μ-Opioid Receptor and CREB Activation Are Required for Nicotine Reward

Neuron, Vol. 46, 933–943, June 16, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.neuron.2005.05.005

␮-Opioid Receptor and CREB Activation Are Required for Nicotine Reward Carrie L. Walters, Jessica N. Cleck, Yuo-chen Kuo, and Julie A. Blendy* Department of Pharmacology University of Pennsylvania Philadelphia, Pennsylvania 19104

Summary Environmental cues associated with nicotine delivery are an important part of the stimulus that sustains smoking behavior and is often coupled with craving and relapse; however, the neuronal circuitry and molecular substrates underlying this process are still poorly understood. Exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in MOR−/− mice. Moreover, a single administration of an opioid receptor antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. Lastly, repeated nicotine administration results in increased expression of MORs. However, this effect, along with rewarding properties of nicotine, is blocked in mice with a targeted disruption in the CREB gene. Together, pharmacologic and genetic manipulations indicate that phosphorylation of CREB and upregulation of functional MORs are required for nicotine-conditioned reward. Introduction Nicotine is believed to be the primary factor responsible for the addictive properties of cigarettes; however, the mechanism underlying its reinforcing effects remains elusive. Tobacco use is the leading preventable cause of death in the United States, with one in every five deaths attributed to smoking (Peto et al., 1992). Current therapeutic interventions for smoking cessation are inadequate (Vaszar et al., 2002); thus, insights into the molecular and genetic mechanisms that underlie the addictive properties of nicotine would prove useful in creating successful treatment programs for smoking cessation. Nicotine stimulates cholinergic receptors located in the ventral tegmental area (VTA) and nucleus accumbens (NAc), which are important neural substrates in the mesolimbic dopamine reward pathway (Dani and Heinemann, 1996). Several studies indicate that an increase in dopamine in the NAc is an integral component underlying the reinforcing properties of many drugs of abuse (Berridge and Robinson, 1998; Di Chiara and Imperato, 1979), including nicotine (Corrigall and Coen, 1991; Corrigall et al., 1994; Di Chiara, 2000). Specifically, studies in mice lacking the β2 subunit of *Correspondence: [email protected]

the neuronal nicotinic acetylcholine receptor show decreased nicotine-induced dopamine release in the ventral striatum and decreased nicotine self-administration (Picciotto et al., 1998). While the role of dopamine in mediating reinforcing properties of nicotine is fairly well established, less is known about the influence of other neurotransmitter systems. Recently, the role of endogenous opioids has been investigated in nicotine action. Nicotine has been shown to cause a release of endogenous opioids in the NAc of rat and striatum of mouse (Davenport et al., 1990; Dhatt et al., 1995; Houdi et al., 1991; Pierzchala et al., 1987; Pomerleau and Pomerleau, 1984), and interference with the endogenous opioid system has consequences for a number of nicotine-mediated responses. For example, the opioid receptor antagonist naloxone has been shown to block or attenuate nicotine-induced increases in food responding (Corrigall et al., 1988), nicotine-induced analgesia (Aceto et al., 1993), as well as withdrawal or aversion in nicotinedependent animals (Ise et al., 2000; Malin et al., 1993). Likewise, studies in the ␮-opioid receptor knockout mouse (MOR−/−) demonstrate that this receptor is required for nicotine-induced antinociception and rewarding effects of nicotine (Berrendero et al., 2002). In humans, smoking appears to be closely linked to opioid drug use (Mello et al., 1980), and endogenous opioid levels are increased following smoking (Pomerleau et al., 1983). Finally, humans that carry a ␮-opioid receptor (MOR) variant (Asn40Asp) are more likely to remain abstinent at the end of nicotine cessation treatment (Lerman et al., 2004). Taken together, these studies suggest that MORs have a critical role in mediating nicotine reward; therefore, smoking cessation therapies that target the MOR should be highly effective. The transcription factor CREB is thought to play a major role in the rewarding properties of many drugs of abuse. Several studies have correlated changes in CREB protein and activation with morphine administration (Guitart et al., 1992; Lane-Ladd et al., 1997; Windell et al., 1996), and mice that harbor a mutation in the CREB gene (CREBα⌬ mutant mice) (Blendy et al., 1996) do not find morphine reinforcing (Walters and Blendy, 2001). Chronic cocaine increases CREB phosphorylation in the NAc (Kano et al., 1995a; Terwilliger et al., 1991); however, viral-mediated overexpression of CREB in this region decreases the reinforcing properties of cocaine (Carlezon et al., 1998). Moreover, overexpression of a mutant form of CREB increased the reinforcing properties of cocaine (Carlezon et al., 1998). Similar effects are seen in CREBaD mutant mice, which are more responsive to the reinforcing effects of cocaine compared to wild-type littermates (Walters and Blendy, 2001). Few studies have examined the relationship between reinforcing properties of nicotine and CREB. Chronic nicotine administration in mice results in decreased CREB phosphorylation in the NAc but increased CREB phosphorylation in the prefrontal cortex, while nicotine withdrawal increases CREB phosphorylation in the VTA

Neuron 934

(Brunzell et al., 2003). In contrast, withdrawal from chronic nicotine in rats decreases CREB, phosphorylated CREB, and CRE-DNA binding in the cortex and amygdala (Pandey et al., 2001). Thus, the role of CREB in mediating the long-term plasticity that may underlie the development of nicotine addiction is poorly understood. Human imaging, clinical, and laboratory studies indicate that nicotine craving and smoking relapse can be triggered by environmental cues associated with smoking behavior (Brody et al., 2002; Childress et al., 1999). As smokers self-administer nicotine within the context of specific environmental stimuli, cues may not only influence the degree to which nicotine is self administered, but they may become as important to smoking behavior as nicotine itself (Caggiula et al., 2002). Therefore, the goals of the present studies were to determine what molecular mechanisms are activated during nicotine administration, if these mechanisms are similarly engaged during reexposure to environmental cues associated with nicotine delivery, and whether these mechanisms are necessary and sufficient to sustain the behavioral responses associated with nicotine reward. Results To investigate the mechanisms underlying nicotine reinforcement, we examined the activation of CREB via phosphorylation (pCREB). Twenty minutes following acute nicotine treatment, pCREB-positive cells are markedly increased in the VTA (Figure 1). To evaluate the effect of opioid receptor blockade on this response, we examined the levels of pCREB following acute nicotine in animals that had been pretreated with naloxone. Nicotine-induced increases in pCREB are blocked in the VTA with naloxone pretreatment (Figure 1). Naloxone has some affinity for all subtypes of opioid receptors. Therefore, to establish the specificity of this antagonist, we utilized MOR−/− mice and examined pCREB induction in the VTA after an acute injection of nicotine. In the wild-type animals, nicotine treatment resulted in an increase in pCREB-positive cells in the VTA (Figure 2) as seen previously; however, nicotineinduced increases in pCREB were absent in the MOR−/− mice (Figure 2). These data confirm that the ability of naloxone to attenuate nicotine-induced increases in pCREB occurs through the MOR. To examine the extent to which nicotine increases CREB phosphorylation throughout the brain, we measured pCREB levels in a number of brain regions associated with reward, dopamine signaling, and learning and conditioning. In addition to the VTA, pCREB levels were increased in the NAc, striatum, and pedunculopontine tegmental nucleus (PPT) following a single administration of nicotine (Figure 3). In contrast, pCREB levels were not affected in the substantia nigra, hippocampus, or cingulate cortex by acute nicotine administration (Figure 3). The rewarding or aversive effects of drugs can be evaluated through the use of conditioned place preference. During the course of the experiment, mice are confined to one side of a testing chamber or the other and receive nicotine (for details, see Experimental Pro-

cedures). On test day, animals are drug free and allowed to roam between the two sides. If a drug has been experienced as reinforcing, animals will spend considerably more time on the side of the chamber that was paired with the drug. To examine the effects of repeated nicotine administration on pCREB levels, we utilized the same number and frequency of nicotine injections used in the behavioral assessment of nicotine reward. Of interest, pCREB levels were increased only in the VTA and striatum 20 min following repeated nicotine (Figure 3). However, 24 hr following repeated nicotine administration there were no increases in pCREB levels in any brain regions examined (Figure 3). Although acute and repeated nicotine administration clearly increases CREB phosphorylation in some brain regions, it is not known whether exposure to an environment previously paired with nicotine is able to induce a similar effect. Therefore, we trained mice in a conditioned place preference paradigm with 1.0 mg/kg or 2.0 mg/kg nicotine. Lower doses of nicotine (0.1, 0.25, and 0.5 mg/kg) did not produce any significant preference or aversion (data not shown), and levels of pCREB were not evaluated following these doses. Levels of pCREB were examined immediately following exposure to the place preference chambers, 24 hr following the last nicotine administration. While levels of pCREB were not increased in animals that received nicotine in their home cage at this time, pCREB levels were significantly increased in the VTA, cingulate cortex, NAc, and PPT in animals that had prior exposure to nicotine in the conditioning boxes (Figure 3). The increase in the NAc and PPT may be due in part to the significant increase in pCREB levels in response to saline conditioning alone, while increases in VTA and cingulate cortex appear to be specific to exposure to the nicotine-conditioned environment (Figure 3). As acute nicotine administration induced increases in pCREB that were blocked by pretreatment with naloxone, the effects of naloxone on environment-induced increase in pCREB were examined as well. A single injection of naloxone administered 24 hr after the last nicotine injection blocked the conditioned increase in pCREB in both the VTA and NAc (Figures 4A and 4B). The behavioral significance of blocking CREB phosphorylation after exposure to an environment previously paired with nicotine was determined in conditioned place preference experiments. Animals in which pCREB was elevated showed significant preference to the nicotine-paired side of the conditioning chamber (Figure 4C). In contrast, at a higher dose (2.0 mg/kg), animals found nicotine aversive as indicated by the fact that animals spend significantly less time on the side of the chamber that was paired with nicotine. Of interest, a dose of 2.0 mg/kg of nicotine did not produce a change in pCREB levels in the VTA (data not shown). Pretreatment with naloxone on test day only blocked the reinforcing effects of nicotine at 1.0 mg/kg but not the aversive effects seen at 2.0 mg/kg. These data reveal that activation of CREB is associated with the conditioned behavioral reward expressed following nicotine place preference. Moreover, a single injection of naloxone on the test day was sufficient to attenuate this increase in environment-conditioned CREB phos-

␮-Opioid Receptor and CREB in Nicotine Reward 935

Figure 1. The Opioid Antagonist Naloxone Blocks the Nicotine-Induced Increase in phospho-CREB-Positive Cells in the VTA after an Acute Injection Animals were pretreated with an injection of either saline or naloxone (1 mg/kg s.c.) followed 5 min later by an injection of saline or nicotine (1 mg/kg i.p.) and then sacrificed after 15 min. (A) Saline pretreatment with saline treatment (black bar indicates 100 ␮m). (B) Naloxone pretreatment with saline treatment. (C) Saline pretreatment with nicotine treatment. (D) Naloxone pretreatment with nicotine treatment. (E) Stereotaxic atlas representation of the section corresponding to bregma −3.88 mm. Shaded square indicates the region of the high-power micrographs that surround the border of the VTA where the phospho-CREB-positive cells were counted. (F) Quantification of phospho-CREBpositive cells in the VTA. Open bars are saline-pretreated animals, and closed bars are naloxone-pretreated animals. *p < 0.05 from corresponding saline pretreatment group and corresponding nicotine-treated group [F(3, 20) = 97.646; ANOVA with a BonferroniDunn post hoc; n = 6 per group; mean counts ± SEM].

phorylation and to block the rewarding effects of nicotine. To examine the specificity of the effects of MOR blockade, including changes in pCREB and subsequent alterations in the expression of conditioned place

Figure 2. pCREB in the VTA Is Increased after an Acute Nicotine Injection in Wild-Type but Not ␮-Opioid Knockout Mice Mice were treated with nicotine (1 mg/kg) and perfused 20 min later, and brains were processed for pCREB immunohistochemistry. The x axis represents treatment, and the y axis represents the number of pCREB-positive cells counted in the VTA. The open bars are wild-type, and the closed bars are ␮-opioid receptor knockout mice. *p < 0.05 from wild-type saline group and mutant nicotine group [F(3, 12) = 11.427; ANOVA with a Bonferroni-Dunn post hoc; n = 4 per group; mean counts ± SEM].

preference, naloxone was administered on test day of a cocaine-conditioned place preference paradigm. Behavioral responses and changes in pCREB levels in the VTA and NAc were examined. In the VTA, there was no effect of cocaine-conditioned place preference on pCREB levels, nor was there an effect of naloxone pretreatment (Figure 5A). In contrast, there was an increase in pCREB levels in the NAc on test day, but unlike nicotine conditioning, this increase was not affected by pretreatment with naloxone (Figure 5B). Naloxone administration on test day did not block the expression of cocaine-conditioned place preference (Figure 5C). These data suggest that the effects of naloxone on pCREB levels as well as conditioned behavior are specific to nicotine and not generalized to cocaine. Pharmacologic tools, such as naloxone, allow us to manipulate the activity of CREB indirectly through modulation of the opioid receptor. To directly examine CREB activity, and the functional significance of these increases in pCREB, we utilized CREBaD mutant mice in conditioned place preference for nicotine. We previously found that the rewarding properties of morphine are completely absent in these mice (Walters and Blendy, 2001). This is not due to a general deficit in reward-mediated behavior in these animals, as both cocaine and food CPP are present in CREBaD mutant mice (Walters and Blendy, 2001; and unpublished data). Here, we demonstrate that the rewarding properties of nicotine at 1.0 mg/kg were absent in the CREBaD mu-

Neuron 936

Figure 3. Phospho-CREB-Positive Cell Counts in Several Brain Areas after Nicotine Treatment The y axis represents the number of pCREBpositive cells. The x axis represents various drug treatment paradigms. Home cage 1X represents single injection (i.p.) of saline or nicotine in the home cage, with the animal killed 20 min after injection. Home cage 4X (20 min) represents repeated injection (i.p.) of saline or nicotine, every other day in the home cage, for 8 days, with the animal killed 20 min after last injection. Home cage 4X (24 hr) represents repeated injection (i.p.) of saline or nicotine, every other day in the home cage, for 8 days, with the animal killed 24 hr after last injection. CPP box 4X (24 hr) represents repeated injection (i.p.) of saline or nicotine, every other day, in conditioning place preference boxes, for 8 days, with the animal killed 24 hr after last injection, immediately following exposure to conditioning boxes. *p < 0.05 from corresponding saline group, +p < 0.05 from corresponding home cage group. All statistics were done by ANOVA with a Fisher’s post hoc; n = 6–8 per group; mean counts ± SEM. [VTA, F(7, 41) = 85.996; NAc, F(7, 41) = 9.480; cingulate cortex, F(7, 33) = 1.133; substantia nigra, F(7, 33) = 0.724; striatum, F(7, 33) = 5.738; PPT: F(7, 33) = 9.285; hippocampus, F(7,41) = 4.907].

tant mice (Figure 6). In contrast, the aversive properties of nicotine, evident at 2.0 mg/kg, were not altered by CREB deficiency and were comparable to the response seen in wild-type controls. The present data demonstrate that a genetic deficiency (CREB mutation) and a pharmacological manipulation (MOR antagonism) have remarkably similar effects on rewarding properties of nicotine. However, one caveat of genetic models such as CREBaD mutant mice is that functional compensation can occur due to the absence of the gene product throughout development. Thus, putative CREB target genes, such as the MOR, which contains a CRE sequence in its promoter (Min et al., 1994), could be particularly susceptible to altered regulation in these mice. However, there was no difference in MOR mRNA levels in the VTA and NAc between wild-type and CREBaD mutant mice (Figure 7A), suggesting that constitutive loss of CREB had not altered any systems responsible for the basal regulation of the MOR gene in these areas. In contrast, repeated nicotine administration in the conditioned place preference paradigm (1.0 mg/kg, once every other day for 8 days) re-

sulted in an increase in MOR mRNA in the VTA in wildtype mice but not in CREBaD mutant mice (Figure 7A). This upregulation of MOR mRNA appears to be specific for nicotine, as MOR mRNA levels were not significantly different in wild-type or CREBaD mutant mice following cocaine administration. Furthermore, within the mesolimbic dopamine reward pathway, this increase in MOR mRNA was specific to the VTA, as levels were not altered in the NAc (Figure 7B). While cocaine administration induced changes in MOR mRNA in the NAc, this effect is equivalent in wild-type and CREBaD mutant mice. To determine if the nicotine-dependent changes in MOR mRNA levels are due to a direct activation of the MOR gene by CREB, we performed in vivo chromatin immunoprecipitation (ChIP) assays. ChIPs were performed using chromatin obtained from the VTA of saline- and nicotine-treated mice. To quantify the degree of MOR promoter enrichment, we performed real-time PCR of the immunoprecipitated material with primers surrounding the CRE. Chromatin precipitation with the CREB-specific antibody resulted in 1.8-fold enrichment

␮-Opioid Receptor and CREB in Nicotine Reward 937

Figure 4. Pretreatment with the Opioid Antagonist Naloxone on Test Day Blocked the Nicotine-Induced Increase in pCREB in the VTA and NAc and the Corresponding Reinforcing Effects in Wild-Type Mice (A) pCREB-positive cells in the VTA on test day of conditioned place preference. Open bars represent saline pretreatment on test day, and closed bars represent naloxone pretreatment on test day. *p < 0.05 from corresponding saline group and corresponding nicotine group [F(3, 20) = 97.646; ANOVA with a Bonferroni-Dunn post hoc; n = 4 per group; mean counts ± SEM]. (B) pCREB-positive cells in the NAc on test day of conditioned place preference. Open bars represent saline pretreatment on test day, and closed bars represent naloxone pretreatment on test day. *p < 0.05 from all groups [F(3, 20) = 5.539; ANOVA with a Bonferroni-Dunn post hoc; n = 4 per group; mean counts ± SEM]. (C) Nicotine-conditioned place preference with naloxone pretreatment on test day only. Open bars represent saline pretreatment, and dark bars represent pretreatment with naloxone. *p < 0.05 from corresponding saline-paired group. **p < 0.05 from corresponding naloxone pretreatment group [F(5, 30) = 17.884; ANOVA with a Bonferroni-Dunn post hoc; n = 6 per group; mean ± SEM].

of MOR promoter DNA from nicotine-treated VTA chromatin (Figure 7C), whereas minimal enrichment was obtained when a control IgG was used. In contrast, no enrichment occurred in saline-treated mice with either

Figure 5. Naloxone Pretreatment on Test Day Only Does Not Affect pCREB Induction in the VTA and NAc or Cocaine-Conditioned Place Preference (A) pCREB-positive cells in the VTA after test day of conditioned place preference. Open bars represent saline pretreatment on test day, and closed bars represent naloxone pretreatment on test day. There are no significant differences [F(3, 17) = 0.262; ANOVA with a Bonferroni-Dunn post hoc; n = 4–6 per group; mean counts ± SEM]. (B) pCREB-positive cells in the NAc after test day of conditioned place preference. Open bars represent saline pretreatment on test day, and closed bars represent naloxone pretreatment on test day. *p < 0.05 from corresponding saline-paired groups [F(3, 17) = 5.499; ANOVA with a Bonferroni-Dunn post hoc; n = 4–6 per group; mean counts ± SEM]. (C) Cocaine-conditioned place preference with naloxone pretreatment on test day only. Open bars represent saline pretreatment, and dark bars represent pretreatment with naloxone. *p < 0.05 from corresponding saline-paired group [F(5, 30) = 6.679; ANOVA with a Bonferroni-Dunn post hoc; n = 6 per group; mean ± SEM].

CREB- or IgG-immunoprecipitated material (Figure 7C). These data indicate that CREB does not interact in vivo with the MOR CRE under basal conditions but does so following nicotine stimulation. These findings are consistent with the mRNA expression data, which demonstrate similar levels of MOR in wild-type and CREBα⌬ mutant mice, but a lack of upregulation in these mu-

Neuron 938

Figure 6. CREBα⌬ Mutant Mice Do Not Find Nicotine Rewarding at 1.0 mg/kg Nicotine but Find It Aversive at 2.0 mg/kg Nicotine Open bars represent wild-type mice, and dark bars represent CREBα⌬ mutant mice. The y axis is expressed as time spent on nicotine-paired side minus time spent on unpaired side in seconds. *p < 0.05 from corresponding saline-paired group. **p < 0.05 from corresponding mutant group [F(5, 30) = 18.127; ANOVA with a Bonferroni-Dunn post hoc; n = 6 per group; mean ± SEM].

tants following nicotine administration. Taken together, these results demonstrate that CREB is required for nicotine-mediated MOR upregulation, but not basal gene transcription. Nicotine-mediated increases in MOR expression levels may be related to the behavioral manifestation of reward. To determine if blockade of the MOR prior to nicotine administration affects the upregulation of this receptor, we injected wild-type mice with naloxone prior to drug treatment using the same injection regimen as was used in the place conditioning paradigm for nicotine or cocaine. Saline-pretreated animals showed an increase in MOR in the VTA when administered nicotine; however, this increase was attenuated in animals pretreated with naloxone (Figure 8A). In the NAc, pretreatment with saline resulted in an increase in MOR after cocaine administration that was not affected by pretreatment with naloxone (Figure 8B). Discussion Drug-associated cues elicit conditioned neuronal activation in several brain regions detected by the expression of the immediate-early gene c-fos (Brown et al., 1992; Franklin and Druhan, 2000; Schroeder et al., 2001; Schroeder et al., 2000; Schroeder and Kelley, 2002). Specifically, nicotine-associated sensory cues have been shown to elicit an increase in Fos expression in prefrontal cortical and limbic regions (Schroeder et al., 2001). Clinical studies indicate that exposure to visual imagery of people smoking in various situations or cues related to smoking, such as cigarettes or cigarette-related items, can reliably induce smoking urges in the laboratory (Morgan et al., 1999; Mucha et al., 1999) and are accompanied by altered glucose metabolism in brain regions associated with arousal, sensory integration, compulsive repetitive behaviors, and memory (Brody et al., 2002). The present results demonstrate that nicotine-associated environmental stimuli

Figure 7. CREB Binds Directly to ␮-Opioid Receptor Promoter and Regulates mRNA Levels (A) CREBα⌬ mutant mice do not show an increase in ␮-opioid receptor (MOR) mRNA in the VTA after nicotine treatment. MOR mRNA expression in the VTA is increased after nicotine treatment but not cocaine treatment in wild-type animals only. Mice were given a saline, nicotine (1 mg/kg), or cocaine (10 mg/kg) injection once a day every other day for a total of 8 days (four injections). Twenty-four hours after the last injection, mice were sacrificed, the VTA was dissected out, and QPCR was performed for the MOR. The x axis represents the treatment, and the y axis is the ratio of MOR concentration to the housekeeping gene HPRT concentration. Open bars represent wild-type animals, and closed bars represent CREBα⌬ mutant animals. *p < 0.05 from wild-type groups and mutant nicotine group [F(5, 34) = 1.909; ANOVA with a Bonferroni-Dunn post hoc; n = 5–9 per group; mean ± SEM]. (B) MOR mRNA expression in the NAc is not changed after nicotine injections but is increased in wild-type and CREBα⌬ mutant animals after cocaine treatment. Animals were treated as described above, only NAc was dissected and PCR was performed on this region. *p < 0.05 from corresponding saline groups [F(5, 18) = 3.612; ANOVA with a Bonferroni-Dunn post hoc; n = 4 per group; mean ± SEM]. (C) (Top) CREB is bound to the CRE element in the promoter of the MOR 20 min following an acute injection of 1 mg/kg nicotine, but not saline. Chromatin was immunoprecipitated with an antibody specific to CREB or a nonrelevant IgG. After purification of the DNA from the ChIP material, a fragment of the MOR promoter spanning the CRE was amplified by PCR. PCR products were then visualized on an ethidium bromide-stained agarose gel. The IgG control shows no binding, confirming the specificity of the assay. The 28S ribosomal RNA loci were used as a loading control along with input genomic DNA as a positive control for the PCR conditions. (Bottom) The CRE site at the MOR promoter was enriched in mice that received nicotine, but not saline. Fold enrichment was obtained from QPCR using the loci encoding the 28S rRNAs as the control. The x axis represents treatment, and the y axis represents the fold change. Open bars represent saline treatment, and dark bars represent nicotine treatment. A fold change of 1 indicates no enrichment, whereas fold changes greater than 1 indicate enrichment.

␮-Opioid Receptor and CREB in Nicotine Reward 939

Figure 8. Pretreatment with Naloxone Blocks the Nicotine-Induced Increase in ␮-Opioid Receptor mRNA in the VTA (A) ␮-opioid receptor mRNA expression in the VTA is increased after nicotine treatment but not in animals pretreated with naloxone. Mice were given an injection of naloxone (1 mg/kg s.c.) and 5 min later were treated with saline, nicotine (1 mg/kg), or cocaine (10 mg/kg) injection once a day every other day for a total of 8 days (four injections). Twenty-four hours after the last injection, mice were sacrificed, the VTA was dissected out, and QPCR was performed for the ␮-opioid receptor. The x axis represents the treatment, and the y axis is the ratio of ␮-opioid receptor concentration to the housekeeping gene HPRT concentration. Open bars represent wild-type animals, and closed bars represent CREBα⌬ mutant animals. *p < 0.05 from wild-type groups and mutant nicotine group [F(5, 19) = 3.763; ANOVA with a Bonferroni-Dunn post hoc; n = 5–9 per group; mean ± SEM]. (B) ␮-opioid receptor mRNA expression in the NAc is increased in wild-type mice after cocaine treatment and is not affected after pretreatment with naloxone. Animals were treated as described above, only NAc was dissected and PCR was performed on this region. *p < 0.05 from corresponding saline groups [F(5, 18) = 3.601; ANOVA with a Bonferroni-Dunn post hoc; n = 4 per group; mean ± SEM].

can activate the same signal transduction molecules as the drug itself. An increase in pCREB is evident not only after acute and repeated nicotine administration, but also following exposure to an environment in which the animal has previously received nicotine. The ability of naloxone to block both the conditioned increase in pCREB as well as behavioral manifestation of reward suggests that activation of an endogenous opioid receptor is necessary for these effects. Indeed, nicotine-stimulated increases in dopamine in the NAc are dependent upon the activity of MORs located in the VTA (Tanda and Di Chiara, 1998). Furthermore, previous results indicate that nicotine is not rewarding in mice

lacking MORs (Berrendero et al., 2002), and the present data demonstrate that nicotine-mediated increases in pCREB are also absent in these mice. The present series of results extend these finding and demonstrate that the MOR is required to sustain rewarding properties of nicotine during reexposure to environmental cues associated with this drug. The similarity between effects of a mutation in the Creb gene and pharmacologic manipulation of the endogenous opioid system on behavioral responses to nicotine is striking. For example, the effects of naloxone administered on test day parallel those of the CREB deficiency, in that both are capable of blocking reinforcing, but not aversive properties of nicotine (compare Figures 4C and 6; nicotine at 2.0 mg/kg). In addition, a dose of nicotine that elicits reward is accompanied by increases of pCREB, which are blocked by naloxone, while a dose of nicotine that is aversive does not alter pCREB levels and is not attenuated by naloxone. Previous studies have suggested that alternate neuroanatomical pathways are responsible for mediating the reinforcing and aversive properties of nicotine (Laviolette and van der Kooy, 2003a; Laviolette and van der Kooy, 2003b), and these results confirm a molecular distinction between reward and aversion. The results presented above suggest that activation of both the endogenous opioid system and CREB are critical for the expression of conditioned nicotine reward; however, the question remains as to how specific this mechanism is for nicotine. Cocaine shares many of the downstream effects of nicotine and produces at least some of its acute reinforcing properties via actions on the mesolimbic dopamine system (Nestler, 1994). In addition, administration of cocaine has been shown to induce CREB phosphorylation (Kano et al., 1995b; Konradi et al., 1994). However, our data indicate that the mechanism of conditioned drug reward for cocaine does not rely on the endogenous opioid system. Cocaine-conditioned place preference was unaffected by pretreatment with naloxone on test day. Furthermore, pretreatment with naloxone on pairing days does not prevent the development of conditioned place preference for cocaine, as was the case for nicotine (data not shown). In addition, drug-mediated increases in pCREB, which in this case occur primarily in the NAc rather than the VTA, are not affected by naloxone. Lastly, CREB-deficient mice show increases rather than reductions in cocaine-conditioned place preference (Walters and Blendy, 2001). Thus, a strong correlation has been established between a molecular response, activation of CREB, and a behavioral response, nicotine-conditioned reward. While this correlation is specific for nicotine and not cocaine, further studies with additional drugs of abuse will be necessary to determine the extent of this paradigm. Mechanisms underlying the nicotine-opioid interaction are not well characterized. Previous studies demonstrate that chronic nicotine infusions result in a downregulation of nicotinic receptor function (Marks et al., 1993); however, some of the chronic effects of nicotine may be attributed to effects on opioid receptor levels and/or signaling as well. Chronic administration of nicotine has been shown to upregulate MORs in the striatum (Wewers et al., 1999). This upregulation could

Neuron 940

be due to increased release of endogenous opioid peptides (Davenport et al., 1990) and subsequent compensatory mechanisms of postsynaptic receptors. A putative downstream target of CREB activation is the MOR. DNA sequences in the promoter of the MOR gene contain cAMP response elements that are potential CREB binding sites (Min et al., 1994). However, the presence of the CRE sequence in the promoter or enhancer of a gene alone is not sufficient to characterize it as a direct target of CREB, nor does it predict that CREB mediates its regulation. There are no differences in basal levels of MOR mRNA expression between wildtype and CREBα⌬ mutant mice that could account for the difference in nicotine reward in these animals. This is consistent with the role of CREB in activity-dependent, and not basal regulation of gene expression and confirmed by our in vivo ChIP data demonstrating that CREB is not bound to the MOR promoter in basal conditions. After nicotine treatment, CREB occupies the MOR promoter, and MOR mRNA expression is increased in wild-type animals. These data demonstrate that CREB binding in vivo is regulated by nicotine. Nicotine administration causes a release of endogenous opioids in the NAc of rat and striatum of mouse (Davenport et al., 1990; Dhatt et al., 1995; Houdi et al., 1991; Pierzchala et al., 1987; Pomerleau and Pomerleau, 1984). Hence, it is possible that this increase in endogenous opioids after repeated nicotine treatment is responsible for the subsequent compensatory mechanisms of increased MOR mRNA in the VTA. We hypothesize that this upregulation is critical for conditioned reward behavior. Support for this hypothesis comes from the fact that MOR expression is not increased in CREBα⌬ mutant mice following nicotine treatment, and nicotine reward is absent in these mice. Furthermore, pretreatment with naloxone blocks the nicotine-induced increase in MOR in the VTA with no effect on the cocaine-induced increase in MOR in the NAc. This lack of upregulation of MOR mRNA could be an underlying mechanism for the deficit observed in nicotine reward in the CREBα⌬ mutant mice as well as in mice treated with naloxone; however, additional experiments are required to further elucidate the exact mechanism by which this occurs. The role of the opioid system in mediating rewarding properties of nicotine is poorly understood. In humans, smoking appears to be closely linked to opioid drug use (Mello et al., 1980), and endogenous opioid levels are increased following smoking, paralleling increases in plasma nicotine levels (Pomerleau et al., 1983). However, clinical studies examining the efficacy of opioid receptor antagonists to block cigarette craving so far have produced mixed results ranging from ineffectiveness at smoking cessation to mild reductions in the desire to smoke (Covey et al., 1999; King and Meyer, 2000; Wong et al., 1999). In our studies, we trained animals to develop a preference for nicotine and were able to block that preference with naloxone when animals were reexposed to the environment associated with nicotine, but in the absence of the drug itself. These data suggest that the timing and context of opioid receptor antagonist administration are critical for determining the effectiveness of blocking nicotine reward. Studies reporting a negative effect of opioid antago-

nists on smoking behavior in humans have administered opioid antagonists either chronically (Wong et al., 1999) or 1 (Nemeth-Coslett and Griffiths, 1986) to 24 hr (Sutherland et al., 1995) prior to smoking a cigarette, and subsequent smoking behavior was evaluated in a hospital or laboratory setting, clearly not environments associated with cigarette smoking. Given the results reported here, clinical studies designed to evaluate administration of opioid antagonists just prior to cues associated with smoking could lead to a more promising treatment regimen. Experimental Procedures Subjects CREBaD mice were generated as previously described (Blendy et al., 1996; Hummler et al., 1994) and are maintained as F1 hybrids of 129SvEvTac:C57/BL/6. The parental strains for this hybrid line have been backcrossed with vendor-supplied wild-type strains for several generations. For all experiments, mutants and wildtype controls are obtained from crossing heterozygote CREBaD 129SvEvTac N10 with heterozygote CREBaD C57BL/6 N12. This breeding scheme allows us to rigorously control for a uniform genetic background of experimental animals over time. MOR knockout mice and wild-type control mice were maintained in a C57BL/6 background. MOR knockout mice were generously supplied by Dr. John Pintar (Rutgers University). pCREB Immunohistochemistry For acute studies, wild-type mice were given an injection of either saline or naloxone (1.0 mg/kg s.c.; Sigma, St. Louis, MO) followed 5 min later by a single injection of saline or nicotine (1.0 mg/kg i.p.; nicotine hydrogen tartrate salt; Sigma, St. Louis, MO). Twenty minutes after this injection, mice were anesthetized and perfused with 4% paraformaldehyde. Brains were removed and sectioned coronally on a cryostat at 40 ␮M. Processing for pCREB-positive cells was performed as previously described (Walters et al., 2003). Briefly, sections were blocked in BSA and incubated in primary phospho-CREB antibody (1:1000; Upstate Biotechnologies, Lake Placid, NY) for two nights at 4°C. Sections were then incubated in biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) followed by Avidin-Biotin Complex (1:500; Vector Laboratories, Burlingame, CA). Bound antibody was visualized through incubation in 0.02% 3,3#-diaminobenzidine (Sigma, St. Louis, MO) with 0.02% hydrogen peroxide for 5 min. Slices were mounted, dehydrated, and cover-slipped with Permount mounting medium. Brain regions counted were identified by their approximate stereotaxic coordinates (Franklin and Paxinos, 1997), which are as follows: VTA, bregma −3.64; NAc, bregma 1.10; cingulated cortex, bregma 1.10; substantia nigra, bregma −3.64; striatum, bregma 1.10; PPT, bregma −4.36; and hippocampus, bregma −1.70. For specific atlas identification of regions counted, see Figure S1 in the Supplemental Data available with this article online. Quantification of pCREB-immunolabeled nuclear profiles was performed using Image-Pro Plus (Media Cybernetics Inc., Silver Spring, MD) by an individual blind to the treatments. The appropriate area was digitally imaged, and pCREB staining was subject to a threshold and counted. Profiles were counted in representative serial sections with comparable sections chosen for comparison between drug and saline groups. For place conditioning studies, place conditioning was performed as described below. Immediately following the test, mice were removed from the cages, anesthetized, and perfused with 4% paraformaldehyde. Brains were then processed for pCREB as described previously (Walters et al., 2003). Place Conditioning Place conditioning boxes consist of two distinct sides (20 cm × 20 cm × 20 cm). A partition separates the two sides with an opening that allows access to either side of the chamber, and this partition can be closed off for pairing days.

␮-Opioid Receptor and CREB in Nicotine Reward 941

Preconditioning Phase On day 1, animals were placed in the boxes and allowed to roam freely from side to side for 15 min, and time spent in each side was recorded. These data were used to separate the animals into groups of approximately equal bias. Conditioning Phase Animals were paired for 30 min with the saline group receiving saline in both sides of the boxes and drug groups receiving nicotine at 1.0 mg/kg or 2.0 mg/kg (nicotine hydrogen tartrate salt; Sigma, St. Louis, MO) or cocaine at 10.0 mg/kg (NIDA Drug Supply, Research Triangle Park, NC) on one of the sides and saline on the opposite side. Drug-paired sides were randomized among all groups. Conditioning lasted for 8 days, with animals in the drug group receiving drug every other day. Test Phase On the test day, animals were all given a saline injection and allowed to roam freely between the two sides. Time spent on each side was recorded, and data are expressed as time spent on drugpaired side minus time spent on saline-paired side. A positive number indicates a preference for the drug-paired side, while a negative number indicates an aversion to the drug-paired side. A number at or near zero indicates no preference for either side. For studies with naloxone pretreatment, place conditioning was performed as described previously. Animals received saline or naloxone (1.0 mg/kg s.c.) 5 min prior to saline injection on the test day only. Real-Time Quantitative PCR Animals were sacrificed during the light phase of the light/dark cycle. Following cervical dislocation, brains were dissected for RNA preparation. RNA was isolated using guanidine isothiocyanate (Chomczynski and Sacchi, 1987). The quality of the RNA samples was determined by ethidium bromide staining of 18S and 28S rRNAs following fractionation on denaturing agarose gels. Highquality total RNA samples from the VTA and NAc of individual animals were reverse transcribed using 1 ␮g Oligo (dT) primer, Superscript II Reverse Transcriptase, and accompanying reagents (Invitrogen) at 42°C for 1 hr. VTA and NAc cDNA was used at a 1:3 dilution in water in subsequent real-time PCR reactions. PCR reaction mixes were assembled using the Brilliant SYBR Green QPCR Master Mix (Stratagene), 10 ␮M primers, and the included reference dye at a 1:200 dilution according to the manufacturer’s instructions, except that the total reaction volume was scaled down from 50 ␮l to 25 ␮l. Reactions were performed using the SYBR Green (with Dissociation Curve) program on the Prism7000 Multiplex Quantitative PCR System (ABI). Cycling parameters were 95°C for 10 min and then 40 cycles of 95°C (30 s), 58°C or 60°C (1 min), and 72°C (30 s) followed by a melting curve analysis. All reactions were performed in triplicate with reference dye normalization, and the median cycle threshold value was used for analysis. Primer sequences are available upon request. ChIP Twenty minutes following an acute injection of saline or nicotine (1.0 mg/kg i.p.; nicotine hydrogen tartrate salt; Sigma, St. Louis, MO), wild-type mice were killed, the brains were removed, and the VTA was dissected out and immediately flash frozen in liquid nitrogen. Following dissection, four macrodissected VTAs were pooled, minced in cold PBS, and then crosslinked using 1% formaldehyde/ PBS for 10 min. The crosslinking was quenched by the addition of glycine to a final concentration of 0.125 M. The samples were washed once with cold PBS and Dounce homogenized in ChIP lysis buffer (10 mM NaCl/3 mM MgCl2/0.5% NP-40/10 mM Tris-HCl [pH 8.1] plus protease inhibitors). After 5 min on ice, nuclei were sedimented by centrifugation. The pellet was then resuspended in nuclear lysis buffer (1% SDS/5 mM EDTA [pH 8.0]/50 mM Tris-HCl [pH 8.1]/protease inhibitors). Following a 5 min incubation on ice, the lysate was sonicated (Sonic Dismembrator Model 100-Fisher) using three pulses for 20 s at 4–6 W. Insoluble debris was removed by centrifugation, and the supernatant was collected and flash frozen in liquid nitrogen. An input fraction was generated by uncrosslinking nonimmunoprecipitated chromatin material. Appropriate frag-

mentation of the chromatin was assessed by agarose gel electrophoresis, with a goal of DNA fragments of approximately 300– 700 bp in length. Chromatin DNA concentrations were calculated by measuring A260 on a Nanodrop ND-1000 (Nanodrop). Two micrograms of chromatin was used for each immunoprecipitation. The chromatin was precleared by incubating with protein-G agarose at 4°C for 1 hr. Ten micrograms of antiserum raised against CREB (Upstate) or 2 ␮g of preimmune IgG (Santa Cruz) was added, and the samples were rotated at 4°C overnight. Immunoprecipitates were isolated by incubating with blocked protein-G agarose and washed at room temperature with the following buffers: lowsalt wash buffer (0.1% SDS/1% Triton X-100/2 mM EDTA/20 mM Tris-HCl [pH 8.1]/150 mM NaCl), high-salt wash buffer (0.1% SDS/ 1% Triton X-100/2 mM EDTA/20 mM Tris-HCl [pH 8.1]/500 mM NaCl), LiCl wash buffer (0.25 M LiCl/1% Nonidet P-40/1% deoxycholate/1 mM EDTA/10 mM Tris-HCl [pH 8.1]) and 1× TE buffer. Chromatin was eluted from the antibody by incubation for 10 min at room temperature with elution buffer (0.1 M NaHCO3/1% SDS). Chromatin was uncrosslinked by adding NaCl to 0.2 M and incubating at 65°C for at least 4 hr. Samples were then digested with 40 ng of proteinase K and DNA isolated by Quiquick columns (Qiagen). Promoter enrichment was visualized on ethidium bromide-stained agarose gels and quantified by real-time quantitative PCR. Promoter enrichment was calculated by comparing the difference in abundance of the control DNA sequences (28S rRNA loci) to the promoter sequence of interest (MOR) in genomic DNA (input) to the immunoprecipitated DNA. The loci encoding the 28S ribosomal RNA were used as our normalization sequence, as these sequences are not bound by CREB. Primer sequences are available upon request.

Supplemental Data One supplemental figure can be found with this article online at http://www.neuron.org/cgi/content/full/46/6/933/DC1/. Acknowledgments This work was supported in part by National Institute on Drug Abuse grants DA-11649-01A2 (J.A.B.) and 1F31 DA 015949 (C.L.W.) and a TTURC grant from the National Cancer Institute and National Institute on Drug Abuse (P5084718). Received: May 17, 2004 Revised: January 14, 2005 Accepted: May 2, 2005 Published: June 15, 2005 References Aceto, M.D., Scates, S.M., Ji, Z., and Bowman, E.R. (1993). Nicotine’s opioid and anti-opioid interactions: proposed role in smoking behavior. Eur. J. Pharmacol. 248, 333–335. Berrendero, F., Kieffer, B.L., and Maldonado, R. (2002). Attenuation of nicotine-induced antinociception, rewarding effects, and dependence in ␮-opioid receptor knock-out mice. J. Neurosci. 22, 10935–10940. Berridge, K.C., and Robinson, T.E. (1998). What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res. Brain Res. Rev. 28, 309–369. Blendy, J.A., Kaestner, K.H., Schmid, W., Gass, P., and Schutz, G. (1996). Targeting of the CREB gene leads to up-regulation of a novel CREB mRNA isoform. EMBO J. 15, 1098–1106. Brody, A.L., Mandelkern, M.A., London, E.D., Childress, A.R., Lee, G.S., Bota, R.G., Ho, M.L., Saxena, S., Baxter, L.R., Jr., Madsen, D., and Jarvik, M.E. (2002). Brain metabolic changes during cigarette craving. Arch. Gen. Psychiatry 59, 1162–1172. Brown, E.E., Robertson, G.S., and Fibiger, H.C. (1992). Evidence for conditional neuronal activation following exposure to a cocainepaired environment: role of forebrain limbic structures. J. Neurosci. 12, 4112–4121.

Neuron 942

Brunzell, D.H., Russell, D.S., and Picciotto, M.R. (2003). In vivo nicotine treatment regulates mesocorticolimbic CREB and ERK signaling in C57Bl/6J mice. J. Neurochem. 84, 1431–1441. Caggiula, A.R., Donny, E.C., Chaudhri, N., Perkins, K.A., EvansMartin, F.F., and Sved, A.F. (2002). Importance of nonpharmacological factors in nicotine self-administration. Physiol. Behav. 77, 683– 687.

Kano, T., Suzuki, Y., Shibuya, M., Kiuchi, K., and Hagiwara, M. (1995b). Cocaine-induced CREB phosphorylation and cFos expression are suppressed in Parkinsonism model mice. Neuroreport 6, 2197–2200. King, A.C., and Meyer, P.J. (2000). Naltrexone alteration of acute smoking response in nicotine-dependent subjects. Pharmacol. Biochem. Behav. 66, 563–572.

Carlezon, W.A., Jr., Thome, J., Olson, V.G., Lane-Ladd, S.B., Brodkin, E.S., Hiroi, N., Duman, R.S., Neve, R.L., and Nestler, E.J. (1998). Regulation of cocaine reward by CREB. Science 282, 2272–2275.

Konradi, C., Cole, R.L., Heckers, S., and Hyman, S.E. (1994). Amphetamine regulates gene expression in rat striatum via transcription factor CREB. J. Neurosci. 14, 5623–5634.

Childress, A.R., Mozley, P.D., McElgin, W., Fitzgerald, J., Reivich, M., and O’Brien, C.P. (1999). Limbic activation during cue-induced cocaine craving. Am. J. Psychiatry 156, 11–18.

Lane-Ladd, S.B., Pineda, J., Boundy, V.A., Pfeuffer, T., Krupinski, J., Aghajanian, G.K., and Nestler, E.J. (1997). CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence. J. Neurosci. 17, 7890–7901.

Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Corrigall, W.A., and Coen, K.M. (1991). Selective dopamine antagonists reduce nicotine self-administration. Psychopharmacology (Berl.) 104, 171–176. Corrigall, W.A., Herling, S., and Coen, K.M. (1988). Evidence for opioid mechanisms in the behavioral effects of nicotine. Psychopharmacology (Berl.) 96, 29–35. Corrigall, W.A., Coen, K.M., and Adamson, K.L. (1994). Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area. Brain Res. 653, 278–284. Covey, L.S., Glassman, A.H., and Stetner, F. (1999). Naltrexone effects on short-term and long-term smoking cessation. J. Addict. Dis. 18, 31–40. Dani, J.A., and Heinemann, S. (1996). Molecular and cellular aspects of nicotine abuse. Neuron 16, 905–908. Davenport, K.E., Houdi, A.A., and Van Loon, G.R. (1990). Nicotine protects against mu-opioid receptor antagonism by beta-funaltrexamine: evidence for nicotine-induced release of endogenous opioids in brain. Neurosci. Lett. 113, 40–46. Dhatt, R.K., Gudehithlu, K.P., Wemlinger, T.A., Tejwani, G.A., Neff, N.H., and Hadjiconstantinou, M. (1995). Preproenkephalin mRNA and methionine-enkephalin content are increased in mouse striatum after treatment with nicotine. J. Neurochem. 64, 1878–1883. Di Chiara, G. (2000). Role of dopamine in the behavioural actions of nicotine related to addiction. Eur. J. Pharmacol. 393, 295–314. Di Chiara, G., and Imperato, A. (1979). Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl. Acad. Sci. USA 85, 5274–5278. Franklin, T.R., and Druhan, J.P. (2000). Expression of Fos-related antigens in the nucleus accumbens and associated regions following exposure to a cocaine-paired environment. Eur. J. Neurosci. 12, 2097–2106. Franklin, K.B.J., and Paxinos, G. (1997). The Mouse Brain in Stereotaxic Coordinates (San Diego, CA: Academic Press). Guitart, X., Thompson, M.A., Mirante, C.K., Greenberg, M.E., and Nestler, E.J. (1992). Regulation of cyclic AMP response elementbinding protein (CREB) Phosphorylation by acute and chronic morphine in the rat locus coeruleus. J. Neurochem. 58, 1168–1171.

Laviolette, S.R., and van der Kooy, D. (2003a). Blockade of mesolimbic dopamine transmission dramatically increases sensitivity to the rewarding effects of nicotine in the ventral tegmental area. Mol. Psychiatry 8, 50–59. Laviolette, S.R., and van der Kooy, D. (2003b). The motivational valence of nicotine in the rat ventral tegmental area is switched from rewarding to aversive following blockade of the α7-subunitcontaining nicotinic acetylcholine receptor. Psychopharmacology (Berl.) 166, 306–313. Lerman, C., Wileyto, E.P., Patterson, F., Rukstalis, M., AudrainMcGovern, J., Restine, S., Shields, P.G., Kaufmann, V., Redden, D., Benowitz, N., and Berrettini, W.H. (2004). The functional mu opioid receptor (OPRM1) Asn40Asp variant predicts short-term response to nicotine replacement therapy in a clinical trial. Pharmacogenomics J. 4, 184–192. Malin, D.H., Lake, J.R., Carter, V.A., Cunningham, J.S., and Wilson, O.B. (1993). Naloxone precipitates nicotine abstinence syndrome in the rat. Psychopharmacology (Berl.) 112, 339–342. Marks, M.J., Grady, S.R., and Collins, A.C. (1993). Downregulation of nicotinic receptor function after chronic nicotine infusion. J. Pharmacol. Exp. Ther. 266, 1268–1276. Mello, N.K., Mendelson, J.H., Sellers, M.L., and Kuehnle, J.C. (1980). Effects of heroin self-administration on cigarette smoking. Psychopharmacology (Berl.) 67, 45–52. Min, B.H., Augustin, L.B., Felsheim, R.F., Fuchs, J.A., and Loh, H.H. (1994). Genomic structure analysis of promoter sequence of a mouse ␮ opioid receptor gene. Proc. Natl. Acad. Sci. USA 91, 9081–9085. Morgan, M.J., Davies, G.M., and Willner, P. (1999). The Questionnaire of Smoking Urges is sensitive to abstinence and exposure to smoking-related cues. Behav. Pharmacol. 10, 619–626. Mucha, R.F., Geier, A., and Pauli, P. (1999). Modulation of craving by cues having differential overlap with pharmacological effect: evidence for cue approach in smokers and social drinkers. Psychopharmacology (Berl.) 147, 306–313. Nemeth-Coslett, R., and Griffiths, R.R. (1986). Naloxone does not affect cigarette smoking. Psychopharmacology (Berl.) 89, 261–264. Nestler, E.J. (1994). Hard target: Understanding dopaminergic neurotransmission. Cell 79, 923–926.

Houdi, A.A., Pierzchala, K., Marson, L., Palkovits, M., and Van Loon, G.R. (1991). Nicotine-induced alteration in Tyr-Gly-Gly and Metenkephalin in discrete brain nuclei reflects altered enkephalin neuron activity. Peptides 12, 161–166.

Pandey, S.C., Roy, A., Xu, T., and Mittal, N. (2001). Effects of protracted nicotine exposure and withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat brain. J. Neurochem. 77, 943–952.

Hummler, E., Cole, T.J., Blendy, J.A., Ganss, R., Aguzzi, A., Schmid, W., Beermann, F., and Schutz, G. (1994). Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Proc. Natl. Acad. Sci. USA 91, 5647–5651.

Peto, R., Lopez, A.D., Boreham, J., Thun, M., and Heath, C., Jr. (1992). Mortality from tobacco in developed countries: indirect estimation from national vital statistics. Lancet 339, 1268–1278.

Ise, Y., Narita, M., Nagase, H., and Suzuki, T. (2000). Modulation of opioidergic system on mecamylamine-precipitated nicotine-withdrawal aversion in rats. Psychopharmacology (Berl.) 151, 49–54. Kano, T., Suzuki, Y., Shibuya, M., Kiuchi, K., and Hagiwara, M. (1995a). Cocaine-induced CREB phosphorylation and c-Fos expression are suppressed in Parkinsonism model mice. Neuroreport 6, 2197–2200.

Picciotto, M.R., Zoli, M., Rimondini, R., Lena, C., Marubio, L.M., Pich, E.M., Fuxe, K., and Changeux, J.P. (1998). Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine. Nature 391, 173–177. Pierzchala, K., Houdi, A.A., and Van Loon, G.R. (1987). Nicotineinduced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin. Peptides 8, 1035–1043. Pomerleau, O.F., and Pomerleau, C.S. (1984). Neuroregulators and

␮-Opioid Receptor and CREB in Nicotine Reward 943

the reinforcement of smoking: towards a biobehavioral explanation. Neurosci. Biobehav. Rev. 8, 503–513. Pomerleau, O.F., Fertig, J.B., Seyler, L.E., and Jaffe, J. (1983). Neuroendocrine reactivity to nicotine in smokers. Psychopharmacology (Berl.) 81, 61–67. Schroeder, B.E., and Kelley, A.E. (2002). Conditioned Fos expression following morphine-paired contextual cue exposure is environment specific. Behav. Neurosci. 116, 727–732. Schroeder, B.E., Holahan, M.R., Landry, C.F., and Kelley, A.E. (2000). Morphine-associated environmental cues elicit conditioned gene expression. Synapse 37, 146–158. Schroeder, B.E., Binzak, J.M., and Kelley, A.E. (2001). A common profile of prefrontal cortical activation following exposure to nicotine- or chocolate-associated contextual cues. Neuroscience 105, 535–545. Sutherland, G., Stapleton, J.A., Russell, M.A., and Feyerabend, C. (1995). Naltrexone, smoking behaviour and cigarette withdrawal. Psychopharmacology (Berl.) 120, 418–425. Tanda, G., and Di Chiara, G. (1998). A dopamine-mu1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non-psychostimulant drugs of abuse. Eur. J. Neurosci. 10, 1179– 1187. Terwilliger, R.Z., Beitner-Johnson, D., Sevarino, K.A., Crain, S.M., and Nestler, E.J. (1991). A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function. Brain Res. 548, 100–110. Vaszar, L.T., Sarinas, P.S., and Lillington, G.A. (2002). Achieving tobacco cessation: current status, current problems, future possibilities. Respiration (Herrlisheim) 69, 381–384. Walters, C.L., and Blendy, J.A. (2001). Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse. J. Neurosci. 21, 9438– 9444. Walters, C.L., Kuo, Y.C., and Blendy, J.A. (2003). Differential distribution of CREB in the mesolimbic dopamine reward pathway. J. Neurochem. 87, 1237–1244. Wewers, M.E., Dhatt, R.K., Snively, T.A., and Tejwani, G.A. (1999). The effect of chronic administration of nicotine on antinociception, opioid receptor binding and met-enkephalin levels in rats. Brain Res. 822, 107–113. Windell, K.L., Self, D.W., Lane, S.B., Russell, D.S., Vaidya, V.A., Miserendino, M.J.D., Rubin, C.S., Duman, R.S., and Nestler, E.J. (1996). Regulation of CREB expression: in vivo evidence for a functional role in morphine action in the nucleus accumbens. J. Pharmacol. Exp. Ther. 276, 306–315. Wong, G.Y., Wolter, T.D., Croghan, G.A., Croghan, I.T., Offord, K.P., and Hurt, R.D. (1999). A randomized trial of naltrexone for smoking cessation. Addiction 94, 1227–1237.