Drug experience epigenetically primes Fosb gene inducibility in rat nucleus accumbens

Abstract

ΔFosB, a Fosb gene product, is induced in nucleus accumbens (NAc) and caudate-putamen (CPu) by repeated exposure to drugs of abuse such as cocaine. This induction contributes to aberrant patterns of gene expression and behavioral abnormalities seen with repeated drug exposure. Here, we assessed whether a remote history of cocaine exposure in rats might alter inducibility of the Fosb gene elicited by subsequent drug exposure. We show that prior chronic cocaine administration, followed by extended withdrawal, increases inducibility of Fosb in NAc, as evidenced by greater acute induction of ΔFosB mRNA and faster accumulation of ΔFosB protein after repeated cocaine reexposure. No such primed Fosb induction was observed in CPu; in fact, subsequent acute induction of ΔFosB mRNA was suppressed in CPu. These abnormal patterns of Fosb expression are associated with chromatin modifications at the Fosb gene promoter. Prior chronic cocaine administration induces a long-lasting increase in RNA polymerase II (Pol II) binding at the Fosb promoter in NAc only, suggesting that Pol II "stalling" primes Fosb for induction in this region upon reexposure to cocaine. A cocaine challenge then triggers the release of Pol II from the gene promoter, allowing for more rapid Fosb transcription. A cocaine challenge also decreases repressive histone modifications at the Fosb promoter in NAc, but increases such repressive marks and decreases activating marks in CPu. These results provide new insight into the chromatin dynamics at the Fosb promoter and reveal a novel mechanism for primed Fosb induction in NAc upon reexposure to cocaine.

Figure 1.

Effect of prior chronic cocaine exposure on locomotor activity and Fosb induction in NAc and CPu upon reexposure to the drug. A, Rats were injected intraperitoneally twice daily with either saline (Drug Naive) or cocaine (15 mg/kg; Drug Experienced) for 10 d and, after 28 d of withdrawal, were treated with one (ΔFosB and FosB mRNA) or several (Locomotor activity; ΔFosB protein) challenge injections of cocaine (15 mg/kg, i.p). B, Animals were habituated in a locomotor chamber without any challenge injection for 1 h, and then monitored for locomotor activity after one saline injection (Injection 0, n = 16) or cocaine challenge injections (Injection 1, n = 12; Injection 2, n = 8). Data are expressed as sum of total number of beam breaks over 60 min after the saline or cocaine injection. C, After 28 d of withdrawal, cocaine-naive and -experienced rats were injected intraperitoneally once with saline (Injection 0, n = 3) and with 1, 3, or 6 cocaine challenge injections (n = 5 per group). Animals were killed 24 h after the last injection and ΔFosB protein was quantified immunohistochemically in NAc and CPu. Bonferroni posttests: ∧p < 0.001 (B), p < 0.05 (C), different from Drug Naive with no cocaine challenge; *p < 0.001 (B), p < 0.05 (C), different from Drug Naive with cocaine challenges. Images displayed are from NAc shell. D, Animals were killed 45, 90, or 180 min after a single saline or cocaine challenge (n = 7 per group). Bonferroni posttests: ∧p < 0.05, different from Drug Naive-Saline; *p < 0.05, ^%p = 0.08, different from Drug Naive-Cocaine. Data are expressed as mean ± SEM.

Figure 2.

Effect of prior chronic cocaine exposure on upstream molecular signaling cascades in NAc and CPu. A, Rats were injected intraperitoneally twice daily with either saline (Drug Naive) or cocaine (15 mg/kg; Drug Experienced) for 10 d. After 28 d of withdrawal, animals were given one injection of saline or cocaine (15 mg/kg, i.p.) and killed 20 min later to collect NAc and CPu for Western blotting of B, SRF, pSRF, and pSRF/SRF; CREB, pCREB, and pCREB/CREB; ERK42, pERK42, pERK42/ERK42, ERK44, pERK44, and pERK44/ERK44; and AKT, pAKT, and pAKT/AKT, which were analyzed and presented as C, mean ± SEM of protein fold change compared with Drug Naive Saline-treated animals n = 6 per group. Two-tailed Student's t test, p < 0.05. Bold italic highlights significant changes; underlined = decreases; not underlined = increases.

Figure 3.

Effect of prior chronic cocaine exposure on epigenetic priming of the Fosb gene in NAc and CPu. A, Rats were injected intraperitoneally twice daily with either saline (Drug Naive) or cocaine (15 mg/kg; Drug Experienced) for 10 d. After 28 d of withdrawal, animals were either not injected (Drug Naive-No Challenge) or given one injection of cocaine (15 mg/kg, i.p.) and killed 1 h later (Drug Experienced-Challenge +1 h) to collect NAc and CPu for ChIP analysis. B, Graphical map (not to scale) to show relative position of the four sets of primers designed upstream and downstream of the Fosb TSS: primer 1 is situated 200 bp upstream of the TSS; primer 2 is centered ±55 bp around the TSS; primers 3 and 4 are located 693 and 1417 bp within the Fosb gene body, respectively. ChIP was performed for C, H3K9me2 using primers 1, 2, and 4; D, H3K4me3 and H3K27me3 using primer 1 only; and E, Pol II-pSer5 across all 4 primers. n = 5–12 per group; two-tailed Student's t tests: *p < 0.05, ^%p = 0.1, ^#p = 0.2, compared with respective Drug Naïve controls. Data are expressed as mean ± SEM.