Darlene H. Brunzell

The Role of α6-Containing Nicotinic Acetylcholine Receptors in Nicotine Reward and Withdrawal

The α6 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 α6-selective antagonist α-conotoxin H9A;L15A (MII[H9A;L15A]), we determined the role of α6* 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 withdrawal-induced somatic signs or hyperalgesia. These results suggest a role for the α6 nAChR subunit in nicotine reward and affective nicotine withdrawal but not acute nicotine-induced or physical withdrawal behaviors.

α -Conotoxin MII-Sensitive Nicotinic Acetylcholine Receptors in the Nucleus Accumbens Shell Regulate Progressive Ratio Responding Maintained by Nicotine

β2 subunit containing nicotinic acetylcholine receptors (β2*nAChRs; asterisk (*) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The α6 subunit assembles with β2 on DA neurons where α6β2*nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of α-conotoxin MII (α-CTX MII), an antagonist with selectivity for α6β2*nAChRs, the purpose of these experiments was to determine if α6β2*nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light+tone cues (NIC) or for light+tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of α-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. α-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate cortex. Concentrations of α-CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion α-CTX MII did not affect locomotor activity in an open field. These data suggest that α6β2*nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation.

Alpha7 nicotinic acetylcholine receptors modulate motivation to self-administer nicotine: implications for smoking and schizophrenia.

Individuals diagnosed with schizophrenia have an exceptionally high risk for tobacco dependence. Postmortem studies show that these individuals have significant reductions in α7 nicotinic acetylcholine receptors (nAChRs) in several brain areas. Decreased α7-mediated function might not only be linked to schizophrenia but also to increased tobacco consumption. The purpose of this study was to determine whether pharmacological blockade of α7 nAChRs would increase motivation of rats to intravenously self-administer nicotine (NIC) during a progressive ratio schedule of reinforcement (PR). Before PR, rats received local infusions of 0, 10, or 20 pmol of a selective α7 nAChR antagonist, α-conotoxin ArIB [V11L,V16D] (ArIB) into the nucleus accumbens (NAc) shell or the anterior cingulate cortex, brain areas that contribute to motivation for drug reward. We additionally sought to determine whether local infusion of 0, 10, or 40 nmol of a selective α7 nAChR agonist, PNU 282987, into these brain areas would decrease motivation for NIC use. Infusion of ArIB into the NAc shell and anterior cingulate cortex resulted in a significant increase in active lever pressing, breakpoints, and NIC intake, suggesting that a decrease in α7 nAChR function increases motivation to work for NIC. In contrast, PNU 282987 infusion resulted in reductions in these measures when administered into the NAc shell, but had no effect after administration into the anterior cingulate cortex. These data identify reduction of α7 nAChR function as a potential mechanism for elevated tobacco use in schizophrenia and also identify activation of α7 nAChRs as a potential strategy for tobacco cessation therapy.

Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference

The ability of nicotine to alter firing of dopamine neurons is the first step leading to nicotine reward, but activation of intracellular signaling pathways downstream of nicotinic acetylcholine receptors is likely to be critical for longer-term consequences of nicotine exposure, including conditioned reward. The transcription factor cyclic AMP-response element binding protein (CREB) is important for new gene transcription and in its phosphorylated form (pCREB) promotes long-term changes in synaptic strength. Previous studies have implicated nucleus accumbens (NAc) CREB activity in the modulation of cocaine and morphine reward, and have shown that nicotine conditioned place preference (CPP) is associated with NAc CREB activation. It is not clear whether CPP elicits phosphorylation of CREB or if elevations in pCREB support nicotine CPP. In the current study, we investigated levels of CREB and pCREB during Pavlovian conditioning with nicotine in a novel context in the absence of chamber choice. Nicotine context conditioning resulted in elevated pCREB levels in the NAc shell but not the NAc core of mice following placement in the nicotine-paired chamber in the absence of nicotine. To test if CREB activity in the NAc shell contributes to cue-induced responses that may precipitate nicotine-seeking, we used viral-mediated gene transfer of a dominant-negative CREB construct in the NAc shell of C57BL/6J mice and found that disruption of CREB activation before training blocked nicotine place preference across a range of doses. Taken together, these studies identify the NAc shell as a brain region where CREB activity is essential for nicotine CPP.

Galanin Protects Against Behavioral and Neurochemical Correlates of Opiate Reward

The mechanisms underlying responses to drugs of abuse have been widely investigated; however, less is known about pathways normally protective against the development of drug reinforcement. These pathways are also important since they may regulate individual differences in vulnerability to addiction. The neuropeptide galanin and its binding sites are expressed in brain areas important for drug reward. Previous studies have shown that centrally infused galanin attenuates morphine place preference and peripheral injection of galnon, a galanin agonist, decreases opiate withdrawal signs. The current studies in galanin knockout (GKO) mice examined the hypothesis that galanin is an endogenous negative regulator of opiate reward and identified downstream signaling pathways regulated by galanin. We show that GKO mice demonstrate increased locomotor activation following morphine administration, which is inhibited by acute administration of galnon. GKO mice also show enhanced morphine place preference, supporting the idea that galanin normally antagonizes opiate reward. In addition, morphine-induced ERK1/2 phosphorylation was increased in the VTA of both wild-type and GKO mice, but only the GKO mice showed increases in ERK1/2 and CREB phosphorylation in the amygdala or nucleus accumbens. Furthermore, a single systemic injection of galnon in GKO mice was sufficient to reverse some of the biochemical changes brought about by morphine administration. These data suggest that galanin normally attenuates behavioral and neurochemical effects of opiates; thus, galanin agonists may represent a new class of therapeutic targets for opiate addiction.

Low Dose Nicotine and Antagonism of β2 Subunit Containing Nicotinic Acetylcholine Receptors Have Similar Effects on Affective Behavior in Mice

Nicotine leads to both activation and desensitization (inactivation) of nicotinic acetylcholine receptors (nAChRs). This study tested the hypothesis that nicotine and a selective antagonist of β2*nAChRs would have similar effects on affective behavior. Adult C57BL/6J male mice were tested in a conditioned emotional response (CER) assay which evaluates the ability of an aversive stimulus to inhibit goal-directed behavior. Mice lever-pressed for a saccharin reinforcer according to a variable schedule of reinforcement during sessions in which two presentations of a compound light/tone conditioned stimulus (CS) co-terminated with a 0.1 or 0.3 mA, 0.5 s footshock unconditioned stimulus (US). During testing in the absence of the US, mice received doses of i.p. nicotine (0, 0.0032, 0.01, 0.032, 0.1 mg/kg) or a selective β2 subunit containing nAChR (β2*nAChR) antagonist dihydro-beta-erythroidine (0, 0.1, 0.3, 1.0, 3.0 mg/kg DHβE). There was a dose-dependent effect of nicotine revealing that only low doses (0.01, 0.032 mg/kg) increased CER suppression ratios (SR) in these mice. DHβE also dose-dependently increased SR at the 3 mg/kg dose. In ethological measures of fear−/anxiety-like behavior, these doses of nicotine and DHβE significantly reduced digging behavior in a marble burying task and 0.3 mg/kg DHβE promoted open-arm activity in the elevated plus maze. Doses of nicotine and DHβE that altered affective behavior had no effect on locomotor activity. Similar to previous reports with anxiolytic drugs, low dose nicotine and DHβE reversed SR in a CER assay, decreased digging in a marble burying assay and increased open arm activity in the elevated plus maze. This study provides evidence that inactivation of β2*nAChRs reduces fear-like and anxiety-like behavior in rodents and suggests that smokers may be motivated to smoke in part to desensitize their β2*nAChRs. These data further identify β2*nAChR antagonism as a potential therapeutic strategy for relief of negative affect and anxiety.

Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine- and Cocaine-Conditioned Reward in Mice

Mesolimbic α6* 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 α6*-nAChRs subtypes in the rewarding effects of nicotine. In this report, we investigated and compared the role of α6*-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 α6β2* nAChRs and genetic deletion of the α6 or α4 subunits in mice. We found that α6 KO mice exhibited a rightward shift in the nicotine dose–response curve compared with WT littermates but that α4 KO failed to show nicotine preference, suggesting that α6α4β2*-nAChRs are involved. Furthermore, α6β2* nAChRs in nucleus accumbens were found to have an important role in nicotine-conditioned reward as the intra-accumbal injection of the selective α6β2* α-conotoxin MII [H9A; L15A], blocked nicotine CPP. In contrast to nicotine, α6 KO failed to condition to cocaine, but cocaine CPP in the α4 KO was preserved. Intriguingly, α-conotoxin MII [H9A; L15A], blocked cocaine conditioning in α4 KO mice, implicating α6β2* nAChRs in cocaine reward. Importantly, these effects did not generalize as α6 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 α6 KO or WT mice. These data illustrate that the subjective rewarding effects of both nicotine and cocaine may be mediated by mesolimbic α6β2* nAChRs and that antagonists of these receptor subtypes may exhibit therapeutic potential.

Preclinical evidence that activation of mesolimbic alpha 6 subunit containing nicotinic acetylcholine receptors supports nicotine addiction phenotype.

INTRODUCTION Nicotine is a major psychoactive ingredient in tobacco yet very few individuals quit smoking with the aid of nicotine replacement therapy. Targeted therapies with more selective action at nicotinic acetylcholine receptors (nAChRs) that contain a β2 subunit (β2*nAChRs; *denotes assembly with other subunits) have enjoyed significantly greater success, but exhibit potential for unwanted cardiac, gastrointestinal, and emotive side effects. DISCUSSION This literature review focuses on the preclinical evidence that suggests that subclasses of β2*nAChRs that assemble with the α6 subunit may provide an effective target for tobacco cessation. α6β2*nAChRs have a highly selective pattern of neuroanatomical expression in catecholaminergic nuclei including the ventral tegmental area and its projection regions. α6β2*nAChRs promote dopamine (DA) neuron activity and DA release in the mesolimbic dopamine system, a brain circuitry that is well-studied for its contributions to addiction behavior. A combination of genetic and pharmacological studies indicates that activation of α6β2*nAChRs is necessary and sufficient for nicotine psychostimulant effects and nicotine self-administration. α6β2*nAChRs support maintenance of nicotine use, support the conditioned reinforcing effects of drug-associated cues, and regulate nicotine withdrawal. CONCLUSIONS These data suggest that α6β2*nAChRs represent a critical pool of high affinity β2*nAChRs that regulates nicotine dependence phenotype and suggest that inhibition of these receptors may provide an effective strategy for tobacco cessation therapy.

Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs

α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain‐of‐function α6β2* nAChRs (α6L9′S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9′S NAc slices compared to control, non‐transgenic (non‐Tg) slices. Extracellular DA levels in both non‐Tg and α6L9′S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α‐conotoxin MII completely abolished evoked DA release in α6L9′S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9′S NAc tissue contained significantly more DA, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid compared to non‐Tg NAc tissue. Serotonin (5‐HT), 5‐hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9′S compared to non‐Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9′S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels.

Diverse strategies targeting α7 homomeric and α6β2* heteromeric nicotinic acetylcholine receptors for smoking cessation

Preclinical studies suggest that a diversity of nicotinic acetylcholine receptors (nAChRs) with different sensitivities to nicotine may contribute to tobacco addiction. Using rodent intravenous nicotine self‐administration as a preclinical model with good predictive validity for therapeutic efficacy for tobacco cessation, investigators have identified heteromeric α6β2* and homomeric α7 nAChRs as promising novel therapeutic targets to promote smoking abstinence (*denotes possible assembly with other subunits). The data suggest that diverse strategies that target these subclasses of nAChRs, namely inhibition of α6β2* nAChRs and stimulation of α7 nAChRs, will support tobacco cessation. α6β2* nAChRs, members of the high‐affinity family of β2* nAChRs, function similarly to α4β2* nAChRs, the primary target of the FDA‐approved drug varenicline, but have a much more selective neuroanatomical pattern of expression in catecholaminergic nuclei. Although activation of β2* nAChRs facilitates nicotine self‐administration, stimulation of α7 nAChRs appears to negatively modulate both nicotine reinforcement and β2* nAChR function in the mesolimbic dopamine system. Although challenges and caveats must be considered in the development of therapeutics that target these nAChR subpopulations, an accumulation of data suggests that α7 nAChR agonists, partial agonists, or positive allosteric modulators and α6β2* nAChR antagonists, partial agonists, or negative allosteric modulators may prove to be effective therapeutics for tobacco cessation.