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Background and Purpose: Many of the addictive and rewarding effects of nicotine are due to its actions on the neuronal nicotinic ACh receptor (nAChR) subtypes expressed in dopaminergic mesocorticolimbic cells. The partial agonists, cytisine and varenicline, are helpful smoking cessation aids. These drugs have a number of side effects that limit their usefulness. The aim of this study was to investigate the preclinical pharmacology of the cytisine dimer1,2-bisN-cytisinylethane (CC4).Experimental Approach: The effects of CC4 on nAChRs were investigated using in vitro assays and animal behaviours.Key Results: When electrophysiologically tested using heterologously expressed human subtypes, CC4 was less efficacious than cytisine on neuronal α4β2, α3β4, α7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through α4β2 and α6β2 nAChRs, CC4 is a partial agonist of nAChR-mediated striatal dopamine release and, when co-incubated with nicotine, prevented nicotine's maximal effect on this response. In addition, it had low affinity for, and was less efficacious than nicotine and cytisine on the α3β4 and α7-nAChR subtypes. Like cytisine and nicotine, CC4-induced conditioned place preference (CPP), and its self-administration shows an inverted-U dose-response curve. Pretreatment with non-reinforcing doses of CC4 significantly reduced nicotine-induced self-administration and CPP without affecting motor functions.Conclusion and Implications: Our in vitro and in vivo findings reveal that CC4 selectively reduces behaviours associated with nicotine addiction consistent with the partial agonist selectivity of CC4 for β2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation. [ABSTRACT FROM AUTHOR]

A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene-environment interaction might impact nicotine intake in developmental nicotine-exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 μg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2-bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 μg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine-stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype-matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene-environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring., (© 2018 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

The α6 nicotinic acetylcholine receptor (nAChR) subunit is an attractive drug target for treating nicotine addiction because it is present at limited sites in the brain including the reward pathway. Lynx1 modulates several nAChR subtypes; lynx1-nAChR interaction sites could possibly provide drug targets. We found that dopaminergic cells from the substantia nigra pars compacta (SNc) express lynx1 mRNA transcripts and, as assessed by co-immunoprecipitation, α6 receptors form stable complexes with lynx1 protein, although co-transfection with lynx1 did not affect nicotine-induced currents from cell lines transfected with α6 and β2. To test whether lynx1 is important for the function of α6 nAChRs in vivo, we bred transgenic mice carrying a hypersensitive mutation in the α6 nAChR subunit (α6L9'S) with lynx1 knockout mice, providing a selective probe of the effects of lynx1 on α6* nAChRs. Lynx1 removal reduced the α6 component of nicotine-mediated rubidium efflux and dopamine (DA) release from synaptosomal preparations with no effect on numbers of α6β2 binding sites, indicating that lynx1 is functionally important for α6* nAChR activity. No effects of lynx1 removal were detected on nicotine-induced currents in slices from SNc, suggesting that lynx1 affects presynaptic α6* nAChR function more than somatic function. In the absence of agonist, lynx1 removal did not alter DA release in dorsal striatum as measured by fast scan cyclic voltammetry. Lynx1 removal affected some behaviors, including a novel-environment assay and nicotine-stimulated locomotion. Trends in 24-hour home-cage behavior were also suggestive of an effect of lynx1 removal. Conditioned place preference for nicotine was not affected by lynx1 removal. The results show that some functional and behavioral aspects of α6-nAChRs are modulated by lynx1.

(E)-5-(Pyrimidin-5-yl)-1,2,3,4,7,8-hexahydroazocine (TC299423) is a novel agonist for nicotinic acetylcholine receptors (nAChRs). We examined its efficacy, affinity, and potency for α6β2 ∗ (α6β2-containing), α4β2 ∗ , and α3β4 ∗ nAChRs, using [ 125 I]-epibatidine binding, whole-cell patch-clamp recordings, synaptosomal 86 Rb + efflux, [ 3 H]-dopamine release, and [ 3 H]-acetylcholine release. TC299423 displayed an EC 50 of 30-60 nM for α6β2 ∗ nAChRs in patch-clamp recordings and [ 3 H]-dopamine release assays. Its potency for α6β2 ∗ in these assays was 2.5-fold greater than that for α4β2 ∗ , and much greater than that for α3β4 ∗ -mediated [ 3 H]-acetylcholine release. We observed no major off-target binding on 70 diverse molecular targets. TC299423 was bioavailable after intraperitoneal or oral administration. Locomotor assays, measured with gain-of-function, mutant α6 (α6L9'S) nAChR mice, show that TC299423 elicits α6β2 ∗ nAChR-mediated responses at low doses. Conditioned place preference assays show that low-dose TC299423 also produces significant reward in α6L9'S mice, and modest reward in WT mice, through a mechanism that probably involves α6(non-α4)β2 ∗ nAChRs. However, TC299423 did not suppress nicotine self-administration in rats, indicating that it did not block nicotine reinforcement in the dosage range that was tested. In a hot-plate test, TC299423 evoked antinociceptive responses in mice similar to those of nicotine. TC299423 and nicotine similarly inhibited mouse marble burying as a measure of anxiolytic effects. Taken together, our data suggest that TC299423 will be a useful small-molecule agonist for future in vitro and in vivo studies of nAChR function and physiology.

The adult with congenital heart disease (CHD) is at risk of developing atherosclerotic cardiovascular disease (ASCVD). We performed a cross-sectional study to describe established ASCVD risk factors and estimate 10-year and lifetime risk of ASCVD in adults over age 18 with CHD of moderate or great complexity using 3 validated risk assessment tools-the Framingham Study Cardiovascular Disease Risk Assessment, the Reynolds Risk Score, and the ASCVD Risk Estimator. We obtained extensive clinical and survey data on 178 enrolled patients, with average age 37.1 ± 12.6 years, 51% men. At least 1 modifiable ASCVD risk factor was present in 70%; the 2 most common were overweight/obesity (53%) and systemic hypertension (24%). Laboratory data were available in 103 of the 178 patients. Abnormal levels of glycated hemoglobin, high-sensitivity C-reactive protein, and high-density lipoprotein were each found in around 30% of patients. The 10-year ASCVD predicted risk using all 3 tools was relatively low (i.e., at least 90% of patients <10% risk), yet the median estimated lifetime risk was 36%. In conclusion, ASCVD risk factors are prevalent in adults with CHD. The risk estimation tools suggest that this population is particularly vulnerable to ASCVD with aging and should undergo guideline-based screening and management of modifiable risk factors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

Introduction: Chronic treatment with nicotine is known to increase the α4β2-nAChR sites in brain, to decrease α6β2-nAChR sites and to have minimal effect on α3β4-and α7-nAChR populations. Varenicline is now used as a smoking cessation treatment, with and without continued smoking or nicotine replacement therapy. Varenicline, like nicotine, upregulates the α4β2-nAChR sites; however, it is not known whether varenicline treatment changes expression of the other nAChR subtypes., Methods: Using a mouse model, chronic treatments (10 days) with varenicline (0.12  mg/kg/h) and/or nicotine (1 mg/kg/hr), alone or in combination, were compared for plasma and brain levels of drugs, tolerance to subsequent acute nicotine and expression of four subtypes of nAChR using autoradiography., Results: The upregulation of α4β2-nAChR sites elicited by chronic varenicline was very similar to that elicited by chronic nicotine. Treatment with both drugs somewhat increased up-regulation, indicating that these doses were not quite at maximum effect. Similar down-regulation was seen for α6β2-nAChR sites. Varenicline significantly increased both α3β4-and α7-nAChR sites while nicotine had less effect on these sites. The drug combination was similar to varenicline alone for α3β4-nAChR sites, while for α7 sites the drug combination was less effective than varenicline alone. Varenicline had small but significant effects on tolerance to acute nicotine., Conclusions: Effects of varenicline in vivo may not be limited to the α4β2*-nAChR subtype. In addition, smoking cessation treatment with varenicline may not allow receptor numbers to be restored to baseline and may, in addition, change expression of other receptor subtypes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The expression of high-affinity α4β2* nicotinic acetylcholine receptors (nAChR) increases following chronic exposure to nicotinic agonists. While, nAChR antagonists can also produce upregulation, these changes are often less pronounced than achieved with agonists. It is unknown if nAChR agonists and antagonists induce receptor upregulation by the same mechanisms. In this study, primary neuronal cultures prepared from cerebral cortex, hippocampus, diencephalon, and midbrain/hindbrain of C57BL/6J mouse embryos were treated chronically with nicotine (agonist), mecamylamine (noncompetitive antagonist) or dihydro-β-erythroidine (competitive antagonist) or the combination of nicotine with each antagonist. The distribution of intracellular and surface [(125)I]epibatidine-binding sites were subsequently measured. Treatment with 1 μmol/L nicotine upregulated intracellular and cell surface [(125)I]epibatidine binding after 96 h. Chronic dihydro-β-erythroidine (10 μmol/L) treatment also increased [(125)I]epibatidine binding on the cell surface; however, mecamylamine was ineffective in upregulating receptors by itself. The combination of 1 μmol/L nicotine plus 10 μmol/L mecamylamine elicited a significantly higher upregulation than that achieved by treatment with nicotine alone due to an increase of [(125)I]epibatidine binding on the cell surface. This synergistic effect of mecamylamine and nicotine was found in neuronal cultures from all four brain regions. Chronic treatment with nicotine concentrations as low as 10 nmol/L produced upregulation of [(125)I]epibatidine binding. However, the effect of mecamylamine was observed only after coincubation with nicotine concentrations equal to or greater than 100 nmol/L. Vesicular trafficking was required for both nicotine and nicotine plus mecamylamine-induced upregulation. Results presented here support the idea of multiple mechanisms for nAChR upregulation.

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [(3)H]-GABA and [(3)H]-dopamine ((3)H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABAB receptors with (R)-baclofen decreased both [(3)H]-GABA and [(3)H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [(3)H]-DA release, but potently inhibited ACh-evoked [(3)H]-GABA release. Inhibition of nAChR-evoked [(3)H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABAB agonist. The early inhibitory effect of GABAB activation on ACh-evoked [(3)H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [(3)H]-GABA release. These results suggest that α4β2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABAB autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4β2*-type nAChRs on striatal GABAergic nerve terminals by GABAB autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are down-regulated following chronic nicotine exposure (unlike other subtypes that have been investigated - most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR down-regulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than up-regulation of α4β2*-nAChR. In contrast, nAChR-mediated [(3) H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [(3) H]-DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function. This study examined dose-response relationships for murine α6β2*-nicotinic acetylcholine receptor (nAChR) down-regulation by chronic nicotine treatment. The ID50 value for α6β2* down-regulation (35 nM) is ≈ 3x lower than the ED50 value for α4β2* nAChR up-regulation (95 nM), both well within the range reached by human smokers. Chronic nicotine treatment altered α6β2*- and α4β2*-nAChR-mediated [(3) H]-dopamine release from striatal and olfactory tubercle synaptosomes, but dopaminergic parameters were largely unaffected. We conclude that functional changes are primarily driven by altered nAChR activity., (© 2014 International Society for Neurochemistry.)

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., (© 2013 International Society for Neurochemistry.)

Introduction: Nicotine interacts with nicotinic acetylcholine receptors (nAChRs) and modifies neuronal functions. The net result of nicotine exposure is difficult to assess because multiple nAChR subtypes exist and are expressed on multiple classes of neurons. Nicotine, unlike the natural agonist acetylcholine, remains in tissues for hours, and during this extended exposure nAChRs desensitize. Therefore, agonists can block the natural functions of nAChRs. Higher nicotine concentrations are required to desensitize α4β2-nAChRs containing the α5 subunit. The aim of these experiments was to determine if this property holds true for compounds other than nicotine., Methods: [(3)H]-dopamine release from crude mouse striatal synaptosomal preparations was used to measure activation and desensitization of the [(α4β2)2β2] and [(α4β2)2α5] nAChR subtypes. Affinity was measured by competition with [(125)I]-epibatidine., Results: Nine compounds of varying affinity and efficacy were tested. All compounds partially desensitized both subtypes; concentration necessary for desensitization correlated with binding site affinity but not efficacy. All compounds showed a similar, significant shift in concentration necessary for a 50% effect when the α5 subunit was included (averaging 8-fold higher). The extent of desensitization produced by a 10-min exposure did not correlate with affinity or efficacy of compound., Conclusion: Full or partial nicotinic agonists used as medications may effectively desensitize α4β2-nAChRs. However, significantly higher concentrations of all compounds tested were required to elicit desensitization of α4α5β2-nAChRs than α4β2-nAChRs. If desensitization is the important property for a smoking cessation drug, basic screening at both subtypes may provide a mechanistic foundation for effectiveness.

Accumulating evidence supports the idea that drugs acting at nicotinic acetylcholine receptors (nAChRs) may be beneficial for Parkinson's disease, a neurodegenerative movement disorder characterized by a loss of nigrostriatal dopaminergic neurons. Nicotine administration to parkinsonian animals protects against nigrostriatal damage. In addition, nicotine and nAChR drugs improve L-dopa-induced dyskinesias, a debilitating side effect of L-dopa therapy which remains the gold-standard treatment for Parkinson's disease. Nicotine exerts its antidyskinetic effect by interacting with multiple nAChRs. One approach to identify the subtypes specifically involved in L-dopa-induced dyskinesias is through the use of nAChR subunit null mutant mice. Previous work with β2 and α6 nAChR knockout mice has shown that α6β2* nAChRs were necessary for the development/maintenance of L-dopa-induced abnormal involuntary movements (AIMs). The present results in parkinsonian α4 nAChR knockout mice indicate that α4β2* nAChRs also play an essential role since nicotine did not reduce L-dopa-induced AIMs in such mice. Combined analyses of the data from α4 and α6 knockout mice suggest that the α6α4β2β3 subtype may be critical. In contrast to the studies with α4 and α6 knockout mice, nicotine treatment did reduce L-dopa-induced AIMs in parkinsonian α7 nAChR knockout mice. However, α7 nAChR subunit deletion alone increased baseline AIMs, suggesting that α7 receptors exert an inhibitory influence on L-dopa-induced AIMs. In conclusion, α6β2*, α4β2* and α7 nAChRs all modulate L-dopa-induced AIMs, although their mode of regulation varies. Thus drugs targeting one or multiple nAChRs may be optimal for reducing L-dopa-induced dyskinesias in Parkinson's disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nicotinic acetylcholine receptors (nAChRs) containing either the α4 and/or α6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including α4 and α6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the α4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the α4 nAChR subunit, Leu9'Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9'Ala mice with the α4β2 nAChR antagonist dihydro-β-erythroidine (DHβE). Interestingly, in Leu9'Ala mice, DHβE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHβE-induced motor impairment in Leu9'Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHβE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9'Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9'Ala striatal synaptosomes revealed agonist hypersensitivity only at α4(non-α6)* nAChRs. Similarly, α6 nAChR subunit deletion in an α4 hypersensitive nAChR (Leu9'Ala/α6 KO) background had little effect on the DHβE-induced phenotype, suggesting an α4(non-α6)* nAChR-dependent mechanism. Together, these data indicate that α4(non-α6)* nAChR have an impact on motor output and may be potential molecular targets for treatment of disorders associated with motor impairment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4β2-containing (α4β2(∗)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4β2(∗) nAChRs in a genedose dependent pattern by immunopurifying β2(∗) nAChRs from mice that differ in α4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2(∗) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4β2 nAChRs and their downstream signaling mechanisms., (Copyright © 2013. Production and hosting by Elsevier Ltd.)

Several mutations in α4 or β2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the β2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (β2VL). Previous studies indicated that the β2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of β2VL mice. No differences in β2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the β2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

L-Dopa-induced dyskinesias are a serious side effect that develops in most Parkinson's disease patients on dopamine replacement therapy. Few treatment options are available to manage dyskinesias; however,recent studies show that nicotine reduces these abnormal involuntary movements (AIMs) in parkinsonian animals by acting at nicotinic acetylcholine receptors (nAChRs). Identification of the nAChR subtypes that mediate this reduction in AIMs is important as it will help in the development of nAChR subtype selective drugs for their treatment. Here we investigate the role of α6β2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using a6 nAChR subunit null mutant (α6⁻/⁻) mice.Wildtype and α6⁻/⁻ mice were lesioned by unilateral injection of 6-hydroxydopamine (3 mg/ml) into the medial forebrain bundle. They were then given L-dopa (3 mg/kg) plus benserazide (15 mg/kg) 2e3 wk later. L-dopa-induced AIMs developed to a similar extent in α6⁻/⁻ and wildtype mice.However, AIMs in α6⁻/⁻ mice declined to ~50% of that in wildtype mice with continued L-dopa treatment. Nicotine treatment also decreased AIMs by ~50% in wildtype mice, although not in α6⁻/⁻ mice. There were no effects on parkinsonism under any experimental condition. To conclude, the similar declines in L-dopa-induced AIMs in nicotine-treated wildtype mice and in α6⁻/⁻ mice treated with and without nicotine indicate an essential role for α6β2* nAChRs in the maintenance of L-dopa-induced AIMs.These findings suggest that α6β2* nAChR drugs have potential for reducing L-dopa-induced dyskinesias in Parkinson's disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chronic nicotine produces up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) (* denotes that an additional subunit may be part of the receptor). However, the extent of up-regulation to persistent ligand exposure varies across brain regions. The aim of this work was to study the cellular distribution and function of nAChRs after chronic nicotine treatment in primary cultures of mouse brain neurons. Initially, high-affinity [(125)I]epibatidine binding to cell membrane homogenates from primary neuronal cultures obtained from diencephalon and hippocampus of C57BL/6J mouse embryos (embryonic days 16-18) was measured. An increase in α4β2*-nAChR binding sites was observed in hippocampus, but not in diencephalon, after 24 h of treatment with 1 μM nicotine. However, a nicotine dose-dependent up-regulation of approximately 3.5- and 0.4-fold in hippocampus and diencephalon, respectively, was found after 96 h of nicotine treatment. A significant fraction of total [(125)I]epibatidine binding sites in both hippocampus (45%) and diencephalon (65%) was located on the cell surface. Chronic nicotine (96 h) up-regulated both intracellular and surface binding in both brain regions without changing the proportion of those binding sites compared with control neurons. The increase in surface binding was not accompanied by an increase in nicotine-stimulated Ca(2+) influx, suggesting persistent desensitization or inactivation of receptors at the plasma membrane occurred. Given the differences observed between hippocampus and diencephalon neurons exposed to nicotine, multiple mechanisms may play a role in the regulation of nAChR expression and function.

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.

Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Introduction: The smoking cessation aid, varenicline, has higher affinity for the alpha4beta2-subtype of the nicotinic acetylcholine receptor (α4β2*-nAChR) than for other subtypes of nAChRs by in vitro assays. The mechanism of action of acute varenicline was studied in vivo to determine (a) subtype activation associated with physiological effects and (b) dose relationship as an antagonist of nicotine., Methods: Acute doses of saline, nicotine, and varenicline were given to mice, and locomotor depression and hypothermia were measured. Subunit null mutant mice as well as selective antagonists were used to study mode of action of varenicline as an agonist. Varenicline as an antagonist of nicotine was also investigated., Results: Varenicline evokes locomotor depression and hypothermia at higher doses than necessary for nicotine. Null mutation of the α7- or β2-nAChR subunit did not decrease the effectiveness of varenicline; however, null mutation of the β4 subunit significantly decreased the magnitude of the varenicline effect. Effects of the highest dose studied were blocked by mecamylamine (general nAChR antagonist) and partially antagonized by hexamethonium (largely peripheral nAChR antagonist). No significant block was seen with ondansetron antagonist of 5-hydroxytryptamine 3 receptor. Using a dose of nicotine selective for β2*-nAChR subtype effects with these tests, dose-dependent antagonism by varenicline was seen. Effective inhibitory doses were determined and appear to be in a range consistent with binding affinity or desensitization of β2*-nAChRs., Conclusions: Varenicline acts as a functional antagonist of β2*-nAChRs, blocking certain effects of nicotine. At higher doses, varenicline is an agonist of β4*-nAChRs producing physiological changes in mice.

Desensitization is a complex property of nicotinic acetylcholine receptors (nAChR). Several subtypes of nAChR have high sensitivity to nicotine and mediate effects of nicotine at concentrations found in blood of tobacco smokers. Desensitization of some of these receptor subtypes has been studied in model systems, however, other subtypes have been difficult to express heterologously in native forms. In addition, model systems may not have the same accessory molecules and post-translational modifications found in native populations. We have used wild-type and subunit null mutant mice to study desensitization properties of the high sensitivity α4β2-nAChRs including those that have α5 subunits at both GABAergic and dopaminergic nerve terminals. In addition, we have studied the desensitization of one subtype of α6β2-nAChRs at dopaminergic terminals using α4 subunit null mutant mice. Exposure to low nicotine concentrations, leads to rapid, but partial desensitization of activity mediated by these receptors. α4β2-nAChRs including α5 subunits show faster rates of recovery from desensitization than α4β2-nAChRs without α5. Inclusion of the α5 subunit significantly shifts the concentration response for desensitization to higher values, indicating that receptors with α5 subunits are less desensitized by a 10-min exposure to low concentrations of nicotine. Receptors with α6 subunits appear to desensitize to a lesser degree than those with α4 subunits, indicating that α6β2-nAChRs are somewhat resistant to desensitization by nicotine. These results highlight the importance of studying various receptor subtypes in native systems and how they may differentially respond to nicotine and to nicotinic drugs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of α6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) α6 subunits. α6 L9'S mice harboring six or more copies of the hypersensitive α6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S α6 genes had locomotor activity resembling that of control mice. α6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy α6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy α6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy α6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of α6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in α6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of α6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Although a relative newcomer to the nicotinic acetylcholine receptor (nAChR) family, substantial evidence suggests that α6 containing nAChRs play a key role in CNS function. This subtype is unique in its relatively restricted localization to the visual system and catecholaminergic pathways. These latter include the mesolimbic and nigrostriatal dopaminergic systems, which may account for the involvement of α6 containing nAChRs in the rewarding properties of nicotine and in movement. Here, we review the literature on the role of α6 containing nAChRs with a focus on the striatum and nucleus accumbens. This includes molecular, electrophysiological and behavioral studies in control and lesioned animal models, as well as in different genetic models. Converging evidence suggest that the major α6 containing nAChRs subtypes in the nigrostriatal and mesolimbic dopamine system are the α6β2β3 and α6α4β2β3 nAChR populations. They appear to have a dominant role in regulating dopamine release, with consequent effects on nAChR-modulated dopaminergic functions such as reinforcement and motor behavior. Altogether these data suggest that drugs directed to α6 containing nAChRs may be of benefit for the treatment of addiction and for neurological disorders with locomotor deficits such as Parkinson's disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

L-DOPA-induced dyskinesias or abnormal involuntary movements (AIMs) are a debilitating adverse complication associated with prolonged L-DOPA administration for Parkinson's disease. Few treatments are currently available for dyskinesias. Our recent data showed that nicotine reduced L-DOPA-induced AIMs in parkinsonian animal models. An important question is the nicotinic acetylcholine receptor (nAChR) subtypes through which nicotine exerts this beneficial effect, because such knowledge would allow for the development of drugs that target the relevant receptor population(s). To address this, we used β2 nAChR subunit knockout [β2(-/-)] mice because β2-containing nAChRs are key regulators of nigrostriatal dopaminergic function. All of the mice were lesioned by intracranial injection of 6-hydroxydopamine into the right medial forebrain bundle. Lesioning resulted in a similar degree of nigrostriatal damage and parkinsonism in β2(-/-) and wild-type mice. All of the mice then were injected with L-DOPA (3 mg/kg) plus benserazide (15 mg/kg) once daily for 4 weeks until AIMs were fully developed. L-DOPA-induced AIMs were approximately 40% less in the β2(-/-) mice compared with the wild-type mice. It is interesting to note that nicotine (300 μg/ml in drinking water) reduced L-DOPA-induced AIMs by 40% in wild-type mice but had no effect in β2(-/-) mice with partial nigrostriatal damage. The nicotine-mediated decline in AIMs was much less pronounced in wild-type mice with near-complete degeneration, suggesting that presynaptic nAChRs on dopaminergic terminals have a major influence. These data demonstrate an essential role for β2* nAChRs in the antidyskinetic effect of nicotine and suggest that drugs targeting these subtypes may be useful for the management of L-DOPA-induced dyskinesias in Parkinson's disease.

Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors.

Nicotine activation of nicotinic acetylcholine receptors (nAChRs) within the dopaminergic (DAergic) neuron-rich ventral tegmental area (VTA) is necessary and sufficient for nicotine reinforcement. In this study, we show that rewarding doses of nicotine activated VTA DAergic neurons in a region-selective manner, preferentially activating neurons in the posterior VTA (pVTA) but not in the anterior VTA (aVTA) or in the tail VTA (tVTA). Nicotine (1 μM) directly activated pVTA DAergic neurons in adult mouse midbrain slices, but had little effect on DAergic neurons within the aVTA. Quantification of nAChR subunit gene expression revealed that pVTA DAergic neurons expressed higher levels of α4, α6, and β3 transcripts than did aVTA DAergic neurons. Activation of nAChRs containing the α4 subunit (α4(*) nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: nicotine failed to activate pVTA DAergic neurons in α4 knockout animals; in contrast, pVTA α4(*) nAChRs were selectively activated by nicotine in mutant mice expressing agonist-hypersensitive α4(*) nAChRs (Leu9'Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by α4(*) nAChRs and were significantly larger in pVTA neurons than in aVTA neurons. Infusion of an α6(*) nAChR antagonist into the VTA blocked activation of pVTA DAergic neurons in WT mice and in Leu9'Ala mice at nicotine doses, which only activate the mutant receptor indicating that α4 and α6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA through α4α6(*) nAChRs. These receptors represent novel targets for smoking-cessation therapies.

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express alpha6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive alpha6(L9'S)* receptors. alpha6(L9'S) mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by alpha6alpha4* pentamers, as alpha6(L9'S) mice lacking alpha4 subunits displayed essentially normal behavior. In alpha6(L9'S) mice, receptor numbers are normal, but loss of alpha4 subunits leads to fewer and less sensitive alpha6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires alpha4 subunits, implicating alpha6alpha4beta2* nAChRs in alpha6(L9'S) mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by alpha6(L9'S) nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in alpha6(L9'S), but not WT or alpha4KO/alpha6(L9'S), mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from alpha6(L9'S) presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate alpha6alpha4beta2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR., ((c) 2010 Elsevier Ltd. All rights reserved.)

The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the alpha7, beta2, or beta4 subunit. In agreement with general belief, the alpha4beta2*-nAChR and alpha7-nAChR subtypes are major components of the epibatidine binding sites. alpha4beta2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while alpha7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.

Recent studies suggest that high-affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha4 and beta2 subunits (alpha4beta2*) functionally interact with G-protein-coupled dopamine (DA) D(2) receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9'Ala) rendering alpha4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D(2)-receptor agonist. When challenged with the D(2)R agonist, quinpirole (0.5-10 mg/kg), Leu9'Ala mice, but not wild-type (WT) littermates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole-induced DA depletion did not differ between Leu9'Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c-Fos expression in choline acetyltransferase (ChAT)-positive interneurons. Highlighting the importance of the cholinergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinson's disease, and the data suggest that a D(2)R-alpha4*-nAChR functional interaction regulates cholinergic interneuron activity.

Wild-type and alpha5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate alpha-conotoxin MII (alpha-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration-effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the alpha5 null mutant. Deleting the alpha5 gene also resulted in decreased maximal ACh-stimulated alpha-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and alpha5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHbetaE-sensitive (HS) and DHbetaE-resistant (LS) components were found in both genotypes. These results indicate that alpha-CtxMII-resistant DA release is mediated by alpha4alpha5beta2, (alpha4)(2)(beta2)(3) (HS), and (alpha4)(3)(beta2)(2) (LS) nAChRs.

Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While alpha4beta2 and alpha7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among alpha4beta2*-nAChR, ABT-089 had partial agonist activity (7-23% of nicotine) and high selectivity for alpha4alpha5beta2 nAChR as evidenced by loss of activity in thalamus of alpha5(-/-) mice. ABT-089 stimulated [(3)H]-dopamine release (57%) exceeded the activity at alpha4beta2* nAChR, that could be explained by the activity at alpha6beta2* nAChR. The concentration-response relationship for ABT-089 stimulation of alpha6beta2* nAChR was biphasic. EC(50) and efficacy values for ABT-089, respectively, were 28 microM and 98% at the less sensitive alpha6beta2* nAChR and 0.11 microM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations

Direct comparison of pyridine versus pyrimidine substituents on a small but diverse set of ligands indicates that the pyrimidine substitution has the potential to enhance affinity and/or functional activity at alpha6 subunit-containing neuronal nicotinic receptors (NNRs) and decrease activation of ganglionic nicotinic receptors, depending on the scaffold. The ramifications of this structure-activity relationship are discussed in the context of the design of small molecules targeting smoking cessation.

High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo-interpeduncular (Hb-IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (+/+) and beta2 knock-out (-/-) mice to establish that the Hb and IPn contain significant beta2* and beta4* populations of nAChR receptors (each of which is heterogeneous). The beta4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (alpha2beta2*, alpha4beta3beta2*, alpha3beta3beta4*, alpha6beta3beta4*). Our studies on IPn synaptosomes obtained from +/+ and alpha2, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4(-/-) mice show that only the alpha3beta4 and alpha3beta3beta4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in beta3(-/-) mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and alpha3beta4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the beta3 subunit may be important for transporting the alpha3beta4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb-IPn system and identify alpha3beta4 and alpha3beta3beta4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.

The discovery that mammalian brain expresses the mRNAs for nine different nicotinic cholinergic receptor subunits (alpha2-alpha7, beta2-beta4) that form functional receptors when expressed in Xenopus laevis oocytes suggests that many different types of nicotinic cholinergic receptors (nAChRs) might be expressed in the mammalian brain., Using an historical approach, this chapter reviews some of the progress made in identifying the nAChR subtypes that seem to play a vital role in modulating dopaminergic function. nAChR subtypes that are expressed in dopamine neurons, as well as neurons that interact with dopamine neurons (glutamatergic, GABAergic), serve as the focus of this review. Subjects that are highlighted include the discovery of a low affinity alpha4beta2* nAChR, the identity of recently characterized alpha6* nAChRs, and the finding that these alpha6* receptors have the highest affinity for receptor activation of any of the native receptors that have been characterized to date. Topics that have been ignored in other recent reviews of this area, such as the discovery and potential importance of alternative transcripts, are presented along with a discussion of their potential importance.

Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.

Nicotine treatment has long been associated with alterations in alpha4beta2(*) nicotinic acetylcholine receptor (nAChR) expression that modify dopaminergic function. However, the influence of long-term nicotine treatment on the alpha6beta2(*) nAChR, a subtype specifically localized on dopaminergic neurons, is less clear. Here we used voltammetry, as well as receptor binding studies, to identify the effects of nicotine on striatal alpha6beta2(*) nAChR function and expression. Long-term nicotine treatment via drinking water enhanced nonburst and burst endogenous dopamine release from rat striatal slices. In control animals, alpha6beta2(*) nAChR blockade with alpha-conotoxin MII (alpha-CtxMII) decreased release with nonburst stimulation but not with burst firing. These data in control animals suggest that varying stimulus frequencies differentially regulate alpha6beta2(*) nAChR-evoked dopamine release. In contrast, in nicotine-treated rats, alpha6beta2(*) nAChR blockade elicited a similar pattern of dopamine release with nonburst and burst firing. To elucidate the alpha6beta2(*) nAChR subtypes altered with long-term nicotine treatment, we used the novel alpha-CtxMII analog E11A in combination with alpha4 nAChR knockout mice. (125)I-alpha-CtxMII competition studies in striatum of knockout mice showed that nicotine treatment decreased the alpha6alpha4beta2(*) subtype but increased the alpha6(nonalpha4)beta2(*) nAChR population. These data indicate that alpha6beta2(*) nAChR-evoked dopamine release in nicotine-treated rats is mediated by the alpha6(nonalpha4)beta2(*) nAChR subtype and suggest that the alpha6alpha4beta2(*) nAChR and/or alpha4beta2(*) nAChR contribute to the differential effect of higher frequency stimulation on dopamine release under control conditions. Thus, alpha6beta2(*) nAChR subtypes may represent important targets for smoking cessation therapies and neurological disorders involving these receptors such as Parkinson's disease.

This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and alpha-conotoxin MII (alpha-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the alpha6 subunit (alpha4alpha6beta2beta3, alpha6beta2beta3, alpha6beta2) and bind alpha-CtxMII with high affinity. One of these three subtypes (alpha4alpha6beta2beta3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for alpha-CtxMII (alpha4beta2, alpha4alpha5beta2) are somewhat more numerous than the alpha6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.

We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Parkinson's disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the alpha6beta2(*) nicotinic acetylcholine receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum ((*)denotes the presence of possible additional subunits). In this study, we used a novel alpha-conotoxin MII (alpha-CtxMII) analog E11A to further investigate alpha6beta2(*) nAChR subtypes in mouse, monkey, and human striatum. Receptor competition studies with (125)I-alpha-CtxMII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct alpha6beta2(*) nAChR subtypes. These include a very high (femtomolar) and a high (picomolar) affinity site, with approximately 40% of the sites in the very high affinity form. It is noteworthy that only the high-affinity form was detected in alpha4 nAChR-null mutant mice. Because (125)I-alpha-CtxMII binds primarily to alpha6alpha4beta2beta3 and alpha6beta2beta3 nAChR subtypes in mouse striatum, these data suggest that the population lost in the alpha4 knockout mice was the alpha6alpha4beta2beta3 subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal alpha6beta2(*) populations in two animal models and in Parkinson's disease. There was a preferential loss of the very high affinity subtype in striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), monkeys treated with MPTP, and patients with Parkinson's disease. These data suggest that dopaminergic terminals expressing the alpha6alpha4beta2beta3 population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with alpha-CtxMII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson's disease.

Subtypes of nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds alpha-conotoxin MII (alpha-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the alpha6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of alpha6beta2* nAChRs expressed in vivo for assessing pharmacology of alpha6beta2* nAChRs. Binding to striatal membranes and function in synaptosomes from (alpha4-/-)(beta3+/+) and (alpha4-/-)(beta3-/-) mice were measured and compared with wild-type (alpha4+/+)(beta3+/+) mice. Gene deletions (alpha4 and beta3) decreased binding of (125)I-alpha-CtxMII without affecting affinity for alpha-CtxMII or inhibition of alpha-CtxMII binding by epibatidine or nicotine. Deletion of the alpha4 subunit substantially increased EC(50) values for both nicotine- and cytisine-stimulated alpha-CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC(50) values was seen upon the additional deletion of the beta3 subunit. The data indicate that one alpha-CtxMII-sensitive nAChR subtype, prevalent on wild-type dopaminergic terminals, has the lowest EC(50) for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of alpha6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.

Rationale: This review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure., Objectives: This review capitalizes on the authors' collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models., Results: Seven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses., Conclusions: The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.

Nicotinic acetylcholine receptors (nAChRs) are decreased in the striata of patients with Parkinson's disease (PD) or in experimental models after nigrostriatal damage. Because presynaptic nAChRs on striatal dopamine terminals mediate dopamine release, receptor loss may contribute to behavioral deficits in PD. The present experiments were done to determine whether nAChR function is affected by nigrostriatal damage in nonhuman primates, because this model shares many features with PD. Initial characterization of nicotine-evoked [3H]dopamine release from monkey striatal synaptosomes revealed that release was calcium-dependent and inhibited by selective nAChR antagonists. It is noteworthy that a greater proportion (approximately 70%) of release was inhibited by the alpha3*/alpha6* antagonist alpha-conotoxinMII (alpha-CtxMII) compared with rodents. Monkeys were lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and [3H]dopamine release, dopamine transporter, and nAChRs were measured. As anticipated, lesioning decreased the transporter and alpha3*/alpha6* nAChRs in caudate and putamen. In contrast, alpha3*/alpha6* nAChR-evoked [3H]dopamine release was reduced in caudate but not putamen, demonstrating a dissociation between nAChR sites and function. A different pattern was observed in the mesolimbic dopamine system. Dopamine transporter levels in nucleus accumbens were not reduced after MPTP, as expected; however, there was a 50% decline in alpha3*/alpha6* nAChR sites with no decrease in alpha3*/alpha6* receptor-evoked dopamine release. No declines in alpha-CtxMII-resistant nAChR (alpha4*) binding or nicotine-evoked release were observed in any region. These results show a selective preservation of alpha3*/alpha6* nAChR-mediated function in the nigrostriatal and mesolimbic dopamine systems after nigrostriatal damage. Maintenance of function in putamen, a region with a selective loss of dopaminergic terminals, may be important in PD.

Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) are distinct from other subtypes in their relatively restricted localization to the striatum and some other brain regions. The effect of nicotine treatment on nAChR subtypes has been extensively investigated, with the exception of changes in alpha-conotoxin MII-sensitive receptor expression. We therefore determined the consequence of long-term nicotine administration on this subtype and its function. Nicotine was given in drinking water to provide a long-term yet intermittent treatment. Consistent with previous studies, nicotine exposure increased 125I-epibatidine and 125I-A85380 (3-[2-(S)-azetidinylmethoxy]pyridine), but not 125I-alpha-bungarotoxin, receptors in cortex and striatum. We observed an unexpected reduction (30%) in striatal 125I-alpha-conotoxin MII sites, which occurred because of a decrease in B(max). This decline was more robust in older (>8-month-old) compared with younger (2-4-month-old) mice, suggesting age is important for nicotine-induced disruption of nAChR phenotype. Immunoprecipitation experiments using nAChR subunit-directed antibodies indicate that alterations in subunit-immunoreactivity with nicotine treatment agree with those in the receptor binding studies. To determine the relationship between striatal nAChR sites and function, we measured nicotine-evoked [3H]dopamine release. A decline was obtained with nicotine treatment that was caused by a selective decrease in alpha-conotoxin MII-sensitive but not alpha-conotoxin MII-resistant dopamine release. These results may explain previous findings that nicotine treatment decreased striatal nAChR-mediated dopamine function, despite an increase in [3H]nicotine (alpha4*) sites. The present data suggest that the alpha6* nAChR subtype represents a key factor in the control of dopamine release from striatum, which adapts to long-term nicotine treatment by down-regulation of alpha6* receptor sites and function.

The subunit composition and pharmacology of alpha-Conotoxin MII-binding (alpha-CtxMII) nicotinic acetylcholine receptors (nAChR) was studied by an improved [(125)I]-alpha-CtxMII membrane binding method. This binding method facilitates pharmacological studies that have been difficult to accomplish with [(125)I]-alpha-CtxMII autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding. Binding densities and K(d)-values obtained by this [(125)I]-alpha-CtxMII membrane binding were similar to the values obtained by autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding, verifying that each of these approaches measures the same nAChR population. Binding results with nAChR subunit-null mutant mice confirm and extend observations from earlier studies: [(125)I]-alpha-CtxMII binding measures two sets of alpha6beta2* nAChR (alpha4alpha6beta2beta3 or alpha6beta2beta3). Most nicotinic agonists and antagonists show monophasic inhibition of [(125)I]-alpha-CtxMII binding, indicating that alpha4alpha6beta2beta3 and alpha6beta2beta3 have similar binding properties. Comparison of the binding and activation profiles of alpha6beta2* nAChR to those of other nAChR subtypes (alpha4beta2* and beta4*) indicates that these receptors have distinctly different pharmacology indicating that it may be possible to target alpha6beta2* nAChR selectively to develop compounds that might be therapeutically useful.