Disruption of Astrocyte-Dependent Dopamine Control in the Developing Medial Prefrontal Cortex Leads to Excessive Grooming in Mice.

BACKGROUND: Astrocytes control synaptic activity by modulating perisynaptic concentrations of ions and neurotransmitters including dopamine (DA) and, as such, could be involved in the modulating aspects of mammalian behavior. METHODS: We produced a conditional deletion of the vesicular monoamine transporter 2 (VMAT2) specifically in astrocytes (aVMTA2cKO mice) and studied the effects of the lack of VMAT2 in prefrontal cortex (PFC) astrocytes on the regulation of DA levels, PFC circuit functions, and behavioral processes. RESULTS: We found a significant reduction of medial PFC (mPFC) DA levels and excessive grooming and compulsive repetitive behaviors in aVMAT2cKO mice. The mice also developed a synaptic pathology, expressed through increased relative AMPA versus NMDA receptor currents in synapses of the dorsal striatum receiving inputs from the mPFC. Importantly, behavioral and synaptic phenotypes were rescued by re-expression of mPFC VMAT2 and L-DOPA treatment, showing that the deficits were driven by mPFC astrocytes that are critically involved in developmental DA homeostasis. By analyzing human tissue samples, we found that VMAT2 is expressed in human PFC astrocytes, corroborating the potential translational relevance of our observations in mice. CONCLUSIONS: Our study shows that impairment of the astrocytic control of DA in the mPFC leads to symptoms resembling obsessive-compulsive spectrum disorders such as trichotillomania and has a profound impact on circuit function and behaviors.

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments.

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.

Biomonitoring of nutrient and toxic element concentrations in the Sarno River through aquatic plants.

The Sarno River is considered the most polluted river in Europe and one of the ten most polluted rivers in the world. So far, its quality has been usually evaluated by water and sediment analyses of either inorganic or organic pollutants. However, a biomonitoring approach would be of paramount importance in the evaluation of river quality, since it integrates pollutant temporal fluctuations, as in the case of discontinuous inputs from urban, industrial and agricultural activities. To this end, a passive biomonitoring study of the Sarno River was carried out, using two native aquatic plants accumulators of inorganic pollutants. The spring area was monitored analysing the roots of the semi-submerged Apium nodiflorum, whereas the whole river course was monitored analysing the shoots of the submerged Potamogeton pectinatus. The information on the four macronutrient (Ca, K, Mg, P), the six micronutrient (Cu, Fe, Mn, Na, Ni, Zn) and the four toxic element (Cd, Cr, Pb, V) concentrations were separately combined in the Nemerow Pollution Index. Results evidenced a severe pollution degree of the Sarno River, attributable to toxic elements > micronutrients > macronutrients. In particular, the spring area showed high K concentrations, as well as high concentrations of several micronutrients and toxic elements. A generalized Zn contamination and a progressive macronutrient (above all Ca and P), micronutrient (above all Ni, Cu and Fe) and toxic element (above all Cr and Pb) accumulation toward the mouth was related to pollution from agricultural and urban activities. Industrial sources, especially tanneries along the Solofrana tributary, accounted for high Mn concentrations, whereas the volcanic origin of the substrate accounted for a generalized V contamination.

Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes.

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.

SHANK3 controls maturation of social reward circuits in the VTA.

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of autism spectrum disorder. How SHANK3 insufficiency affects specific neural circuits and how this is related to specific symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the ventral tegmental area of mice. We identified dopamine (DA) and GABA cell-type-specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate behavioral deficits, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors mGluR1 during the first postnatal week restored DA neuron excitatory synapse transmission and partially rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired ventral tegmental area function to social behaviors and identify mGluR1 modulation during postnatal development as a potential treatment strategy.

Astrocytes and Microglia and Their Potential Link with Autism Spectrum Disorders.

The cellular mechanism(s) underlying autism spectrum disorders (ASDs) are not fully understood although it has been shown that various genetic and environmental factors contribute to their etiology. As increasing evidence indicates that astrocytes and microglial cells play a major role in synapse maturation and function, and there is evidence of deficits in glial cell functions in ASDs, one current hypothesis is that glial dysfunctions directly contribute to their pathophysiology. The aim of this review is to summarize microglia and astrocyte functions in synapse development and their contributions to ASDs.

Design of novel 3,6-diazabicyclo[3.1.1]heptane derivatives with potent and selective affinities for α4ß2 neuronal nicotinic acetylcholine receptors.

New analogues (3a-l) of the previously described α4ß2 selective ligand 3-(6-halopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptanes (2a,b) have been synthesized and their binding activity for neuronal acetylcholine receptor subtypes α4ß2 and α7 were assayed. Six of these compounds (3a,b,c,j,k and l) showed high affinity and selectivity for α4ß2 receptors. The phenylpyridyl-diazabicycloheptane 3c displayed Ki value of 11.17 pM for α4ß2, in line with that of the halogenated homologues 3a,b, although it was characterized by an improved selectivity (Ki = 17 µM for α7 receptors). The influence of substitutions on the phenylpyridyl moiety on binding at both α4ß2 and α7 receptors has been examined through the Topliss decision tree analysis. Substitution with electron-donating groups (as CH3 and OCH3) resulted in a good affinity for α4ß2 receptors and substantially no affinity for α7. Amongst all the tested phenyl-substituted compounds, the p-NO2-phenyl substituted analogue 3j exhibited the highest α4ß2 affinity, with Ki value comparable to that of 3c. Intrinsic α4ß2 receptor mediated activity in [(3)H]-DA release assay was showed by compound 3a as well as by the reference analogue 2a, whereas phenyl substituted derivative 3c exhibited α4ß2 antagonist activity.

Bifunctional compounds targeting both D2 and non-α7 nACh receptors: design, synthesis and pharmacological characterization.

We designed, prepared and tested a set of structural analogs 1-4 as new hybrid compounds by incorporating, through a common alkyl chain of variable length, the pharmacophoric elements of N-n-alkyl nicotinium salts (non-α7 nicotinic acetylcholine receptors antagonists) and of 7-hydroxy-2-(aminomethyl)chromanes (dopaminergic D2 receptor agonists). The target compounds, which were assayed in binding experiments and electrophysiological, functional and Erk1/2 activation tests, essentially combined the pharmacological profiles of their individual receptor ligands. Among the studied derivatives, hybrid 2, one of the shortest homologs, in addition to the antagonist nicotinic profile similar to the other three congeners, behaved as a high affinity ligand at the investigated heteromeric nAChRs and as a low efficacy agonist at D2Rs. These bifunctional derivatives represent novel pharmacological tools in the study of nicotine addiction.

Which lesson can be learnt from a historical contamination analysis of the most polluted river in Europe?

The Sarno River trend analysis during the last 60 years was traced focusing on the socio-economic and environmental issues. The river, originally worshiped as a god by Romans, is affected by an extreme level of environmental degradation, being sadly reputed as the most polluted river in Europe. This is the "not to be followed" example of the worst way a European river can be managed. Data about water, sediment, soil, biota and air contamination were collected from scientific papers, monitoring surveys, and technical reports depicting a sick river. Originally, the river was reputed as a source of livelihood, now it is considered a direct threat for human health. Wastewater can still flow through the river partially or completely untreated, waste production associated with the manufacture of metal products and leather tanning continues to suffer from the historical inadequacy of regional wastewater treatment plants (WWTPs), associated with the partial or no reuse of effluents. All efforts should be devoted to solving the lack of wastewater and waste management, the gap in land planning, improving the capacity of existing WWTPs also via the construction of new sewer sections, restoring Sarno River minimum vital-flow, keeping to a minimum uncontrolled discharges as well as supporting river contracts. The 2015 goal stated by the Water Framework Directive (2000/60/EC) is still far to be reached. The lesson has not been learnt yet.

The cytisine derivatives, CC4 and CC26, reduce nicotine-induced conditioned place preference in zebrafish by acting on heteromeric neuronal nicotinic acetylcholine receptors.

RATIONALE: Cigarette smoking is one of the most serious health problems worldwide and people trying to stop smoking have high rates of relapse. Zebrafish (Danio rerio), by combining pharmacological and behavioral assays, is a promising animal model for rapidly screening new compounds to induce smoking cessation. OBJECTIVES: This study aims to identify possible acetylcholine nicotinic receptors (nAChRs) involved in mediating nicotine (NIC)-induced conditioned place preference (CPP) in zebrafish and investigate the effect of the CC4 and CC26 cytisine derivatives in reducing NIC-induced CPP. METHODS: CPP was evaluated using a two-compartment chamber, and the zebrafish were given CC4 (0.001-5 mg/kg), CC26 (0.001-1 mg/kg), cytisine (0.1-2.5 mg/kg), and varenicline (1-10 mg/kg) alone or with NIC (0.001 mg/kg). Swimming activity was evaluated using a square observational chamber. The affinity of the nicotinic ligands for native zebrafish brain nAChRs was evaluated by binding studies using [(3)H]-Epibatidine (Epi) and [(125)I]-αBungarotoxin (αBgtx) radioligands, and their subtype specificity was determined by means of electrophysiological assay of oocyte-expressed α4ß2 and α7 subtypes. RESULTS: CC4 and CC26 induced CPP with an inverted U-shaped dose-response curve similar to that of NIC. However, when co-administered with NIC, they blocked its reinforcing or slightly aversive effect. Binding and electrophysiological studies showed that this effect was due to binding to high-affinity heteromeric but not α7-containing receptors. CONCLUSIONS: We have further characterized CC4 and identified a new compound (CC26) that may be active in inducing smoking cessation. Zebrafish is a very useful model for screening new compounds that can affect the rewarding properties of NIC.

Analysis of synaptic-like microvesicle exocytosis of B-cells using a live imaging technique.

Pancreatic ß-cells play central roles in blood glucose homeostasis. Beside insulin, these cells release neurotransmitters and other signaling molecules stored in synaptic-like microvesicles (SLMVs). We monitored SLMV exocytosis by transfecting a synaptophysin-pHluorin construct and by visualizing the cells by Total Internal Reflection Fluorescence (TIRF) microscopy. SLMV fusion was elicited by 20 mM glucose and by depolarizing K(+) concentrations with kinetics comparable to insulin secretion. SLMV exocytosis was prevented by Tetanus and Botulinum-C neurotoxins indicating that the fusion machinery of these organelles includes VAMP-2/-3 and Syntaxin-1, respectively. Sequential visualization of SLMVs by TIRF and epifluorescence microscopy showed that after fusion the vesicle components are rapidly internalized and the organelles re-acidified. Analysis of single fusion episodes revealed the existence of two categories of events. While under basal conditions transient fusion events prevailed, long-lasting episodes were more frequent upon secretagogue exposure. Our observations unveiled similarities between the mechanism of exocytosis of insulin granules and SLMVs. Thus, diabetic conditions characterized by defective insulin secretion are most probably associated also with inappropriate release of molecules stored in SLMVs. The assessment of the contribution of SLMV exocytosis to the manifestation of the disease will be facilitated by the use of the imaging approach described in this study.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

CC4, a dimer of cytisine, is a selective partial agonist at α4ß2/α6ß2 nAChR with improved selectivity for tobacco smoking cessation.

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 α4ß2, α3ß4, α7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through α4ß2 and α6ß2 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 α3ß4 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 ß2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation.

The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4ß2 and α7 neuronal nicotinic acetylcholine receptors.

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4ß2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.

Synthesis and binding affinity at α4ß2 and α7 nicotinic acetylcholine receptors of new analogs of epibatidine and epiboxidine containing the 7-azabicyclo[2.2.1]hept-2-ene ring system.

A group of novel racemic nicotinic ligands structurally related to epibatidine or epiboxidine [(±)-10-(±)-17] was synthesized through a palladium-catalyzed cross-coupling between the appropriate vinyl triflate and a range of organometallic heterocycles. The target compounds were evaluated for binding affinity at the α4ß2 and α7 neuronal nicotinic receptors (nAChRs). The set of 3-pyridinyl derivatives (±)-10, (±)-11 and (±)-12 exhibited an affinity for the α4ß2 nAChR subtype in the subnanomolar range (K(i) values of 0.20, 0.40 and 0.50nM, respectively) and behaved as α4ß2 versus α7 subtype selective ligands. Interestingly, the epiboxidine-related dimethylammonium iodide (±)-17, which retained a good affinity for the α4ß2 nAChR (K(i)=13.30nM), tightly bound also to the α7 subtype (K(i)=1.60nM), thus displaying a reversal of the affinity trend among the reference and new nicotinic ligands under investigation.

New spirocyclic Δ²-isoxazoline derivatives related to selective agonists of α7 neuronal nicotinic acetylcholine receptors.

A set of structural analogues of spirocyclic quinuclidinyl-Δ(2)-isoxazolines, characterized as potent and selective α7 nicotinic agonists, was prepared and assayed for binding affinity at α7 and α4ß2 neuronal nicotinic acetylcholine receptors (nAChRs). The investigated derivatives (3a-3c, 4a-4c, 5a-5c, 6a-6c, and 7a-7c), synthesized via the 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles, showed an overall reduced affinity at the α7 subtype when compared with their model compounds. Solely Δ(2)-isoxazolines 3a, 3b, and 6c maintained a binding affinity in the nanomolar range at the α7 nAChRs (K(i) = 230, 420 and 700 nM, respectively). The quaternary ammonium salt 6c retained also a noteworthy α7 vs. α4ß2 subtype selectivity, whereas 3a and 3b showed a sharp reduction in selectivity compared with 1a and 1b, their quinuclidinyl higher homologues.

Unichiral 2-(2'-pyrrolidinyl)-1,4-benzodioxanes: the 2R,2'S diastereomer of the N-methyl-7-hydroxy analogue is a potent α4ß2- and α6ß2-nicotinic acetylcholine receptor partial agonist.

A series of unichiral 7-substituted 2-(1'-methyl-2'-pyrrolidinyl)-1,4-benzodioxanes were synthesized and tested for the affinity for the α4ß2 and α7 central nicotinic receptors; the 2R,2'S diastereomer of the 7-OH analogue [(R,S)-7], unique in the series, has a high α4ß2 affinity (12nM K(i)). N-Demethylation and configuration inversion of the stereocenters greatly weaken its α4ß2 affinity, confirming that such a rigid molecule can be considered a new template for α4ß2 ligands. Docking analysis showed how (R,S)-7 is capable of strongly and specifically interacting with the amino acidic counterpart of the α4ß2 receptor binding site. Further pharmacological characterization demonstrated that (R,S)-7 also has a high affinity for the α6ß2 receptor, and in vitro functional tests indicated that it is a potent α4ß2 and α6ß2 partial agonist, with modest affinity and potency for the α3ß4 receptor. Comparison with varenicline, a well-known nicotinic partial agonist used as a smoking cessation aid, interestingly reveals similar nicotinoid profiles.

Engineering of α-conotoxin MII-derived peptides with increased selectivity for native α6ß2* nicotinic acetylcholine receptors.

α6ß2* Nicotinic acetylcholine receptors are expressed in selected central nervous system areas, where they are involved in striatal dopamine (DA) release and its behavioral consequences, and other still uncharacterized brain activities. α6ß2* receptors are selectively blocked by the α-conotoxins MII and PIA, which bear a characteristic N-terminal amino acid tail [arginine (R), aspartic acid (D), and proline (P)]. We synthesized a group of PIA-related peptides in which R1 was mutated or the RDP motif gradually removed. Binding and striatal DA release assays of native rat α6ß2* receptors showed that the RDP sequence, and particularly residue R1, is essential for the activity of PIA. On the basis of molecular modeling analyses, we synthesized a hybrid peptide (RDP-MII) that had increased potency (7-fold) and affinity (13-fold) for α6ß2* receptors but not for the very similar α3ß2* subtype. As docking studies also suggested that E11 of MII might be a key residue engendering α6ß2* vs. α3ß2* selectivity, we prepared MII[E11R] and RDP-MII[E11R] peptides. Their affinity and potency for native α6ß2* receptors were similar to those of their parent analogues, whereas, for the oocyte expressed rat α3ß2* subtype, they showed a 31- and 14-fold lower affinity and 21- and 3.5-fold lower potency. Thus, MII[E11R] and RDP-MII[E11R] are potent antagonists showing a degree of α6ß2* vs. α3ß2* selectivity in vivo.

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors.

A set of racemic spirocyclic quinuclidinyl-Δ(2)-isoxazoline derivatives was synthesized using a 1,3-dipolar cycloaddition-based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4ß2) nicotinic acetylcholine receptors. Δ(2) -Isoxazolines 3 a (3-Br), 6 a (3-OMe), 5 a (3-Ph), 8 a (3-OnPr), and 4 a (3-Me) were the ligands with the highest affinity for the α7 subtype (K(i) values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM, respectively), and showed excellent α7 versus α4ß2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4ß2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)-3-methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene sesquifumarate 6 a were prepared using (+)-dibenzoyl-L- or (-)-dibenzoyl-D-tartaric acid as resolving agents. Enantiomer (R)-(-)-6 a was found to be the eutomer, with K(i) values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.

Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6ß2 properties: discovery of selective antagonists.

BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of α6ß2-containing (α6ß2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6ß2*-nAChRs has delayed the discovery of α6ß2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6ß2 properties, to enable pharmacological characterization and the identification of novel α6ß2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the α6, ß2, ß3, chimeric α6/3 and mutant ß3(V273S) subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized α6ß2*-nAChRs was determined with ¹²5I-epibatidine binding. KEY RESULTS: Functional channels were detected after co-transfection of α6/3, ß2 and ß3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3ß2ß3(V273S)-nAChR pharmacology was unique, and clearly distinct from α4ß2-, α3ß4-, α7- and α1ß1δε-nAChRs. Antagonist potencies in inhibiting α6/3ß2ß3(V273S) -nAChRs was similar to their binding affinity for rat native α6ß2*-nAChRs. Agonist affinities for α6ß2*-nAChRs was higher than their potency in activating α6/3ß2ß3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3ß2ß3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6ß2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6ß2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6ß2-selective antagonists. We also propose a pharmacophore model of α6ß2 antagonists, which offers a starting point for the development of new smoking cessation agents.