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Heidi C O'Neill,
Duncan C Laverty,
Natalie E Patzlaff,
Bruce N Cohen,
Carlos Fonck,
Sheri McKinney,
J Michael McIntosh,
Jon M Lindstrom,
Henry A Lester, Sharon R Grady,
Michael J Marks
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ABSTRACT: 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) 3min 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.
Pharmacology Biochemistry and Behavior 11/2012; · 2.53 Impact Factor
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Mariaelvina Sala,
Daniela Braida,
Luca Pucci,
Irene Manfredi,
Michael J Marks,
Charles R Wageman, Sharon R Grady,
Barbara Loi,
Sergio Fucile,
Francesca Fasoli,
Michele Zoli,
Bruno Tasso,
Fabio Sparatore,
Francesco Clementi,
Cecilia Gotti
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ABSTRACT: BACKGROUND AND PURPOSE: Many of the addictive and rewarding effects of nicotine are due to its actions on the neuronal nicotinic acetylcholine 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 pre-clinical pharmacology of the cytisine dimer CC4. EXPERIMENTAL APPROACH: The effects of CC4 on nAChRs were investigated using in vitro assays and animal behaviors. 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. Pre-treatment with non-reinforcing doses of CC4 significantly reduced nicotine-induced self-administration and CPP without affecting motor functions. CONCLUSIONS 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.
British Journal of Pharmacology 09/2012; · 4.41 Impact Factor
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Elisha D W Mackey,
Staci E Engle,
Mi Ran Kim,
Heidi C O'Neill,
Charles R Wageman,
Natalie E Patzlaff,
Ying Wang, Sharon R Grady,
J Michael McIntosh,
Michael J Marks,
Henry A Lester,
Ryan M Drenan
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ABSTRACT: 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.
Journal of Neuroscience 07/2012; 32(30):10226-37. · 7.11 Impact Factor
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ABSTRACT: 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.
Neuropharmacology 05/2012; 63(3):450-9. · 4.81 Impact Factor
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ABSTRACT: 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.
Journal of Pharmacology and Experimental Therapeutics 04/2012; 342(2):245-54. · 3.83 Impact Factor
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ABSTRACT: Wild-type and α5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate α-conotoxin
MII (α-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration–effect curves for ACh-stimulated
release (20s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the α5 null mutant.
Deleting the α5 gene also resulted in decreased maximal ACh-stimulated α-CtxMII-resistant DA release. When a shorter perfusion
time (5s) was used, biphasic curves were detected in both wild-type and α5 null mutants, indicative of high- and low-sensitivity
(HS and LS) activity. In addition, DHβE-sensitive (HS) and DHβE-resistant (LS) components were found in both genotypes. These
results indicate that α-CtxMII-resistant DA release is mediated by α4α5β2, (α4)2(β2)3 (HS), and (α4)3(β2)2 (LS) nAChRs.
Journal of Molecular Neuroscience 04/2012; 40(1):91-95. · 2.50 Impact Factor
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ABSTRACT: 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 α7, β2, or β4 subunit. In agreement with general belief, the α4β2*-nAChR and α7-nAChR subtypes are major components
of the epibatidine binding sites. α4β2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine
binding sites, while α7-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.
Journal of Molecular Neuroscience 04/2012; 40(1):96-104. · 2.50 Impact Factor
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ABSTRACT: 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.
Journal of Neurochemistry 04/2012; 122(1):48-57. · 4.06 Impact Factor
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ABSTRACT: 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.
Neuropharmacology 04/2012; 62(5-6):1935-43. · 4.81 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Nicotine & Tobacco Research 01/2012; 14(6):711-9. · 2.58 Impact Factor
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ABSTRACT: 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.
Journal of Neuroscience 07/2011; 31(30):10891-902. · 7.11 Impact Factor
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ABSTRACT: 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.
Biochemical pharmacology 06/2011; 82(8):873-82. · 4.25 Impact Factor
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ABSTRACT: 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.
Journal of Pharmacology and Experimental Therapeutics 06/2011; 338(3):932-41. · 3.83 Impact Factor
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ABSTRACT: 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.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 02/2011; 36(5):1021-32. · 6.99 Impact Factor
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ABSTRACT: 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.
Journal of Neuroscience 07/2010; 30(29):9877-89. · 7.11 Impact Factor
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Sharon R Grady,
Ryan M Drenan,
Scott R Breining,
Daniel Yohannes,
Charles R Wageman,
Nikolai B Fedorov,
Sheri McKinney,
Paul Whiteaker,
Merouane Bencherif,
Henry A Lester,
Michael J Marks
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ABSTRACT: 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.
Neuropharmacology 06/2010; 58(7):1054-66. · 4.81 Impact Factor
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ABSTRACT: 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.
The FASEB Journal 09/2009; 24(1):49-57. · 5.71 Impact Factor
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ABSTRACT: 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.
Bioorganic & medicinal chemistry letters 06/2009; 19(15):4359-63. · 2.65 Impact Factor
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ABSTRACT: 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 <or=300 microM at alpha3beta4-nAChR measured by [(3)H]-acetylcholine release from interpeduncular nucleus. Thus, ABT-089 is a beta2* nAChR ligand with demonstrable agonist activity at alpha4beta2* and alpha6beta2* receptors. As one form of alpha6beta2* nAChR is sensitive to sub-muM concentrations, we propose that this receptor in particular may contribute to the enhanced cognitive performance following low doses of ABT-089.
Biochemical pharmacology 05/2009; 78(7):795-802. · 4.25 Impact Factor
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ABSTRACT: 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.
Journal of Neuroscience 03/2009; 29(7):2272-82. · 7.11 Impact Factor
