The ␣6 nicotinic acetylcholine receptor (nAChR) subunit is involved in nicotine-stimulated dopamine release in the striatum. It is expressed in brain regions and coexpressed with nAChR subtypes implicated in nicotine dependence behaviors; hence, this subunit may play a role in nicotine dependence. Using the ␣6-selective antagonist ␣-conotoxin H9A;L15A (MII[H9A; L15A]), we determined the role of ␣6* nAChRs in the pharmacological and behavioral effects of nicotine. We measured effects of pretreatment with MII[H9A;L15A] on analgesia, locomotion, and body temperature after a single injection of nicotine. Effects of MII[H9A;L15A] on nicotine reward were measured using the conditioned place preference (CPP) paradigm. We further measured physical (somatic signs and hyperalgesia) and affective [anxiety-related behavior and conditioned place aversion (CPA)] nicotine withdrawal behaviors after extended nicotine exposure. Results showed that MII[H9A;L15A] did not block acute nicotine effects on the behaviors measured. Conversely, MII[H9A:l15A] blocked the expression of nicotine CPP, as well as withdrawal-associated CPA and anxiety-related behavior in the elevated plus maze, but not withdrawalinduced somatic signs or hyperalgesia. These results suggest a role for the ␣6 nAChR subunit in nicotine reward and affective nicotine withdrawal but not acute nicotine-induced or physical withdrawal behaviors.

The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome.

Mesolimbic a6* nicotinic acetylcholine receptors (nAChRs) are thought to have an important role in nicotine behavioral effects. However, little is known about the role of the various a6*-nAChRs subtypes in the rewarding effects of nicotine. In this report, we investigated and compared the role of a6*-nAChRs subtypes and their neuro-anatomical locus in nicotine and cocaine reward-like effects in the conditioned place preference (CPP) paradigm, using pharmacological antagonism of a6b2* nAChRs and genetic deletion of the a6 or a4 subunits in mice. We found that a6 KO mice exhibited a rightward shift in the nicotine dose-response curve compared with WT littermates but that a4 KO failed to show nicotine preference, suggesting that a6a4b2*-nAChRs are involved. Furthermore, a6b2* nAChRs in nucleus accumbens were found to have an important role in nicotine-conditioned reward as the intra-accumbal injection of the selective a6b2* a-conotoxin MII [H9A; L15A], blocked nicotine CPP. In contrast to nicotine, a6 KO failed to condition to cocaine, but cocaine CPP in the a4 KO was preserved. Intriguingly, a-conotoxin MII [H9A; L15A], blocked cocaine conditioning in a4 KO mice, implicating a6b2* nAChRs in cocaine reward. Importantly, these effects did not generalize as a6 KO showed both a conditioned place aversion to lithium chloride as well as CPP to palatable food. Finally, dopamine uptake was not different between the a6 KO or WT mice. These data illustrate that the subjective rewarding effects of both nicotine and cocaine may be mediated by mesolimbic a6b2* nAChRs and that antagonists of these receptor subtypes may exhibit therapeutic potential.

Tobacco smoking during adolescence has become a prominent preventable health problem faced in the United States. Addictive properties of smoking are thought to have a pronounced effect at a young age, thereby increasing vulnerability to a life-long addiction and decreasing the likelihood of smoking cessation during adulthood. Learning and memory involvement in nicotine reward was assessed in early adolescent (PND 28–34) and adult (PND 70+) male ICR mice by conducting conditioning sessions of nicotine (0.5 mg/kg) acquisition at varying time-spans, and evaluating extinction and reinstatement of nicotine preference using Conditioned Place Preference. Acquisition studies resulted in a significant preference for nicotine after 3 days of conditioning for both age groups, but not after only 1 or 2 conditioning days. In the extinction study, adolescent mice exhibited preference for nicotine 72 h after the last conditioning session, whereas preference for nicotine was extinct in adult mice by 72 h. Reinstatement studies showed adolescent mice, but not adult mice, recovering nicotine preference after a priming injection of 0.1 mg/kg nicotine on day 9 after the mice underwent extinction. No significant differences were found when nAChRs were quantified in both early adolescent and adult mice using binding techniques including cytisine sensitive, α-conotoxin-MII sensitive, and α-bungarotoxin sensitive nAChRs. Levels of striatal dopamine release were measured in both age groups using a dopamine release assay over a range of nicotine doses, which also resulted in no significant differences. More sensitive assays may facilitate in understanding the mechanisms of nicotine reward in adolescent mice.

The influx of Ca2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs) leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB), which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (−/−) mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV −/− mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.