(A) Rats were treated with saline, acute amphetamine (4 mg/kg), or chronic amphetamine (7 days), and allowed to withdraw for 1 to 10 days. Rats that received chronic amphetamine received a challenge dose of either saline or amphetamine (4 mg/kg) and were analyzed 1 hr later. The levels of c-fos mRNA observed after an acute dose of amphetamine were significantly reduced in rats which were previously exposed to chronic amphetamine and withdrawn from the drug for 1 through 5 days (ANOVA: significant effect of drug challenge, F (1,35) = 132.57, P < 0.0001, significant effect of withdrawal, F (5,35) = 9.24, P < 0.0001; Bonferroni post-hoc: 1 day withdrawal vs. acute; *P < 0.05, 3 days withdrawal vs. acute **P < 0.01, 5 day withdrawal vs. acute, ***P < 0.001, n = 3–5). Moreover, at 1 day of withdrawal, the fold induction of c-fos vs. acute was significantly blunted (^†P < 0.05), with a strong trend at 3 days a 5 days of withdrawal. In rats treated with chronic amphetamine, after 5 days of withdrawal, there was a significant reduction in c-fos mRNA compared to naive controls (**P < 0.01). (B) ChIP of striatal lysates found significantly more ΔFosB bound to the c-fos promoter after 1 and 5 days of withdrawal from chronic amphetamine (*P < 0.05, n = 4–5, Student’s t-test). (C) Representative image of mouse striatum from the laser capture microscope before (top) or after (bottom) dissection of the infected region. (D) Mice were given an acute dose of amphetamine and processed for laser capture microdissection. In striatum infected with AAV-ΔFosB compared to that infected with AAV-GFP, there was a significant decrease in c-fos mRNA (**P = 0.01, n = 3, Student’s t-test). No change was observed in β-tubulin mRNA (P > 0.05).

(A) ChIP of striatal lysates revealed a significant increase in acetylated histone H4 on the promoter of c-fos 1hr after a challenge dose of amphetamine in drug naïve rats (ANOVA, significant effect of drug, F (1,12) = 6.26, P < 0.05, Bonferroni post-hoc: *P < 0.05, n =5). This increase was not observed in rats previously exposed to chronic amphetamine (P > 0.05). (B) The HDAC inhibitor, sodium butyrate (400 mg/kg) reversed the amphetamine-induced reduction in c-fos mRNA observed after 5 days of withdrawal (ANOVA, significant effect of butyrate F (1,28) = 5.29, P < 0.05, Bonferroni post-hoc: *P < 0.05, n = 4–9). (C) ChIP of striatal lysates revealed significantly more HDAC1 bound to the c-fos promoter (*P < 0.05, n = 5–6, Student’s t-test), but not the promoter of β-actin (P > 0.05), after 5 days of withdrawal from chronic amphetamine. (D) HDAC1 was transfected into PC12 cells with either full-length FosB or ΔFosB. HDAC1 selectively immunoprecipitated with ΔFosB, not full-length FosB. (E) In rats that received chronic electroconvulsive seizures (7 daily seizures), a condition known to increase ΔFosB several-fold, immunoprecipitation of HDAC1 pulled down significant levels of ΔFosB. This interaction was not observed in sham-treated animals. Blots are representative of 2–3 experiments. (F) Serum stimulation increased c-fos mRNA significantly less in cells transfected with HDAC1 than with GFP (*P < 0.05, n = 3 independent experiments). (G) Floxed HDAC1 mice whose striata were infected with either AAV-GFP or AAV-CreGFP on opposite sides of the brain, were treated with chronic amphetamine (7 days, 4 mg/kg) and 5 days of withdrawal. We found significantly higher c-fos expression in cells infected with AAV-CreGFP, where HDAC1 had been floxed out, than in cells expressing AAV-GFP after a 2 mg/kg amphetamine challenge (*P < 0.05, n = 2–3). Significantly less Hdac1 mRNA was observed in AAV-Cre infected neurons (***P < 0.001), while Hdac2 expression was unaffected (P > 0.05).

(A) ChIP of striatal lysates found significantly more di-methylated histone H3 at lysine 9 (H3K9me2) bound to the c-fos promoter after 5 days of withdrawal from chronic amphetamine (*P < 0.05, n = 3, Student’s t-test), while no change occurred on the β-actin promoter. (B) Rats treated with chronic amphetamine and analyzed after 5 days of withdrawal have significantly higher Kmt1a/Suv39h1 mRNA levels compared to saline treated rats (*P < 0.05, n = 3), while Hdac5 levels remain unchanged (P > 0.05).