior, observed in these mice (**P<0.005, n=16 control, 26 defeated). (b) Naïve mice infused intra-NAc with HSV-GFP or HSV-Dnmt3a were subjected to a submaximal protocol of social defeat. HSV-GFP mice showed a significant increase in interaction with a social target (F_1,42 = 5.219, *P<0.05) as would be expected from control mice25, whereas HSV-Dnmt3a mice lacked this phenotype (P>0.05), a pro-depressive-like response. (c) Intra-NAc injection of HSV-Dnmt3a significantly decreased latency to immobility on Day 2 of the rat forced-swim test, also a pro-depressive-like response (**P<0.001 n=7). (d) Conversely, intra-NAc infusion (10 days) of RG108, initiated one day after the last defeat episode, completely reversed the chronic social defeat-induced social avoidance exhibited by vehicle-infused mice (F_1,44 = 12.876 **P<0.001), an effect equivalent to that seen for chronic (20 mg/kg/day IP, 14 days) fluoxetine administration (P>0.05). Data are mean ± sem.

(a) qPCR analysis of NAc of chronic (28 days) cocaine treated mice (20 mg/kg/day IP) that have undergone 28 days of drug withdrawal demonstrated an increase in Dnmt3a mRNA levels (*P<0.05, n=11). (b) Dnmt3a mRNA expression was significantly increased after 28 days of withdrawal in the NAc of chronic (3 week) self-administering rats (*P<0.05, n=6 control, n=7 self-administration). Data are mean ± sem.

(a) Dnmt3a overexpression mimicked cocaine’s ability to increase dendritic spine density of NAc medium spiny neurons compared to saline GFP controls (*P<0.05, n=5 both HSV-GFP conditions, n=6 both HSV-Dnmt3a conditions). Representative confocal scans of GFP immunostained NAc neurons infected with either GFP or Dnmt3a-GFP viruses. To correspond with the shortened timescale of HSV expression, these analyses were performed 4 hr after 5 cocaine injections (20mg/kg IP). (b) Using an alternate method involving direct injection of Lucifer yellow into identified neurons, we found that chronic (7 days) cocaine treatment also significantly increased spine density at 4 hr (*P<0.05, n=7 saline, n=8 cocaine) and 24 hr (**P<0.01, n=8 saline, n=7 cocaine) after the last cocaine injection. Continuous (7 days) intra-NAc RG108 infusion significantly reduced spine density 24 hr after the last chronic cocaine injection (*P<0.05, n=4 Vehicle infused, n=5 RG108 infused). Data are expressed as % change in spine density relative to saline controls. (c) NeuronStudio spine type analysis of the same images from (b) revealed that chronic cocaine selectively increased thin spines both 4 and 24 hr after the last cocaine injection (*P<0.05, **P<0.01). Likewise, intra-NAc RG108 selectively reduced thin spines of chronic cocaine injected animals (*P<0.05). None of these conditions significantly affected the number of mushroom (mshrm) or stubby (stb) spines. Data are mean ± sem.