In collaboration with Amir Levine, we developed a mouse model that would enable us to explore the behavioral, electrophysiological, and molecular genetic effects of particular drug-use sequences. We examined two addiction-related behaviors, locomotor sensitization and conditioned place preference, and the physiological and molecular markers of the priming effects of one drug on another in the nucleus accumbens, a region of the striatum that is critical for reward and addiction.19

Figure 3. Figure 3. Effects of Priming with Nicotine on Cocaine-Induced Locomotor Sensitization and Conditioned Place Preference in Mice. For sensitization, we gave the mice nicotine (50 μg per milliliter) in their drinking water for either 24 hours (Panel A) or 7 days (Panel B). For the subsequent 4 days, we gave the mice a single injection of cocaine per day (20 mg per kilogram of body weight), along with the same amount of nicotine in their drinking water as received previously (10 to 15 mice per group). In Panel B, data are expressed as the total distance traveled on days 9 through 11, as compared with day 1. Panel A shows the lack of effect of 24 hours of nicotine treatment on cocaine-induced locomotion, as compared with the water and saline control, and Panel B the significant effect of 7 days of nicotine treatment on cocaine-induced locomotion on days 9 through 11. Panel C shows the lack of effect of 7 days of cocaine treatment on nicotine-induced locomotion on day 11. Similarly, for conditioned place preference (Panel D), we gave the mice nicotine for 7 days, followed by 4 days of cocaine and nicotine; Panel D shows the data for conditioned preference for the cocaine chamber on day 11. Preference scores were calculated by subtracting the time spent in the cocaine-paired side after conditioning from the time spent before conditioning (8 mice per group). In all panels, data are means ±SE. Data are from Levine et al.19

Locomotor sensitization showed that priming mice with nicotine can enhance the effect of cocaine. Mice given nicotine in their drinking water were no more active than control mice given plain water. Mice given only cocaine were 58% more active than controls (Figure 3A); mice given nicotine for 1 day, followed by 4 days of nicotine and cocaine, showed no increase in locomotor response, but mice given nicotine for 7 days, followed by 4 days of nicotine and cocaine, were significantly (98%) more active than controls (Figure 3A and 3B). Activity did not increase when the protocol was reversed (7 days of cocaine, followed by 4 days of concurrent cocaine and nicotine) (Figure 3C).

Conditioned place preference is a more naturalistic model of addictive behavior than sensitization. It measures the preference of an animal for a particular place in its environment as that place becomes associated with a reward and assumes some of the pleasurable effects of the reward. As with sensitization, mice primed with 7 days of nicotine and then given both nicotine and cocaine for 4 days had a 78% greater preference for the chamber associated with cocaine than were mice given only water and then cocaine (Figure 3D).

We next examined synaptic plasticity, as measured by changes in long-term potentiation, in the core of the nucleus accumbens, a region of the ventral striatum that integrates rewarding input from dopamine-producing neurons in the ventral tegmental area with excitatory input from glutamate-producing neurons in the amygdala and the prefrontal cortex. Reducing excitatory input to the nucleus accumbens is thought to decrease inhibitory output from the nucleus accumbens to the ventral tegmental area and thereby to contribute, by means of disinhibition, to enhanced reward with drugs of abuse. This disinhibition results in the production of more dopamine and contributes to an enhanced rewarding effect of drugs of abuse.

Figure 4. Figure 4. Effects of Priming with Nicotine and Cocaine-Induced Synaptic Plasticity and FosB Expression in Mice. Panel A shows a schematic illustration of the stimulation and recording sites in a coronal slice of the nucleus accumbens of the mouse. Panel B shows long-term potentiation (LTP) measured 180 minutes after high-frequency stimulation (HFS) applied at 30 minutes (arrow). Experimental groups included six control mice given water followed by saline, six mice given nicotine for 7 days in drinking water, six mice given a single injection of cocaine, and nine mice given nicotine for 7 days followed by a single injection of cocaine. Panel C shows the change in long-term potentiation over time in all the groups. An additional experimental group (five mice given cocaine for 7 days, followed by 24 hours of nicotine) was included. Panel D shows the effect of 24 hours of nicotine followed by cocaine (nine mice per group) on real-time FosB expression, measured in polymerase chain reactions (PCRs; control [water followed by saline] normalized to 1 in Panels D through G). Panel E shows the effects of 7 days of nicotine followed by cocaine (nine mice per group). Panel F shows the results of a single injection of cocaine followed by 24 hours of nicotine (five mice per group). Panel G shows the results of 7 days of cocaine followed by 24 hours of nicotine (seven mice per group). In Panels B through G, data are means ±SE. Data are from Levine et al.19

Since we knew that the repeated administration of cocaine resulted in reduced long-term potentiation in the excitatory synapses of the nucleus accumbens in the mouse, we stimulated those synapses and measured long-term potentiation (Figure 4A). We found that just one injection of cocaine in a mouse given nicotine for 7 days led to a marked reduction in long-term potentiation that started immediately after stimulation and persisted for up to 180 minutes. Nicotine alone, cocaine alone for 7 days, or 7 days of cocaine followed by 24 hours of nicotine did not alter long-term potentiation (Figure 4B and 4C).

As in the behavioral experiments, priming with nicotine enhanced the effects of cocaine — in this case, priming changed synaptic plasticity (i.e., decreased long-term potentiation) in the nucleus accumbens. Priming with nicotine appeared to increase the rewarding properties of cocaine by further disinhibiting dopaminergic neurons in the ventral tegmental area.

Previous studies have shown that an important step in the sequence of molecular events leading to addictive behavior in mice is the increased expression of FosB in the striatum. Colby et al.20 found that the targeted expression of ΔFosB in the nucleus accumbens enhanced cocaine-induced behavior. We therefore asked whether the effects of nicotine on behavior that we had observed (cocaine-induced changes in sensitization and conditioned place preference) and changes in synaptic strength (long-term potentiation) correlated with changes in FosB expression in the striatum. We found that giving mice nicotine in their drinking water for 24 hours and for 7 days caused increases in FosB expression of 50% and 61%, respectively (Figure 4D and 4E). A single injection of cocaine after 7 days of nicotine led to a further 74% increase in FosB expression (Figure 4E), as compared with 7 days of exposure to cocaine alone (Figure 4F). As in behavioral and physiological experiments, our genetic study showed that mice given nicotine for 24 hours did not respond to cocaine as dramatically as mice given nicotine for 7 days before being given cocaine (Figure 4D and 4E). Moreover, nicotine given after cocaine did not increase gene expression (Figure 4F and 4G).

We next wanted to determine whether nicotine enhances FosB expression in the striatum by altering chromatin structure at the FosB promoter and thereby magnifying the effect of cocaine. We examined the acetylation of histones H3 and H4 at the FosB promoter.19 After 7 days of nicotine, the acetylation of histones H3 and H4 had increased. Cocaine alone increased the acetylation of histone H4 only; moreover, a single cocaine injection after 7 days of nicotine did not increase the acetylation of histone H4 further.

The ability of nicotine to produce robust acetylation at the FosB promoter suggested that nicotine-induced enhancement of acetylation could be occurring on a widespread scale, at the promoters of other genes expressed in the striatum. Using immunoblotting, we found that after 7 days of nicotine, the acetylation of histones H3 and H4 increased by 32% and 61%, respectively, everywhere in the striatum. These increases were similar to those we found at the FosB promoter. By contrast, 7 days of cocaine alone did not increase the acetylation of histones H3 and H4 in the striatum.19

Is the hyperacetylation produced by nicotine the result of the activation of one or more acetylases or the inhibition of deacetylases? To address this question, we assayed histone deacetylase (HDAC) activity directly in the nuclear fraction of striatum cells and found a 28% reduction in mice given nicotine for 7 to 10 days; by contrast, the mice given cocaine for 7 days had no decrease in HDAC activity.19 The increased histone acetylation in mice given nicotine seemed to result from reduced HDAC activity in the striatum.

The finding that nicotine inhibited HDAC activity in the striatum, thus inducing global changes in histone acetylation in the nucleus accumbens — changes that are known to alter the transcription of genes other than FosB when cocaine is administered — suggested that nicotine enhanced the transcription of FosB in response to cocaine. As an independent test of the finding that nicotine produces its effect on cocaine responses by inhibiting HDAC activity, we simulated the effect of nicotine using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA),19 which enhances the response to cocaine in conditioned place-preference experiments.21 We gave mice SAHA 2 hours before giving them cocaine and observed a 71% increase in FosB expression, as compared with mice given cocaine alone.19

We then asked whether substituting SAHA for nicotine would produce similar effects on cocaine-induced synaptic plasticity. We found that SAHA fully simulated nicotine, inducing a greater reduction in long-term potentiation in the core of the nucleus accumbens than cocaine alone. This is consistent with the idea that increased histone acetylation in the striatum is responsible for the reduction of long-term potentiation after 7 days of nicotine. Moreover, like nicotine alone (Figure 4B and 4C), SAHA alone did not cause a drop in long-term potentiation. Overall, the effects of SAHA and nicotine were quantitatively and qualitatively similar. However, although SAHA, like nicotine, enhances the behavioral effects of cocaine,21 its electrophysiological effects are unknown. These results support our experimental finding that nicotine inhibits HDAC activity.

Figure 5. Figure 5. Priming Effect of Nicotine on Cocaine-Induced Changes in Wild-Type Mice and Genetically Engineered Mice and in Wild-Type Mice Given Theophylline. Panels A and B show long-term potentiation in wild-type mice and in genetically engineered littermates with mutations in the CREB-binding protein CBP (CBP+/−), respectively. The mice were given saline as a control, nicotine for 7 days, a single injection of cocaine, or 7 days of nicotine followed by an injection of cocaine (5 to 8 mice per group). Panel C shows changes in the long-term potentiation amplitude 180 minutes after HFS in the same groups of mice. Panel D shows histone H4 (K5 to K16) acetylation in the tail domain of histone proteins in striatal lysates of CBP+/− mice and wild-type littermates after 7 days of nicotine (4 mice per group). In Panels D and F, values are normalized protein levels, with β-tubulin as a loading control. The Western raw data are produced by measuring the optical densities (ODs) of the different bands. These values are then transformed by first normalizing with β-tubulin as a loading control and then dividing by the values of the control group. Panel E shows changes in the long-term potentiation amplitude 180 minutes after HFS in mice treated with theophylline for 7 days, mice treated with theophylline followed by a single cocaine injection, mice treated with cocaine alone, and controls (6 to 10 mice in each group). Panel F shows graphs of immunoblots of striatal lysates from mice given saline or theophylline (200 mg per liter) in their drinking water for 7 days and then probed with antibodies against acetylated histone H3 (K9) and acetylated histone H4 (K5 to K16). Data are from Levine et al.19

To test further the idea that histone acetylation and deacetylation are key molecular mechanisms of the effect of nicotine on the murine response to cocaine, we conducted genetic and pharmacologic experiments. We studied genetically modified mice with the Rubinstein–Taybi syndrome that lack one functional allele of the gene for the CREB binding protein (CBP) governing histone acetylation. The lack of this allele results in hypoacetylation (abnormally low histone acetylation) in the striatum. The mutant mice had impaired long-term potentiation, as compared with nonengineered (wild-type) controls (Figure 5A and 5B). After being given nicotine for 7 days, these mice had reduced long-term potentiation in response to cocaine (Figure 5C). Using immunoblots, we found that the mutant mice had roughly a 49% reduction in histone H4 acetylation in the striatum, as compared with the control mice. After 7 days of nicotine, histone H4 acetylation in the mutant mice had increased to values that were similar to those in wild-type mice exposed to nicotine for 7 days (Figure 5D).

We hypothesized that hypoacetylation would weaken the effect of cocaine on wild-type mice and produce the opposite effect of SAHA and nicotine. To spur HDAC activity and create a hypoacetylated state, we gave mice low doses of theophylline, an HDAC stimulator. After 7 days, there was no difference in long-term potentiation between mice given theophylline and control mice given plain water: the two groups had a similar increase in long-term potentiation. However, in the mice given theophylline, long-term potentiation did not decrease as much in response to cocaine as it did in the controls (Figure 5E and 5F). Moreover, the mice given theophylline had less acetylated histone H4 (Figure 5E); specifically, less K12-acetylated H4 and K16-acetylated H4 (Figure 5F).

Figure 6. Figure 6. Proposed Model of Nicotine as a Gateway Drug. Panel A shows the action of cocaine alone. Cocaine leads to the activation of PKA, which phosphorylates CREB-1 and leads to the recruitment of the CREB-binding protein CBP, which acetylates histone H4. CRE denotes cAMP responsive element, mRNA messenger RNA, Pol polymerase, and TBP TATA-box–binding protein. Panel B shows that the action of cocaine alone is rapidly reversed by histone deacetylase (HDAC) activity. Panel C shows that nicotine inhibits HDAC activity, thus increasing histone acetylation throughout the striatum and creating an environment conducive to expression of FOSB.19

These data support the idea that a hypoacetylated state, whether caused genetically or pharmacologically, reduces FosB expression and the depression of long-term potentiation in response to cocaine. This is consistent with the earlier finding of Hiroi et al. that the inactivation of FosB lessened addictive behavior.22 Nicotine reduced HDAC activity, thereby increasing histone acetylation and creating an environment conducive to FosB expression. In this way, nicotine promotes greater FosB expression in response to cocaine than cocaine alone does. Moreover, this gene expression cannot be rapidly reversed, because HDAC activity is inhibited (Figure 6).

To investigate the duration of the priming effect of nicotine, we repeated some of our studies, with one variation: after giving the mice nicotine for 7 days, we waited 14 days before giving them cocaine. We found that the locomotor effect of cocaine was not enhanced — unlike the increase we observed when we gave the mice nicotine for 7 days and then gave them both nicotine and cocaine without a delay. Similarly, the depression of long-term potentiation and FosB expression in response to cocaine were the same in mice given nicotine 14 days previously and in mice given no nicotine. These findings indicate that the priming effect of nicotine does not occur unless nicotine is given repeatedly and in close conjunction with cocaine. We have not defined the duration of the priming effect and suspect that it is influenced by the intensity and duration of nicotine exposure.

Beyond the Striatum — Amygdala and Hippocampus

Given that nicotine enhanced the changes in synaptic plasticity in the striatum induced by cocaine, we next asked whether the gateway effect also applied to the amygdala, the region of the brain that orchestrates emotion and is critical for drug addiction. We found that nicotine enhanced long-term potentiation in the amygdala in response to cocaine and that the effect was unidirectional. Moreover, as in the striatum, SAHA simulated the priming effects of nicotine.23

Finally, we asked whether the dopamine D1/D5 receptor (which is important in reward reinforcement) and histone acetylation played a role in the dentate gyrus of the hippocampus, a brain area that is critical for spatial memory and thus for behaviors related to drug addiction that are commonly cued to the spatial context in which the addictive drug is acquired and consumed. We found that priming with nicotine substantially enhanced the long-term potentiation produced by cocaine in the dentate gyrus, and again, the priming effect was unidirectional (i.e., nicotine primed cocaine but cocaine did not prime nicotine). Moreover, the facilitation induced by nicotine and cocaine was blocked by some receptor antagonists that act on the D1/D5 receptor and enhanced by others. Finally, SAHA simulated the priming effect of nicotine but was blocked in the genetically modified mice that had reduced histone acetylation.24

These results extend the evidence that the priming effect of nicotine is achieved, at least partially, by means of histone acetylation and show that the amygdala and the hippocampus are important in processing the effects of nicotine and cocaine. If similar changes in chromatin acetylation and FOSB expression occur in people after nicotine exposure, and if the magnitude of the changes is sufficient to alter human addictive behavior, these experiments suggest new approaches to the treatment of addiction.

Animal Model–Based Predictions for Tests in Humans

Our findings that more than 1 day of nicotine exposure was required to prime cocaine responses in mice and that the first exposure to cocaine had to occur while the mice were being exposed to nicotine prompted us to return to human populations and ask the following questions: What is the smoking status of cocaine users when they start using cocaine? Does beginning cocaine use while actively smoking enhance the effects of cocaine and result in higher rates of cocaine addiction?

To address these questions, we reexamined existing data from a small group of students followed from 15.7 to 34.2 years of age.25 The majority of cocaine users (75.2%) were smoking during the month they began using cocaine. Furthermore, in a large, longitudinal national sample,26 we found that the rate of cocaine dependence (addiction as measured in the population) was highest (20.2%) among users who started using cocaine after having smoked cigarettes. Dependence was much lower among persons who had begun using cocaine before they started smoking (6.3%) and among those who had never smoked more than 100 cigarettes (10.2%) (P<0.001).