Molecular mechanism for a gateway drug: epigenetic changes initiated by nicotine prime gene expression by cocaine

Abstract

In human populations, cigarettes and alcohol generally serve as gateway drugs, which people use first before progressing to marijuana, cocaine, or other illicit substances. To understand the biological basis of the gateway sequence of drug use, we developed an animal model in mice and used it to study the effects of nicotine on subsequent responses to cocaine. We found that pretreatment of mice with nicotine increased the response to cocaine, as assessed by addiction-related behaviors and synaptic plasticity in the striatum, a brain region critical for addiction-related reward. Locomotor sensitization was increased by 98%, conditioned place preference was increased by 78%, and cocaine-induced reduction in long-term potentiation (LTP) was enhanced by 24%. The responses to cocaine were altered only when nicotine was administered first, and nicotine and cocaine were then administered concurrently. Reversing the order of drug administration was ineffective; cocaine had no effect on nicotine-induced behaviors and synaptic plasticity. Nicotine primed the response to cocaine by enhancing its ability to induce transcriptional activation of the FosB gene through inhibition of histone deacetylase, which caused global histone acetylation in the striatum. We tested this conclusion further and found that a histone deacetylase inhibitor simulated the actions of nicotine by priming the response to cocaine and enhancing FosB gene expression and LTP depression in the nucleus accumbens. Conversely, in a genetic mouse model characterized by reduced histone acetylation, the effects of cocaine on LTP were diminished. We achieved a similar effect by infusing a low dose of theophylline, an activator of histone deacetylase, into the nucleus accumbens. These results from mice prompted an analysis of epidemiological data, which indicated that most cocaine users initiate cocaine use after the onset of smoking and while actively still smoking, and that initiating cocaine use after smoking increases the risk of becoming dependent on cocaine, consistent with our data from mice. If our findings in mice apply to humans, a decrease in smoking rates in young people would be expected to lead to a decrease in cocaine addiction.

Fig. 1

Nicotine priming enhances cocaine-induced behavioral endpoints, sensitization and conditioned place preference (CPP). For sensitization, we treated mice with nicotine (50 μg/ml) in the drinking water either for 24 hours (Fig. 1A) or 7 days (Fig. 1B). For the subsequent 4 days, the mice were treated with a single cocaine injection per day (20 mg/kg), with continued exposure to nicotine in the drinking water (n = 10–15 per group). Data expressed as total distance traveled on day 4 compared with day 1. (A) Lack of effect of 24 hours nicotine treatment on cocaine-induced locomotion. (B) Effect of 7 days of nicotine treatment on cocaine-induced locomotion. (C) Lack of effect of 7 days of cocaine treatment on nicotine-induced locomotion. (D) Effect of nicotine pretreatment on CPP. After 7 days of exposure to nicotine, mice were conditioned to either side of the place preference chamber with cocaine or saline. Preference scores were calculated subtracting the time spent in the cocaine-paired side after conditioning to the time before conditioning (n = 8 per group). Data represent mean ± SEM.

Fig. 2

Synaptic plasticity and gene expression responses are enhanced by pretreatment with nicotine followed by cocaine, but not the reverse. (A) A schematic illustration of stimulation (stim) and recording (rec) sites in the coronal slices. (B) LTP measured 180 min after high-frequency stimulation (HFS). Experimental groups include control (water followed by saline; n = 6), 7 days of nicotine (n = 6), single injection of cocaine (n = 6), and 7 days of nicotine followed by a single cocaine injection (n = 9). (C) Time course of change in LTP for all groups. An additional experimental group is included (7 days of cocaine treatment followed by 24 hours of nicotine; pink, n = 5). (D to G) Real-time PCR measurements of FosB expression (control normalized to 1.0). Data represent means ±SEM. (D) Twenty-four hours of nicotine followed by cocaine (n = 9 per group). (E) Seven days of nicotine followed by cocaine (n = 9 per group). (F) A single injection of cocaine followed by 24 hours of nicotine (n = 5 in each group). (G) Seven days of cocaine followed by 24 hours of nicotine (n = 7 in each group). *P < 0.05.

Fig. 3

Nicotine, but not cocaine, induces histone hyperacetylation in the striatum. (A and B) Chromatin immunoprecipitation assays for acetylation for (A) histone H3 (K9) and (B) H4 (K5 to K16) at the FosB promoter were performed in animals treated with acute cocaine (30 mg/kg), 7 days of nicotine (10 mg/ml), and 7 days of nicotine followed by acute cocaine (control set at 1, n = 3 to 6 per group). Data represent means ± SEM. (C to F) After 7 days of nicotine or cocaine exposure, protein extracted from striatal lysates was incubated with antibodies detecting histone H3 (K9) (C and E) and histone H4 (K5 to K16) (D and F) tail modifications. *P < 0.05

Fig. 4

Nicotine inhibits HDAC activity and its effects are mimicked by an acetylase inhibitor. (A-B) In the nuclear fraction, an HDAC activity assay that detects deacetylation of lysine residues was performed in mice treated with nicotine (10 μg/ml) for 7 – 10 days (A) and cocaine (30 mg/kg) for 7 days (B) (n = 5–7 in each group). Data represent means ± SEM. (C) Real-time RT-PCR measuring FosB expression in animals treated with systemic SAHA (25 mg/kg i.p.) followed by cocaine compared with controls (n = 5–7 in each group). (D) Immunoblots of histone H3 and H4 acetylation after SAHA treatment (n = 3–4 in each group). (E) Real-time RT-PCR measuring FosB expression in animals treated with local SAHA (100 μM) infused to the NAc for 7 days followed by cocaine (30 mg/kg) compared with controls (n = 4–7 in each group). (F) LTP measurement in mice treated with SAHA (25 mg/kg) followed by cocaine (30 mg/kg). SAHA was given 2 hours before cocaine or saline injections (n = 5 to 6). Field potential amplitudes were measured in the core of the NAc. (G) Histogram summary of time course of SAHA and cocaine effects on LTP. *P < 0.05.

Fig. 5

The priming effect of nicotine on cocaine in CBP+/− mice and mice treated with theophylline. (A1–A2) LTP measurement in CBP+/− mice and wild-type littermate controls treated with 7 days of nicotine; acute cocaine; and 7 days of nicotine followed by cocaine injection (n = 5–8). (A3) Histogram summary of changes in LTP amplitude at 180 min after HFS in different groups of mice (as shown in A1–A2). (A4) Input-output curve comparing CBP+/− mice and their wild-type littermates. (A5) Histone H4 (K5–16) tail acetylation in striatal lysates of CBP+/− mice and wild-type littermates after 7 days of nicotine exposure (n=4 per group). (B1) LTP measurement in mice treated with theophylline for 7 days; mice treated with theophylline followed by acute cocaine; mice treated with cocaine alone; and controls (n = 6–10 in each group). (B2) Histogram summary of changes in LTP amplitude at 180 min after HFS in different groups of mice (as shown in B1). (B3 and B4) Immunoblots of striatal lysates from mice treated for 7 days with theophylline (200 mg/liter in drinking water) probed with antibodies against acetylated histone H3 (K9) and acetylated histone H4 (K5 to K16) (B3), and antibodies against specific acetylated lysine residues on histone H4 (K5, K8, K12, and K16) (B4) (n = 4 per group). (B5) Real-time PCR measuring FosB expression in animals treated with local theophylline (0.2 mM) infused into the NAc for 7 days compared with controls (n = 5 per group). Groups as shown in B1. *P < 0.05.

Fig. 6

A molecular model for the nicotine-cocaine gateway sequence of drug usage. (A) FosB promoter region at baseline. (B) Acetylation of the promoter region of FosB after 7 days of nicotine exposure. (C) FosB expression in response to cocaine with previous nicotine exposure. TBP, TATA box–binding protein.

Fig. 7

Priming effect of nicotine on cocaine-induced responses requires continuous nicotine administration. (A) Behavioral assay for locomotor sensitization to 4 days of cocaine (20 mg/kg) following 14 days of water after 7 days of nicotine treatment (50 μg/ml); compared with 4 days of cocaine treatment with no prior nicotine; 14 days of water after 7 days of nicotine treatment; and controls (n = 12–15). (B) LTP measurement in mice treated with a single cocaine injection 14 days after 7 days of nicotine; single cocaine injection without prior nicotine treatment; 14 days of water following 7 days of nicotine; and controls (n = 6–7). (C) Histogram of the effect of nicotine cessation on LTP amplitude 180 min after HFS. (D) Real-time PCR measuring FosB expression in the striata of mice pretreated with nicotine for 7 days followed by water for 14 days, and then treated with a single cocaine injection; cocaine with no previous nicotine exposure; nicotine for 7 days, followed by 14 days of water; and controls (n = 3 to 4 in each group). Data represent means ± SEM.