Meeting Report for: The 3rd Satellite Meeting to the SfN: Nicotinic Acetylcholine Receptors as Therapeutic Targets

CA1 stratum radiatum interneurons (SRIs) express alpha7 nicotinic receptors (nAChRs) and receive inputs from glutamatergic neurons/axons that express alpha3beta4beta2 nAChRs. To test the hypothesis that endogenously active alpha7 and/or alpha3beta4beta2 nAChRs control the excitability of CA1 SRIs in the rat hippocampus, we examined the effects of selective receptor antagonists on spontaneous fast current transients (CTs) recorded from these interneurons under cell-attached configuration. The frequency of CTs, which represent action potentials, increased in the absence of extracellular Mg(2+) and decreased in the presence of the alpha3beta4beta2 nAChR antagonist mecamylamine (3 muM) or the NMDA receptor antagonist APV (50 muM). However, it was unaffected by the alpha7 nAChR antagonist MLA (10 nM) or the AMPA receptor antagonist CNQX (10 muM). Thus, in addition to synaptically and tonically activated NMDA receptors, alpha3beta4beta2 nAChRs that are present on glutamatergic axons/neurons synapsing onto SRIs and are activated by basal levels of acetylcholine contribute to the maintenance of the excitability of these interneurons. Kynurenic acid (KYNA), an astrocyte-derived kynurenine metabolite whose levels are increased in the brains of patients with schizophrenia, also controls the excitability of SRIs. At high micromolar concentrations, KYNA, acting primarily as an NMDA receptor antagonist, decreased the CT frequency recorded from the interneurons. At 2 muM, KYNA reduced the CA1 SRI excitability via mechanisms independent of NMDA receptor block. KYNA-induced reduction of excitability of SRIs may contribute to sensory gating deficits that have been attributed to deficient hippocampal GABAergic transmission and high levels of KYNA in the brain of patients with schizophrenia.

The human alpha7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is a candidate gene for schizophrenia and an important drug target for cognitive deficits in the disorder. Activation of the alpha7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca(2+), that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of alpha7*nAChR, as measured by binding of the ligand [(125)I]-alpha-bungarotoxin (I-BTX). The structure of the gene, CHRNA7, is complex. During evolution, CHRNA7 was partially duplicated as a chimeric gene (CHRFAM7A), which is expressed in the human brain and elsewhere in the body. The association between a 2bp deletion in CHRFAM7A and schizophrenia suggested that this duplicate gene might contribute to cognitive impairment. To examine the putative contribution of CHRFAM7A on receptor function, co-expression of alpha7 and the duplicate genes was carried out in cell lines and Xenopus oocytes. Expression of the duplicate alone yielded protein expression but no functional receptor and co-expression with alpha7 caused a significant reduction of the amplitude of the ACh-evoked currents. Reduced current amplitude was not correlated with a reduction of I-BTX binding, suggesting the presence of non-functional (ACh-silent) receptors. This hypothesis is supported by a larger increase of the ACh-evoked current by the allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596) in cells expressing the duplicate than in the control. These results suggest that CHRFAM7A acts as a dominant negative modulator of CHRNA7 function and is critical for receptor regulation in humans.

Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxinMII, 5[(125)I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [(125)I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each binding site was evaluated semi-quantitatively for samples prepared from wild-type and alpha2, alpha4, alpha6, alpha7, beta2, beta4, alpha5 and beta3 null mutant mice. Deletion of the alpha7 subunit completely and selectively eliminated [(125)I]alpha-bungarotoxin binding. The binding of [(125)I]alpha-conotoxinMII was eliminated in most brain regions by deletion of either the alpha6 or beta2 subunit and is reduced by deletion of either the alpha4 or beta3 subunit. The binding of 5[(125)I]A-85380 was completely eliminated by deletion of the beta2 subunit and significantly reduced by deletion of the alpha4 subunit. Most, but not all, alpha4-independent sites require expression of the alpha6 subunit. The effect of gene deletion on total [(125)I]epibatidine binding was very similar to that on [(125)I]A-85380 binding. [(125)I]Epibatidine also labels beta4* nAChR, which was readily apparent for incubations conducted in the presence of 100nM cytisine. The effects of alpha3 gene deletion could not be evaluated, but persistence of residual sites implies the expression of alpha3* nAChR. Taken together these results confirm and extend previously published evaluations of the effect of nAChR gene deletion and help to define the nAChR subtypes measurable by ligand binding.

The hippocampus is one of only two regions in the adult brain where neurons are generated in significant numbers throughout the lifetime of the animal. Numerous studies have demonstrated that these adult-born neurons are essential for optimal cognitive function with unimpaired memory formation and retrieval. The extent to which adult-born neurons survive through an early "critical period" and become integrated into functional networks has been shown to depend on the richness of stimulation they receive during these formative stages. The dentate gyrus in the hippocampus - home of the adult-born neurons - receives extensive cholinergic innervation, and newly generated neurons in the adult hippocampus express substantial numbers of both major types of neuronal nicotinic acetylcholine receptors. Early studies indicated that nicotinic signaling may be important for the development of adult-born neurons: repeated exposure to nicotine impaired their long-term survival. Recent studies with mutant mice lacking either one of the two major nicotinic receptor subtypes demonstrate that receptor loss results in fewer adult-born neurons surviving the critical period and becoming integrated into neural networks. The key nicotinic receptor mediating the largest effects is one that has a high relative permeability to calcium. In view of this feature, it may not be surprising that excessive exposure to nicotine can have detrimental effects on survival and maturation of adult-born neurons in the dentate; these same receptors appear to be key. The results pose serious challenges for therapeutic strategies targeting an individual class of nicotinic receptors for global treatment in the recipient.

Despite the known adverse health effects associated with tobacco use, over 45 million adults in the United States smoke. Cigarette smoking is the major etiologic factor associated with lung cancer. Cigarettes contain thousands of toxic chemicals, many of which are carcinogenic. Nicotine contributes directly to lung carcinogenesis through the activation of nicotinic acetylcholine receptors (nAChRs). nAChRs are ligand-gated ion channels, expressed in both normal and lung cancer cells, which mediate the proliferative, pro-survival, angiogenic, and metastatic effects of nicotine and its nitrosamine derivatives. The underlying molecular mechanisms involve increases in intracellular calcium levels and activation of cancer signal transduction pathways. In addition, acetylcholine (ACh) acts as an autocrine or paracrine growth factor in lung cancer. Other neurotransmitters and neuropeptides also activate similar growth loops. Recent genetic studies further support a role for nAChRs in the development of lung cancer. Several nAChR antagonists have been shown to inhibit lung cancer growth, suggesting that nAChRs may serve as valuable targets for biomarker-guided lung cancer interventions.

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

Nicotinic acetylcholine receptors (nAChRs) function as ligand-gated ion channels activated by the neurotransmitter acetylcholine. Gene knockout and antisense studies coupled with pharmacological studies with nAChR agonists have documented a role of alpha4beta2 nAChR activation in analgesia. ABT-594, for the first time, provided clinical validation to the nAChR agonist pharmacology as a novel mechanism for treatment of pain. However, ABT-594 was poorly tolerated at the efficacious doses, particularly with respect to the side effects of nausea and emesis, which is thought to be mediated by activation of the ganglionic-type (alpha3-containing) nAChRs. An alternate approach is to selectively modulate the alpha4beta2 nAChR via positive allosteric modulation. Positive allosteric modulators (PAMs) are compounds that do not interact with the agonist binding sites or possess intrinsic activity at the receptor per se, but potentiate the effects of the agonist. NS9283 (also known as A-969933), the first oxadiazole analog, was found to selectively enhance the potency of a range of nAChR agonists at alpha4beta2, but not alpha3beta4, nAChRs. Studies reported here, along with the accompanying manuscript [1] collectively point to the conclusion, based on preclinical models, that the analgesic efficacy of clinically well-tolerated doses of ABT-594 in humans can be significantly enhanced by co-administration with the alpha4beta2 PAM. Additionally, studies in ferrets demonstrate no exaggeration of emetic effect when ABT-594 is co-dosed with NS9283. Cardiovascular studies in anesthetized dogs achieve supra-therapeutic plasma concentrations of ABT-594 (>20-fold) without hemodynamic or electrophysiological effects using the co-administration paradigm.

Recent progress has been made in the understanding of the anatomical distribution, composition, and physiological role of nicotinic acetylcholine receptors containing the alpha6 subunit. Extensive study by many researchers has indicated that a collection of alpha6-containing receptors representing a nicotinic sub-family is relevant in preclinical models of nicotine self-administration and locomotor activity. Due to a number of technical difficulties, the state of the art of in vitro model systems expressing alpha6-containing receptors has lagged behind the state of knowledge of native alpha6 nAChR subunit composition. Several techniques, such as the expression of chimeric and concatameric alpha6 subunit constructs in oocytes and mammalian cell lines have been employed to overcome these obstacles. There remains a need for other critical tools, such as selective small molecules and radioligands, to advance the field of research and to allow the discovery and development of potential therapeutics targeting alpha6-containing receptors for smoking cessation, Parkinson's disease and other disorders.

Altered functional interactions among midbrain dopamine (DA) neurons contribute to the reinforcing properties of environmental stimuli and addictive drugs. To examine correlations among DA neurons, acute nicotine was administrated to rats via an intraperitoneal catheter and unit activity was measured using multi-tetrode in vivo recordings. Nicotine administration enhanced the correlated activity of simultaneously recorded DA neurons from the ventral tegmental area (VTA). The strength of the correlations between DA neuron pairs, as measured by cross covariance among two spike trains, showed dynamic changes over time. Nicotine produced a gradual rise in firing rate and burst activity that reached a stable plateau approximately 20 min after the intraperitoneal nicotine infusion. Shortly after that time the cross correlations measured using 5-ms bins increased significantly above baseline. In addition, nicotine increased the firing rates of DA neurons in the posterior VTA more than in the anterior VTA. Unlike nicotine, eticlopride administration also boosted DA neuron firing activity but did not enhance synchronization, indicating that the cross correlations induced by nicotine were not due to a non-specific increase in firing rate. The overall results show that nicotine induces nearly synchronous firing by a subset of DA neurons, and those changes in correlative firing will enhance the DA signal that contributes to nicotine-induced behavioral reinforcement.

Significant advances in human functional brain imaging offer new opportunities for direct observation of the effects of nicotine, novel nicotinic agonists and nicotinic antagonists on human cognitive and behavioral performance. Careful research over the last decade has enabled investigators to explore the role of nicotinic systems on the functional neuroanatomy and neural circuitry of cognitive tasks in domains such as selective attention, working memory, episodic memory, cognitive control, and emotional processing. In addition, recent progress in understanding functional connectivity between brain regions utilized during cognitive and emotional processes offers new opportunities for examining drug effects on network-related activity. This review will critically summarize available nicotinic functional brain imaging studies focusing on the specific cognitive domains of attention, memory, behavioral control, and emotional processing. Generally speaking, nicotine appears to increase task-related activity in non-smokers and deprived smokers, but not active smokers. By contrast, nicotine or nicotinic stimulation decreases the activity of structures associated with the default mode network. These particular patterns of activation and/or deactivation may be useful for early drug development and may be an efficient and cost-effective method of screening potential nicotinic agents. Further studies will have to be done to clarify whether such activity changes correlate with cognitive or affective outcomes that are clinically relevant. The use of functional brain imaging will be a key tool for probing pathologic changes related to brain illness and for nicotinic drug development.

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The alpha4beta2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for alpha4beta2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the alpha4beta2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the alpha4beta2 receptors. Here we discuss the concept of allosterism as it pertains to the alpha4beta2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.

Smoking is responsible for over 400,000 premature deaths in the United States every year, making it the leading cause of preventable death. In addition, smoking-related illness leads to billions of dollars in healthcare expenditures and lost productivity annually. The public is increasingly aware that successfully abstaining from smoking at any age can add years to one's life and reduce many of the harmful effects of smoking. Although the majority of smokers desire to quit, only a small fraction of attempts to quit are actually successful. The symptoms associated with nicotine withdrawal are a primary deterrent to cessation and they need to be quelled to avoid early relapse. This review will focus on the neuroadaptations caused by chronic nicotine exposure and discuss how those changes lead to a withdrawal syndrome upon smoking cessation. Besides examining how nicotine usurps the endogenous reward system, we will discuss how the habenula is part of a circuit that plays a critical role in the aversive effects of high nicotine doses and nicotine withdrawal. We will also provide an updated summary of the role of various nicotinic receptor subtypes in the mechanisms of withdrawal. This growing knowledge provides mechanistic insights into current and future smoking cessation therapies.

Aberrant amyloid-beta peptide (Abeta) accumulation along with altered expression and function of nicotinic acetylcholine receptors (nAChRs) stand prominently in the etiology of Alzheimer's disease (AD). Since the discovery that Abeta is bound to alpha7 nAChRs under many experimental settings, including post-mortem AD brain, much effort has been expended to understand the implications of this interaction in the disease milieu. This research update will review the current literature on the alpha7 nAChR-Abeta interaction in vitro and in vivo, the functional consequences of this interaction from sub-cellular to cognitive levels, and discuss the implications these relationships might have for AD therapies.

Although a relative newcomer to the nicotinic acetylcholine receptor (nAChR) family, substantial evidence suggests that alpha6 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 alpha6 containing nAChRs in the rewarding properties of nicotine and in movement. Here, we review the literature on the role of alpha6 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 alpha6 containing nAChRs subtypes in the nigrostriatal and mesolimbic dopamine system are the alpha6beta2beta3 and alpha6alpha4beta2beta3 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 alpha6 containing nAChRs may be of benefit for the treatment of addiction and for neurological disorders with locomotor deficits such as Parkinson's disease.

Pharmacological and immunological methods have been valuable for both identifying some native nicotinic acetylcholine receptor (nAChR) subtypes that exist in vivo and determining the neurobiological and behavioral role of certain nAChR subtypes. However, these approaches suffer from shortage of subtype specific ligands and reliable immunological reagents. Consequently, genetic approaches have been developed to complement earlier approaches to identify native nAChR subtypes and to assess the contribution of nAChRs to brain function and behavior. In this review we describe how assembly partners, knock-in mice and targeted lentiviral re-expression of genes have been utilized to improve our understanding of nAChR neurobiology. In addition, we summarize emerging genetic tools in nAChR research.

This review highlights some of the many contributions of the late Dr. Jerry J. Buccafusco to the neurobiology of nicotinic acetylcholine receptors (nAChRs) and cognition over a 25 year period. The article is written by two of Dr. Buccafusco's professional colleagues, one from academia and one from the pharmaceutical industry. While Dr. Buccafusco's expertise in the cholinergic field was extensive, his insights into the practical relevance of his work (with a long-term goal of formulating new drug development strategies) were unique, and a great asset to both the basic science community and pharmaceutical companies. In 1988, Dr. Buccafusco's laboratory was the first to report the cognitive enhancing action of low doses of nicotine in non-human primates. Since that time he studied a large number of novel pro-cognitive agents from several pharmacological classes in rodents as well as monkeys. Based on years of observing paradoxical effects of nicotinic ligands in vitro and in vivo, Dr. Buccafusco made the provocative argument that it might be possible to develop new chemical entities (with pro-cognitive actions) that have the ability to desensitize nAChRs without producing an antecedent agonist action. Some of his more recent work focused on development of single molecular entities that act on multiple CNS targets (including nAChRs) to enhance cognition, provide neuroprotection, and/or provide additional therapeutic actions (e.g., antipsychotic effects). Dr. Buccafusco's influence will live on in the work of the numerous graduate students, postdoctoral fellows, and junior faculty that he mentored over the years who now serve in prestigious positions throughout the world.

Tobacco smoking is one of the leading causes of disease and premature death in the United States. Nicotine is considered the major reinforcing component in tobacco smoke responsible for tobacco addiction. Nicotine acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The predominant nAChR subtypes in mammalian brain are those containing alpha4 and beta2 subunits. The alpha4beta2 nAChRs, particularly those located in the mesoaccumbens dopamine pathway, play a key role in regulating the reinforcing properties of nicotine. Considering that twelve mammalian nAChR subunits have been cloned, it is likely that nAChRs containing subunits in addition to, or other than, alpha4 and beta2 also play a role in the tobacco smoking habit. Consistent with this possibility, human genome-wide association studies have shown that genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster located in chromosome region 15q25, which encode the alpha5, alpha3 and beta4 nAChR subunits, respectively, increases vulnerability to tobacco addiction and smoking-related diseases. Most recently, alpha5-containing nAChRs located in the habenulo-interpeduncular tract were shown to limit intravenous nicotine self-administration behavior in rats and mice, suggesting that deficits in alpha5-containing nAChR signaling in the habenulo-interpeduncular tract increases vulnerability to the motivational properties of nicotine. Finally, evidence suggests that nAChRs may also play a prominent role in controlling consumption of addictive drugs other than nicotine, including cocaine, alcohol, opiates and cannabinoids. The aim of the present review is to discuss recent preclinical findings concerning the identity of the nAChR subtypes that regulate self-administration of nicotine and other drugs of abuse.

A promising drug target currently under investigation to improve cognitive deficits in neuropsychiatric and neurological disorders is the neuronal nicotinic alpha7 acetylcholine receptor (alpha7nAChR). Improving cognitive impairments in diseases such as Alzheimer's (AD) and schizophrenia remains a large unmet medical need, and the alpha7nAChR has many properties that make it an attractive therapeutic target. The alpha7nAChR is a ligand gated ion channel that has particularly high permeability to Ca(2+) and is expressed in key brain regions involved in cognitive processes (e.g., hippocampus). The alpha7nAChRs are localized both pre-synaptically, where they can regulate neurotransmitter release, and post-synaptically where they can activate intracellular signaling cascades and influence downstream processes involved in learning and memory. In particular, activation of the alpha7nAChR with small molecule agonists enhances long-term potentiation, an in vitro model of synaptic plasticity, and improves performance across multiple cognitive domains in rodents, monkeys, and humans. Positive allosteric modulation of the alpha7nAChR offers an alternate approach to direct agonism that could prove to be particularly beneficial in certain disease populations where smoking nicotine is prevalent (e.g., schizophrenia) and could interfere with an orthosteric agonist approach. The current review focuses on the neurobiology of the alpha7nAChR, its role in cognition and the development status of some of the most promising molecules advancing for the treatment of cognitive dysfunction in disease.

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric alpha7-type receptors. In recent years, alpha7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of alpha7-selective agonists and partial agonists. However, unique properties of alpha7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of alpha7 has come from the identification of alpha7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit alpha7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.

Nicotinic acetylcholine receptors (nAChRs) warrant attention, as they play many critical roles in brain and body function and have been implicated in a number of neurological and psychiatric disorders, including nicotine dependence. nAChRs are composed as diverse subtypes containing specific combinations of genetically-distinct subunits and that have different functional properties, distributions, and pharmacological profiles. There had been confidence that the rules that define ranges of assembly partners for specific subunits were well-established, especially for the more prominent nAChR subtypes. However, we review here some newer findings indicating that nAChRs having largely the same, major subunits exist as isoforms with unexpectedly different properties. Moreover, we also summarize our own studies indicating that novel nAChR subtypes exist and/or have distributions not heretofore described. Importantly, the nAChRs that exist as new isoforms or subtypes or have interesting distributions require alteration in thinking about their roles in health and disease.

alpha6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of alpha6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-alpha6 and beta2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 muM ACh in 26% (5/19) of cells with evenly expressed alpha6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional alpha6-eGFPbeta2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into beta2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased alpha6-eGFPbeta2 nAChR current amplitude. alpha6-eGFPbeta2 nAChRs were also activated by nicotine and by TC-2403. The alpha6-eGFPbeta2 currents were desensitized by 1muM nicotine, blocked by alpha-conotoxin MII, partially inhibited by dihydro-beta-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that alpha6-eGFPbeta2 nAChRs had similar single-channel conductance to, but longer open time than, alpha4-eGFPbeta2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of alpha6* nAChRs for both pharmacological study and drug screening.

Positive modulation of the neuronal nicotinic acetylcholine receptor (nAChR) alpha4beta2 subtype by selective positive allosteric modulator NS-9283 has shown to potentiate the nAChR agonist ABT-594-induced anti-allodynic activity in preclinical neuropathic pain. To determine whether this benefit can be extended beyond neuropathic pain, the present study examined the analgesic activity and adverse effect profile of co-administered NS-9283 and ABT-594 in a variety of preclinical models in rats. The effect of the combined therapy on drug-induced brain activities was also determined using pharmacological magnetic resonance imaging. In carrageenan-induced thermal hyperalgesia, co-administration of NS-9283 (3.5 mumol/kg, i.p.) induced a 6-fold leftward shift of the dose-response of ABT-594 (ED(50)=26 vs. 160 nmol/kg, i.p.). In the paw skin incision model of post-operative pain, co-administration of NS-9283 similarly induced a 6-fold leftward shift of ABT-594 (ED(50)=26 vs. 153 nmol/kg). In monoiodo-acetate induced knee joint pain, co-administration of NS-9283 enhanced the potency of ABT-594 by 5-fold (ED(50)=1.0 vs. 4.6 nmol/kg). In pharmacological MRI, co-administration of NS-9283 was shown to lead to a leftward shift of ABT-594 dose-response for cortical activation. ABT-594 induced CNS-related adverse effects were not exacerbated in presence of an efficacious dose of NS-9283 (3.5 mumol/kg). Acute challenge of NS-9283 produced no cross sensitization in nicotine-conditioned animals. These results demonstrate that selective positive allosteric modulation at the alpha4beta2 nAChR potentiates nAChR agonist-induced analgesic activity across neuropathic and nociceptive preclinical pain models without potentiating ABT-594-mediated adverse effects, suggesting that selective positive modulation of alpha4beta2 nAChR by PAM may represent a novel analgesic approach.