Grady SR


Full name : Grady Sharon R

First name : Sharon R

Mail : Institute for Behavioral Genetics, University of Colorado, 447UCB, Boulder, CO 80409

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References (42)

Title : Effectiveness of Nicotinic Agonists as Desensitizers at Presynaptic alpha4beta2- and alpha4alpha5beta2-Nicotinic Acetylcholine Receptors - Wageman_2014_Nicotine.Tob.Res_16_297
Author(s) : Wageman CR , Marks MJ , Grady SR
Ref : Nicotine Tob Res , 16 :297 , 2014
Abstract : INTRODUCTION: Nicotine interacts with nicotinic acetylcholine receptors (nAChRs) and modifies neuronal functions. The net result of nicotine exposure is difficult to assess because multiple nAChR subtypes exist and are expressed on multiple classes of neurons. Nicotine, unlike the natural agonist acetylcholine, remains in tissues for hours, and during this extended exposure nAChRs desensitize. Therefore, agonists can block the natural functions of nAChRs. Higher nicotine concentrations are required to desensitize alpha4beta2-nAChRs containing the alpha5 subunit. The aim of these experiments was to determine if this property holds true for compounds other than nicotine.
METHODS: [(3)H]-dopamine release from crude mouse striatal synaptosomal preparations was used to measure activation and desensitization of the [(alpha4beta2)2beta2] and [(alpha4beta2)2alpha5] nAChR subtypes. Affinity was measured by competition with [(125)I]-epibatidine.
RESULTS: Nine compounds of varying affinity and efficacy were tested. All compounds partially desensitized both subtypes; concentration necessary for desensitization correlated with binding site affinity but not efficacy. All compounds showed a similar, significant shift in concentration necessary for a 50% effect when the alpha5 subunit was included (averaging 8-fold higher). The extent of desensitization produced by a 10-min exposure did not correlate with affinity or efficacy of compound. CONCLUSION: Full or partial nicotinic agonists used as medications may effectively desensitize alpha4beta2-nAChRs. However, significantly higher concentrations of all compounds tested were required to elicit desensitization of alpha4alpha5beta2-nAChRs than alpha4beta2-nAChRs. If desensitization is the important property for a smoking cessation drug, basic screening at both subtypes may provide a mechanistic foundation for effectiveness.
ESTHER : Wageman_2014_Nicotine.Tob.Res_16_297
PubMedSearch : Wageman_2014_Nicotine.Tob.Res_16_297
PubMedID: 24052501

Title : Multiple CNS nicotinic receptors mediate L-dopa-induced dyskinesias: studies with parkinsonian nicotinic receptor knockout mice - Quik_2013_Biochem.Pharmacol_86(8)_1153
Author(s) : Quik M , Campos C , Grady SR
Ref : Biochemical Pharmacology , 86 :1153 , 2013
Abstract : Accumulating evidence supports the idea that drugs acting at nicotinic acetylcholine receptors (nAChRs) may be beneficial for Parkinson's disease, a neurodegenerative movement disorder characterized by a loss of nigrostriatal dopaminergic neurons. Nicotine administration to parkinsonian animals protects against nigrostriatal damage. In addition, nicotine and nAChR drugs improve L-dopa-induced dyskinesias, a debilitating side effect of L-dopa therapy which remains the gold-standard treatment for Parkinson's disease. Nicotine exerts its antidyskinetic effect by interacting with multiple nAChRs. One approach to identify the subtypes specifically involved in L-dopa-induced dyskinesias is through the use of nAChR subunit null mutant mice. Previous work with beta2 and alpha6 nAChR knockout mice has shown that alpha6beta2* nAChRs were necessary for the development/maintenance of L-dopa-induced abnormal involuntary movements (AIMs). The present results in parkinsonian alpha4 nAChR knockout mice indicate that alpha4beta2* nAChRs also play an essential role since nicotine did not reduce L-dopa-induced AIMs in such mice. Combined analyses of the data from alpha4 and alpha6 knockout mice suggest that the alpha6alpha4beta2beta3 subtype may be critical. In contrast to the studies with alpha4 and alpha6 knockout mice, nicotine treatment did reduce L-dopa-induced AIMs in parkinsonian alpha7 nAChR knockout mice. However, alpha7 nAChR subunit deletion alone increased baseline AIMs, suggesting that alpha7 receptors exert an inhibitory influence on L-dopa-induced AIMs. In conclusion, alpha6beta2*, alpha4beta2* and alpha7 nAChRs all modulate L-dopa-induced AIMs, although their mode of regulation varies. Thus drugs targeting one or multiple nAChRs may be optimal for reducing L-dopa-induced dyskinesias in Parkinson's disease.
ESTHER : Quik_2013_Biochem.Pharmacol_86(8)_1153
PubMedSearch : Quik_2013_Biochem.Pharmacol_86(8)_1153
PubMedID: 23831952

Title : A role for alpha4(non-alpha6)* nicotinic acetylcholine receptors in motor behavior - Soll_2013_Neuropharmacol_73_19
Author(s) : Soll LG , Grady SR , Salminen O , Marks MJ , Tapper AR
Ref : Neuropharmacology , 73 :19 , 2013
Abstract : Nicotinic acetylcholine receptors (nAChRs) containing either the alpha4 and/or alpha6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including alpha4 and alpha6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the alpha4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the alpha4 nAChR subunit, Leu9'Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9'Ala mice with the alpha4beta2 nAChR antagonist dihydro-beta-erythroidine (DHbetaE). Interestingly, in Leu9'Ala mice, DHbetaE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHbetaE-induced motor impairment in Leu9'Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHbetaE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9'Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9'Ala striatal synaptosomes revealed agonist hypersensitivity only at alpha4(non-alpha6)* nAChRs. Similarly, alpha6 nAChR subunit deletion in an alpha4 hypersensitive nAChR (Leu9'Ala/alpha6 KO) background had little effect on the DHbetaE-induced phenotype, suggesting an alpha4(non-alpha6)* nAChR-dependent mechanism. Together, these data indicate that alpha4(non-alpha6)* nAChR have an impact on motor output and may be potential molecular targets for treatment of disorders associated with motor impairment.
ESTHER : Soll_2013_Neuropharmacol_73_19
PubMedSearch : Soll_2013_Neuropharmacol_73_19
PubMedID: 23688922

Title : Exploring the nicotinic acetylcholine receptor-associated proteome with iTRAQ and transgenic mice - McClure-Begley_2013_Genomics.Proteomics.Bioinformatics_11_207
Author(s) : McClure-Begley TD , Stone KL , Marks MJ , Grady SR , Colangelo CM , Lindstrom JM , Picciotto MR
Ref : Genomics Proteomics Bioinformatics , 11 :207 , 2013
Abstract : Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha4 and beta2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between alpha4beta2-containing (alpha4beta2( *)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with alpha4beta2( *) nAChRs in a genedose dependent pattern by immunopurifying beta2( *) nAChRs from mice that differ in alpha4 and beta2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the alpha4 or the beta2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of alpha4beta2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a beta2( *) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of alpha4beta2 nAChRs and their downstream signaling mechanisms.
ESTHER : McClure-Begley_2013_Genomics.Proteomics.Bioinformatics_11_207
PubMedSearch : McClure-Begley_2013_Genomics.Proteomics.Bioinformatics_11_207
PubMedID: 23891776

Title : CC4, a dimer of cytisine, is a selective partial agonist at alpha4beta2\/alpha6beta2 nAChR with improved selectivity for tobacco smoking cessation - Sala_2013_Br.J.Pharmacol_168_835
Author(s) : Sala M , Braida D , Pucci L , Manfredi I , Marks MJ , Wageman CR , Grady SR , Loi B , Fucile S , Fasoli F , Zoli M , Tasso B , Sparatore F , Clementi F , Gotti C
Ref : British Journal of Pharmacology , 168 :835 , 2013
Abstract : 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 alpha4beta2, alpha3beta4, alpha7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through alpha4beta2 and alpha6beta2 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 alpha3beta4 and alpha7-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 beta2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation.
ESTHER : Sala_2013_Br.J.Pharmacol_168_835
PubMedSearch : Sala_2013_Br.J.Pharmacol_168_835
PubMedID: 22957729

Title : Mice expressing the ADNFLE valine 287 leucine mutation of the Beta2 nicotinic acetylcholine receptor subunit display increased sensitivity to acute nicotine administration and altered presynaptic nicotinic receptor function - O'Neill_2013_Pharmacol.Biochem.Behav_103_603
Author(s) : O'Neill HC , Laverty DC , Patzlaff NE , Cohen BN , Fonck C , McKinney S , McIntosh JM , Lindstrom JM , Lester HA , Grady SR , Marks MJ
Ref : Pharmacol Biochem Behav , 103 :603 , 2013
Abstract : Several mutations in alpha4 or beta2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the beta2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (beta2VL). Previous studies indicated that the beta2VL 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 beta2VL mice. No differences in beta2 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 beta2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.
ESTHER : O'Neill_2013_Pharmacol.Biochem.Behav_103_603
PubMedSearch : O'Neill_2013_Pharmacol.Biochem.Behav_103_603
PubMedID: 23123803

Title : Role for alpha6 nicotinic receptors in l-dopa-induced dyskinesias in parkinsonian mice - Quik_2012_Neuropharmacol_63_450
Author(s) : Quik M , Park KM , Hrachova M , Mallela A , Huang LZ , McIntosh JM , Grady SR
Ref : Neuropharmacology , 63 :450 , 2012
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 alpha6beta2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using a6 nAChR subunit null mutant (alpha6(-)/(-)) mice.Wildtype and alpha6(-)/(-) 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 alpha6(-)/(-) and wildtype mice.However, AIMs in alpha6(-)/(-) 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 alpha6(-)/(-) 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 alpha6(-)/(-) mice treated with and without nicotine indicate an essential role for alpha6beta2* nAChRs in the maintenance of L-dopa-induced AIMs.These findings suggest that alpha6beta2* nAChR drugs have potential for reducing L-dopa-induced dyskinesias in Parkinson's disease.
ESTHER : Quik_2012_Neuropharmacol_63_450
PubMedSearch : Quik_2012_Neuropharmacol_63_450
PubMedID: 22579614

Title : Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior - Cohen_2012_Neurosci_200_31
Author(s) : Cohen BN , Mackey ED , Grady SR , McKinney S , Patzlaff NE , Wageman CR , McIntosh JM , Marks MJ , Lester HA , Drenan RM
Ref : Neuroscience , 200 :31 , 2012
Abstract : Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of alpha6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) alpha6 subunits. alpha6 L9'S mice harboring six or more copies of the hypersensitive alpha6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S alpha6 genes had locomotor activity resembling that of control mice. alpha6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy alpha6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy alpha6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy alpha6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of alpha6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in alpha6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of alpha6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release.
ESTHER : Cohen_2012_Neurosci_200_31
PubMedSearch : Cohen_2012_Neurosci_200_31
PubMedID: 22079576

Title : alpha6* nicotinic acetylcholine receptor expression and function in a visual salience circuit - Mackey_2012_J.Neurosci_32_10226
Author(s) : Mackey ED , Engle SE , Kim MR , O'Neill HC , Wageman CR , Patzlaff NE , Wang Y , Grady SR , McIntosh JM , Marks MJ , Lester HA , Drenan RM
Ref : Journal of Neuroscience , 32 :10226 , 2012
Abstract : Nicotinic acetylcholine receptors (nAChRs) containing alpha6 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 alpha6-containing nAChRs, we created and studied transgenic mice expressing a variant alpha6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In alpha6-GFP transgenic mice, alpha6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong alpha6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found alpha6 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 alpha6* nAChRs, we confirmed functional, postsynaptic alpha6* nAChR expression. Further, sSC GABAergic neurons expressing alpha6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive alpha6* nAChRs by ACh. alpha6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of alpha6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that alpha6* 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 alpha6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.
ESTHER : Mackey_2012_J.Neurosci_32_10226
PubMedSearch : Mackey_2012_J.Neurosci_32_10226
PubMedID: 22836257

Title : Role of alpha6 nicotinic receptors in CNS dopaminergic function: relevance to addiction and neurological disorders - Quik_2011_Biochem.Pharmacol_82(8)_873
Author(s) : Quik M , Perez XA , Grady SR
Ref : Biochemical Pharmacology , 82 :873 , 2011
Abstract : 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.
ESTHER : Quik_2011_Biochem.Pharmacol_82(8)_873
PubMedSearch : Quik_2011_Biochem.Pharmacol_82(8)_873
PubMedID: 21684266

Title : Poster: Comparison of binding and functional activity profiles for a set of nicotinic acetylcholine receptor ligands across multiple alpha6* expression systems -
Author(s) : Breining SR , Hepler C , Whiteaker P , Quik M , Grady SR , Yohannes D
Ref : Biochemical Pharmacology , 82 :1024 , 2011

Title : Poster: alpha6* nAChR expression and function in brain areas influencing DA transmission probed with alpha6-GFP transgenic mice -
Author(s) : Drenan RM , Mackey EDW , Grady SR , Patzlaff NE , Wageman CR , McIntosh JM , Marks MJ , Lester HA
Ref : Biochemical Pharmacology , 82 :1035 , 2011

Title : Nicotine-mediated activation of dopaminergic neurons in distinct regions of the ventral tegmental area - Zhao-Shea_2011_Neuropsychopharmacology_36_1021
Author(s) : Zhao-Shea R , Liu L , Soll LG , Improgo MR , Meyers EE , McIntosh JM , Grady SR , Marks MJ , Gardner PD , Tapper AR
Ref : Neuropsychopharmacology , 36 :1021 , 2011
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 muM) 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 alpha4, alpha6, and beta3 transcripts than did aVTA DAergic neurons. Activation of nAChRs containing the alpha4 subunit (alpha4(*) nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: nicotine failed to activate pVTA DAergic neurons in alpha4 knockout animals; in contrast, pVTA alpha4(*) nAChRs were selectively activated by nicotine in mutant mice expressing agonist-hypersensitive alpha4(*) nAChRs (Leu9'Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by alpha4(*) nAChRs and were significantly larger in pVTA neurons than in aVTA neurons. Infusion of an alpha6(*) 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 alpha4 and alpha6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA through alpha4alpha6(*) nAChRs. These receptors represent novel targets for smoking-cessation therapies.
ESTHER : Zhao-Shea_2011_Neuropsychopharmacology_36_1021
PubMedSearch : Zhao-Shea_2011_Neuropsychopharmacology_36_1021
PubMedID: 21289604

Title : alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief - McGranahan_2011_J.Neurosci_31_10891
Author(s) : McGranahan TM , Patzlaff NE , Grady SR , Heinemann SF , Booker TK
Ref : Journal of Neuroscience , 31 :10891 , 2011
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 alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although alpha4-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 alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. alpha4-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. alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of alpha4beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-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.
ESTHER : McGranahan_2011_J.Neurosci_31_10891
PubMedSearch : McGranahan_2011_J.Neurosci_31_10891
PubMedID: 21795541

Title : Nicotine reduces L-DOPA-induced dyskinesias by acting at beta2* nicotinic receptors - Huang_2011_J.Pharmacol.Exp.Ther_338_932
Author(s) : Huang LZ , Grady SR , Quik M
Ref : Journal of Pharmacology & Experimental Therapeutics , 338 :932 , 2011
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 beta2 nAChR subunit knockout [beta2(-/-)] mice because beta2-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 beta2(-/-) 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 beta2(-/-) mice compared with the wild-type mice. It is interesting to note that nicotine (300 mug/ml in drinking water) reduced L-DOPA-induced AIMs by 40% in wild-type mice but had no effect in beta2(-/-) 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 beta2* 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.
ESTHER : Huang_2011_J.Pharmacol.Exp.Ther_338_932
PubMedSearch : Huang_2011_J.Pharmacol.Exp.Ther_338_932
PubMedID: 21665941

Title : Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive {alpha}4 nicotinic receptors via a cholinergic-dependent mechanism - Zhao-Shea_2010_FASEB.J_24_49
Author(s) : Zhao-Shea R , Cohen BN , Just H , McClure-Begley TD , Whiteaker P , Grady SR , Salminen O , Gardner PD , Lester HA , Tapper AR
Ref : FASEB Journal , 24 :49 , 2010
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.
ESTHER : Zhao-Shea_2010_FASEB.J_24_49
PubMedSearch : Zhao-Shea_2010_FASEB.J_24_49
PubMedID: 19720621

Title : Mouse striatal dopamine nerve terminals express alpha4alpha5beta2 and two stoichiometric forms of alpha4beta2*-nicotinic acetylcholine receptors - Grady_2010_J.Mol.Neurosci_40_91
Author(s) : Grady SR , Salminen O , McIntosh JM , Marks MJ , Collins AC
Ref : Journal of Molecular Neuroscience , 40 :91 , 2010
Abstract : Wild-type and alpha5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate alpha-conotoxin MII (alpha-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration-effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the alpha5 null mutant. Deleting the alpha5 gene also resulted in decreased maximal ACh-stimulated alpha-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and alpha5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHbetaE-sensitive (HS) and DHbetaE-resistant (LS) components were found in both genotypes. These results indicate that alpha-CtxMII-resistant DA release is mediated by alpha4alpha5beta2, (alpha4)(2)(beta2)(3) (HS), and (alpha4)(3)(beta2)(2) (LS) nAChRs.
ESTHER : Grady_2010_J.Mol.Neurosci_40_91
PubMedSearch : Grady_2010_J.Mol.Neurosci_40_91
PubMedID: 19693710

Title : John Daly's compound, epibatidine, facilitates identification of nicotinic receptor subtypes - Marks_2010_J.Mol.Neurosci_40_96
Author(s) : Marks MJ , Laverty DS , Whiteaker P , Salminen O , Grady SR , McIntosh JM , Collins AC
Ref : Journal of Molecular Neuroscience , 40 :96 , 2010
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 alpha7, beta2, or beta4 subunit. In agreement with general belief, the alpha4beta2*-nAChR and alpha7-nAChR subtypes are major components of the epibatidine binding sites. alpha4beta2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while alpha7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.
ESTHER : Marks_2010_J.Mol.Neurosci_40_96
PubMedSearch : Marks_2010_J.Mol.Neurosci_40_96
PubMedID: 19672723

Title : Structural differences determine the relative selectivity of nicotinic compounds for native alpha 4 beta 2*-, alpha 6 beta 2*-, alpha 3 beta 4*- and alpha 7-nicotine acetylcholine receptors - Grady_2010_Neuropharmacol_58_1054
Author(s) : Grady SR , Drenan RM , Breining SR , Yohannes D , Wageman CR , Fedorov NB , McKinney S , Whiteaker P , Bencherif M , Lester HA , Marks MJ
Ref : Neuropharmacology , 58 :1054 , 2010
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.
ESTHER : Grady_2010_Neuropharmacol_58_1054
PubMedSearch : Grady_2010_Neuropharmacol_58_1054
PubMedID: 20114055

Title : Cholinergic modulation of locomotion and striatal dopamine release is mediated by alpha6alpha4* nicotinic acetylcholine receptors - Drenan_2010_J.Neurosci_30_9877
Author(s) : Drenan RM , Grady SR , Steele AD , McKinney S , Patzlaff NE , McIntosh JM , Marks MJ , Miwa JM , Lester HA
Ref : Journal of Neuroscience , 30 :9877 , 2010
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.
ESTHER : Drenan_2010_J.Neurosci_30_9877
PubMedSearch : Drenan_2010_J.Neurosci_30_9877
PubMedID: 20660270

Title : Evaluation of structurally diverse neuronal nicotinic receptor ligands for selectivity at the alpha6( *) subtype - Breining_2009_Bioorg.Med.Chem.Lett_19_4359
Author(s) : Breining SR , Bencherif M , Grady SR , Whiteaker P , Marks MJ , Wageman CR , Lester HA , Yohannes D
Ref : Bioorganic & Medicinal Chemistry Lett , 19 :4359 , 2009
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.
ESTHER : Breining_2009_Bioorg.Med.Chem.Lett_19_4359
PubMedSearch : Breining_2009_Bioorg.Med.Chem.Lett_19_4359
PubMedID: 19560354

Title : Poster: Selectivity of ABT-089 for alpha4\/beta2* and alpha6\/beta2 nicotinic acetylcholine receptors in brain -
Author(s) : Marks MJ , Wageman CR , Grady SR , Gopalakrishnan M , Briggs CA
Ref : Biochemical Pharmacology , 78 :910 , 2009

Title : Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the alpha3beta4* and alpha3beta3beta4* subtypes mediate acetylcholine release - Grady_2009_J.Neurosci_29_2272
Author(s) : Grady SR , Moretti M , Zoli M , Marks MJ , Zanardi A , Pucci L , Clementi F , Gotti C
Ref : Journal of Neuroscience , 29 :2272 , 2009
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.
ESTHER : Grady_2009_J.Neurosci_29_2272
PubMedSearch : Grady_2009_J.Neurosci_29_2272
PubMedID: 19228980

Title : Selectivity of ABT-089 for alpha4beta2* and alpha6beta2* nicotinic acetylcholine receptors in brain - Marks_2009_Biochem.Pharmacol_78(7)_795
Author(s) : Marks MJ , Wageman CR , Grady SR , Gopalakrishnan M , Briggs CA
Ref : Biochemical Pharmacology , 78 :795 , 2009
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.
ESTHER : Marks_2009_Biochem.Pharmacol_78(7)_795
PubMedSearch : Marks_2009_Biochem.Pharmacol_78(7)_795
PubMedID: 19481067

Title : Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning - Mineur_2009_Genes.Brain.Behav_8_257
Author(s) : Mineur YS , Brunzell DH , Grady SR , Lindstrom JM , McIntosh JM , Marks MJ , King SL , Picciotto MR
Ref : Genes Brain Behav , 8 :257 , 2009
Abstract : High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.
ESTHER : Mineur_2009_Genes.Brain.Behav_8_257
PubMedSearch : Mineur_2009_Genes.Brain.Behav_8_257
PubMedID: 19077117

Title : In vivo activation of midbrain dopamine neurons via sensitized, high-affinity alpha 6 nicotinic acetylcholine receptors - Drenan_2008_Neuron_60_123
Author(s) : Drenan RM , Grady SR , Whiteaker P , McClure-Begley TD , McKinney S , Miwa JM , Bupp S , Heintz N , McIntosh JM , Bencherif M , Marks MJ , Lester HA
Ref : Neuron , 60 :123 , 2008
Abstract : Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.
ESTHER : Drenan_2008_Neuron_60_123
PubMedSearch : Drenan_2008_Neuron_60_123
PubMedID: 18940593

Title : The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum - Grady_2007_Biochem.Pharmacol_74(8)_1235
Author(s) : Grady SR , Salminen O , Laverty DC , Whiteaker P , McIntosh JM , Collins AC , Marks MJ
Ref : Biochemical Pharmacology , 74 :1235 , 2007
Abstract : This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and alpha-conotoxin MII (alpha-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the alpha6 subunit (alpha4alpha6beta2beta3, alpha6beta2beta3, alpha6beta2) and bind alpha-CtxMII with high affinity. One of these three subtypes (alpha4alpha6beta2beta3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for alpha-CtxMII (alpha4beta2, alpha4alpha5beta2) are somewhat more numerous than the alpha6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.
ESTHER : Grady_2007_Biochem.Pharmacol_74(8)_1235
PubMedSearch : Grady_2007_Biochem.Pharmacol_74(8)_1235
PubMedID: 17825262

Title : Nigrostriatal damage preferentially decreases a subpopulation of alpha6beta2* nAChRs in mouse, monkey, and Parkinson's disease striatum - Bordia_2007_Mol.Pharmacol_72_52
Author(s) : Bordia T , Grady SR , McIntosh JM , Quik M
Ref : Molecular Pharmacology , 72 :52 , 2007
Abstract : Parkinson's disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the alpha6beta2(*) nicotinic acetylcholine receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum ((*)denotes the presence of possible additional subunits). In this study, we used a novel alpha-conotoxin MII (alpha-CtxMII) analog E11A to further investigate alpha6beta2(*) nAChR subtypes in mouse, monkey, and human striatum. Receptor competition studies with (125)I-alpha-CtxMII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct alpha6beta2(*) nAChR subtypes. These include a very high (femtomolar) and a high (picomolar) affinity site, with approximately 40% of the sites in the very high affinity form. It is noteworthy that only the high-affinity form was detected in alpha4 nAChR-null mutant mice. Because (125)I-alpha-CtxMII binds primarily to alpha6alpha4beta2beta3 and alpha6beta2beta3 nAChR subtypes in mouse striatum, these data suggest that the population lost in the alpha4 knockout mice was the alpha6alpha4beta2beta3 subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal alpha6beta2(*) populations in two animal models and in Parkinson's disease. There was a preferential loss of the very high affinity subtype in striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), monkeys treated with MPTP, and patients with Parkinson's disease. These data suggest that dopaminergic terminals expressing the alpha6alpha4beta2beta3 population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with alpha-CtxMII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson's disease.
ESTHER : Bordia_2007_Mol.Pharmacol_72_52
PubMedSearch : Bordia_2007_Mol.Pharmacol_72_52
PubMedID: 17409284

Title : Nicotine-induced dystonic arousal complex in a mouse line harboring a human autosomal-dominant nocturnal frontal lobe epilepsy mutation - Teper_2007_J.Neurosci_27_10128
Author(s) : Teper Y , Whyte D , Cahir E , Lester HA , Grady SR , Marks MJ , Cohen BN , Fonck C , McClure-Begley TD , McIntosh JM , Labarca C , Lawrence A , Chen F , Gantois I , Davies PJ , Petrou S , Murphy M , Waddington J , Horne MK , Berkovic SF , Drago J
Ref : Journal of Neuroscience , 27 :10128 , 2007
Abstract : We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.
ESTHER : Teper_2007_J.Neurosci_27_10128
PubMedSearch : Teper_2007_J.Neurosci_27_10128
PubMedID: 17881519

Title : Guidelines on nicotine dose selection for in vivo research - Matta_2007_Psychopharmacology.(Berl)_190_269
Author(s) : Matta SG , Balfour DJ , Benowitz NL , Boyd RT , Buccafusco JJ , Caggiula AR , Craig CR , Collins AC , Damaj MI , Donny EC , Gardiner PS , Grady SR , Heberlein U , Leonard SS , Levin ED , Lukas RJ , Markou A , Marks MJ , McCallum SE , Parameswaran N , Perkins KA , Picciotto MR , Quik M , Rose JE , Rothenfluh A , Schafer WR , Stolerman IP , Tyndale RF , Wehner JM , Zirger JM
Ref : Psychopharmacology (Berl) , 190 :269 , 2007
Abstract : RATIONALE: This review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure. OBJECTIVES: This review capitalizes on the authors' collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.
RESULTS: Seven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.
CONCLUSIONS: The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.
ESTHER : Matta_2007_Psychopharmacology.(Berl)_190_269
PubMedSearch : Matta_2007_Psychopharmacology.(Berl)_190_269
PubMedID: 16896961

Title : Chronic nicotine cell specifically upregulates functional alpha 4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path - Nashmi_2007_J.Neurosci_27_8202
Author(s) : Nashmi R , Xiao C , Deshpande P , McKinney S , Grady SR , Whiteaker P , Huang Q , McClure-Begley TD , Lindstrom JM , Labarca C , Collins AC , Marks MJ , Lester HA
Ref : Journal of Neuroscience , 27 :8202 , 2007
Abstract : Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.
ESTHER : Nashmi_2007_J.Neurosci_27_8202
PubMedSearch : Nashmi_2007_J.Neurosci_27_8202
PubMedID: 17670967

Title : Long-term nicotine treatment decreases striatal alpha 6* nicotinic acetylcholine receptor sites and function in mice - Lai_2005_Mol.Pharmacol_67_1639
Author(s) : Lai A , Parameswaran N , Khwaja M , Whiteaker P , Lindstrom JM , Fan H , McIntosh JM , Grady SR , Quik M
Ref : Molecular Pharmacology , 67 :1639 , 2005
Abstract : Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) are distinct from other subtypes in their relatively restricted localization to the striatum and some other brain regions. The effect of nicotine treatment on nAChR subtypes has been extensively investigated, with the exception of changes in alpha-conotoxin MII-sensitive receptor expression. We therefore determined the consequence of long-term nicotine administration on this subtype and its function. Nicotine was given in drinking water to provide a long-term yet intermittent treatment. Consistent with previous studies, nicotine exposure increased 125I-epibatidine and 125I-A85380 (3-[2-(S)-azetidinylmethoxy]pyridine), but not 125I-alpha-bungarotoxin, receptors in cortex and striatum. We observed an unexpected reduction (30%) in striatal 125I-alpha-conotoxin MII sites, which occurred because of a decrease in B(max). This decline was more robust in older (>8-month-old) compared with younger (2-4-month-old) mice, suggesting age is important for nicotine-induced disruption of nAChR phenotype. Immunoprecipitation experiments using nAChR subunit-directed antibodies indicate that alterations in subunit-immunoreactivity with nicotine treatment agree with those in the receptor binding studies. To determine the relationship between striatal nAChR sites and function, we measured nicotine-evoked [3H]dopamine release. A decline was obtained with nicotine treatment that was caused by a selective decrease in alpha-conotoxin MII-sensitive but not alpha-conotoxin MII-resistant dopamine release. These results may explain previous findings that nicotine treatment decreased striatal nAChR-mediated dopamine function, despite an increase in [3H]nicotine (alpha4*) sites. The present data suggest that the alpha6* nAChR subtype represents a key factor in the control of dopamine release from striatum, which adapts to long-term nicotine treatment by down-regulation of alpha6* receptor sites and function.
ESTHER : Lai_2005_Mol.Pharmacol_67_1639
PubMedSearch : Lai_2005_Mol.Pharmacol_67_1639
PubMedID: 15681595

Title : Decrease in alpha3*\/alpha6* nicotinic receptors but not nicotine-evoked dopamine release in monkey brain after nigrostriatal damage - McCallum_2005_Mol.Pharmacol_68_737
Author(s) : McCallum SE , Parameswaran N , Bordia T , McIntosh JM , Grady SR , Quik M
Ref : Molecular Pharmacology , 68 :737 , 2005
Abstract : Nicotinic acetylcholine receptors (nAChRs) are decreased in the striata of patients with Parkinson's disease (PD) or in experimental models after nigrostriatal damage. Because presynaptic nAChRs on striatal dopamine terminals mediate dopamine release, receptor loss may contribute to behavioral deficits in PD. The present experiments were done to determine whether nAChR function is affected by nigrostriatal damage in nonhuman primates, because this model shares many features with PD. Initial characterization of nicotine-evoked [3H]dopamine release from monkey striatal synaptosomes revealed that release was calcium-dependent and inhibited by selective nAChR antagonists. It is noteworthy that a greater proportion (approximately 70%) of release was inhibited by the alpha3*/alpha6* antagonist alpha-conotoxinMII (alpha-CtxMII) compared with rodents. Monkeys were lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and [3H]dopamine release, dopamine transporter, and nAChRs were measured. As anticipated, lesioning decreased the transporter and alpha3*/alpha6* nAChRs in caudate and putamen. In contrast, alpha3*/alpha6* nAChR-evoked [3H]dopamine release was reduced in caudate but not putamen, demonstrating a dissociation between nAChR sites and function. A different pattern was observed in the mesolimbic dopamine system. Dopamine transporter levels in nucleus accumbens were not reduced after MPTP, as expected; however, there was a 50% decline in alpha3*/alpha6* nAChR sites with no decrease in alpha3*/alpha6* receptor-evoked dopamine release. No declines in alpha-CtxMII-resistant nAChR (alpha4*) binding or nicotine-evoked release were observed in any region. These results show a selective preservation of alpha3*/alpha6* nAChR-mediated function in the nigrostriatal and mesolimbic dopamine systems after nigrostriatal damage. Maintenance of function in putamen, a region with a selective loss of dopaminergic terminals, may be important in PD.
ESTHER : McCallum_2005_Mol.Pharmacol_68_737
PubMedSearch : McCallum_2005_Mol.Pharmacol_68_737
PubMedID: 15933214

Title : Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice - Salminen_2004_Mol.Pharmacol_65_1526
Author(s) : Salminen O , Murphy KL , McIntosh JM , Drago J , Marks MJ , Collins AC , Grady SR
Ref : Molecular Pharmacology , 65 :1526 , 2004
Abstract : Pharmacological evaluation of nicotine-stimulated dopamine release from striatum has yielded data consistent with activation of a single population of nicotinic acetylcholine receptors (nAChR). However, discovery that alpha-conotoxin MII (alpha-CtxMII) partially inhibits the response indicates that two classes of presynaptic nAChRs mediate dopamine release. We have investigated the pharmacology and subunit composition of these two classes of nAChR. Inhibition of nicotine-stimulated dopamine release from mouse striatal synaptosomes by alpha-CtxMII occurs within minutes; recovery is slow. The IC50 is 1 to 3 nM. alpha-CtxMII-sensitive and -resistant components have significant differences in pharmacology. The five agonists tested were more potent at activating the alpha-CtxMII-sensitive nAChRs; indeed, this receptor is the highest affinity functional nAChR found, so far, in mouse brain. In addition, cytisine was more efficacious at the alpha-CtxMII-sensitive sites. Methyllycaconitine was 9-fold more potent at inhibiting the alpha-CtxMII-sensitive sites, whereas dihydro-beta-erythroidine was a 7-fold more potent inhibitor of the alpha-CtxMII-resistant response. Both the transient and persistent phases of nicotine-stimulated dopamine release were partially inhibited by alpha-CtxMII with equal potency. The subunit composition of functional nAChRs, was assessed in mice with null mutations for individual nAChR subunits. The beta2 subunit is an absolute requirement for both classes. In contrast, deletion of beta4 or alpha7 subunits had no effect. The alpha-CtxMII-sensitive response requires beta3 and is partially dependent upon alpha4 subunits, probably alpha6beta3beta2 and alpha4alpha6beta3beta2, whereas the alpha-CtxMII-resistant release requires alpha4 and is partially dependent upon alpha5 subunits, probably alpha4beta2 and alpha4alpha5beta2.
ESTHER : Salminen_2004_Mol.Pharmacol_65_1526
PubMedSearch : Salminen_2004_Mol.Pharmacol_65_1526
PubMedID: 15155845

Title : Conditional expression in corticothalamic efferents reveals a developmental role for nicotinic acetylcholine receptors in modulation of passive avoidance behavior - King_2003_J.Neurosci_23_3837
Author(s) : King SL , Marks MJ , Grady SR , Caldarone BJ , Koren AO , Mukhin AG , Collins AC , Picciotto MR
Ref : Journal of Neuroscience , 23 :3837 , 2003
Abstract : Prenatal nicotine exposure has been linked to attention deficit hyperactivity disorder and cognitive impairment, but the sites of action for these effects of nicotine are still under investigation. High-affinity nicotinic acetylcholine receptors (nAChRs) contain the beta2 subunit and modulate passive avoidance (PA) learning in mice. Using an inducible, tetracycline-regulated transgenic system, we generated lines of mice with expression of high-affinity nicotinic receptors restored in specific neuronal populations. One line of mice shows functional beta2 subunit-containing nAChRs localized exclusively in corticothalamic efferents. Functional, presynaptic nAChRs are present in the thalamus of these mice as detected by nicotine-elicited rubidium efflux assays from synaptosomes. Knock-out mice lacking high-affinity nAChRs show elevated baseline PA learning, whereas normal baseline PA behavior is restored in mice with corticothalamic expression of these nAChRs. In contrast, nicotine can enhance PA learning in adult wild-type animals but not in corticothalamic-expressing transgenic mice. When these transgenic mice are treated with doxycycline in adulthood to switch off nAChR expression, baseline PA is maintained even after transgene expression is abolished. These data suggest that high-affinity nAChRs expressed on corticothalamic neurons during development are critical for baseline PA performance and provide a potential neuroanatomical substrate for changes induced by prenatal nicotine exposure leading to long-term behavioral and cognitive deficits.
ESTHER : King_2003_J.Neurosci_23_3837
PubMedSearch : King_2003_J.Neurosci_23_3837
PubMedID: 12736354

Title : The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors - Cui_2003_J.Neurosci_23_11045
Author(s) : Cui C , Booker TK , Allen RS , Grady SR , Whiteaker P , Marks MJ , Salminen O , Tritto T , Butt CM , Allen WR , Stitzel JA , McIntosh JM , Boulter J , Collins AC , Heinemann SF
Ref : Journal of Neuroscience , 23 :11045 , 2003
Abstract : Nigrostriatal dopaminergic neurons express many nicotinic acetylcholine receptor (nAChR) subunits capable of forming multiple nAChR subtypes. These subtypes are expressed differentially along the neuron and presumably mediate diverse responses. beta3 subunit mRNA has restricted expression but is abundant in the substantia nigra and ventral tegmental areas. To investigate the potential role(s) of nicotinic receptors containing the beta3 subunit in dopaminergic tracts, we generated mice with a null mutation in the beta3 gene. We were thereby able to identify a population of beta3-dependent alpha-conotoxin MII-binding nAChRs that modulate striatal dopamine release. Changes were also observed in locomotor activity and prepulse inhibition of acoustic startle, behaviors that are controlled, in part, by nigrostriatal and mesolimbic dopaminergic activity, respectively, suggesting that beta3-containing nAChRs modulate these behaviors.
ESTHER : Cui_2003_J.Neurosci_23_11045
PubMedSearch : Cui_2003_J.Neurosci_23_11045
PubMedID: 14657161

Title : Characterization of [(125) I]epibatidine binding and nicotinic agonist-mediated (86) Rb(+) efflux in interpeduncular nucleus and inferior colliculus of beta2 null mutant mice - Marks_2002_J.Neurochem_81_1102
Author(s) : Marks MJ , Whiteaker P , Grady SR , Picciotto MR , McIntosh JM , Collins AC
Ref : Journal of Neurochemistry , 81 :1102 , 2002
Abstract : The beta2 nicotinic acetylcholine receptor subunit null mutation eliminated most high affinity [(3) H]epibatidine binding in mouse brain, but significant binding remained in accessory olfactory nucleus, medial habenula, inferior colliculus and interpeduncular nucleus. Residual [(125) I]epibatidine binding sites in the inferior colliculus and interpeduncular nucleus were subsequently characterized. Inhibition of [(125) I]epibatidine binding by 12 agonists and six antagonists was very similar in these regions. Most acetylcholine-stimulated (86) Rb(+) efflux is eliminated in thalamus and superior colliculus of beta2 null mutants, but significant activity remained in inferior colliculus and interpeduncular nucleus. This residual activity was subsequently characterized. The 12 nicotinic agonists tested elicited concentration-dependent (86) Rb(+) efflux. Epibatidine was the most potent agonist. Cytisine was also potent and efficacious. EC(50) values for quaternary agonists were relatively high. Cytisine-stimulated (86) Rb(+) efflux was inhibited by six classical nicotinic antagonists. Mecamylamine and D-tubocurarine were most potent, while decamethonium was the least potent. Agonists and antagonists exhibited similar potency in both brain regions. Alpha-bungarotoxin (100 nm) did not significantly inhibit cytisine-stimulated (86) Rb(+) efflux, while the alpha3beta4 selective antagonist, alphaConotoxinAuIB, inhibited a significant fraction of the response in both brain regions. Thus, beta2 null mutant mice express residual nicotinic activity with properties resembling those of alpha3beta4*-nAChR.
ESTHER : Marks_2002_J.Neurochem_81_1102
PubMedSearch : Marks_2002_J.Neurochem_81_1102
PubMedID: 12065623

Title : Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum - Grady_2001_J.Neurochem_76_258
Author(s) : Grady SR , Meinerz NM , Cao J , Reynolds AM , Picciotto MR , Changeux JP , McIntosh JM , Marks MJ , Collins AC
Ref : Journal of Neurochemistry , 76 :258 , 2001
Abstract : Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.
ESTHER : Grady_2001_J.Neurochem_76_258
PubMedSearch : Grady_2001_J.Neurochem_76_258
PubMedID: 11145999

Title : Pharmacological and null mutation approaches reveal nicotinic receptor diversity - Whiteaker_2000_Eur.J.Pharmacol_393_123
Author(s) : Whiteaker P , Marks MJ , Grady SR , Lu Y , Picciotto MR , Changeux JP , Collins AC
Ref : European Journal of Pharmacology , 393 :123 , 2000
Abstract : We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [125I]alpha-Bungarotoxin-, (-)-[3H]nicotine-, and [3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [3H]dopamine, [3H]gamma-aminobutyric acid ([3H]GABA), and 86Rb(+) efflux techniques. Additionally, the effects of beta2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [125I]alpha-bungarotoxin-binding nicotinic acetylcholine receptors; beta2-subunit-dependent and -independent high-affinity (-)-[3H]nicotine-binding sites; beta2-dependent and -independent cytisine-resistant [3H]epibatidine-binding sites; and a beta2-dependent low-affinity [3H]epibatidine binding site. Comparative pharmacology suggested that [3H]GABA and dihydro-beta-erythroidine (DHbetaE)-sensitive 86Rb(+) efflux are mediated by the same (probably alpha4beta2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [3H]dopamine and DHbetaE-resistant 86Rb(+) efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was beta2 dependent. The majority of beta2-independent [3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.
ESTHER : Whiteaker_2000_Eur.J.Pharmacol_393_123
PubMedSearch : Whiteaker_2000_Eur.J.Pharmacol_393_123
PubMedID: 10771005

Title : Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype - Marks_2000_Neuropharmacol_39_2632
Author(s) : Marks MJ , Stitzel JA , Grady SR , Picciotto MR , Changeux JP , Collins AC
Ref : Neuropharmacology , 39 :2632 , 2000
Abstract : Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.
ESTHER : Marks_2000_Neuropharmacol_39_2632
PubMedSearch : Marks_2000_Neuropharmacol_39_2632
PubMedID: 11044733

Title : International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits -
Author(s) : Lukas RJ , Changeux JP , Le Novere N , Albuquerque EX , Balfour DJ , Berg DK , Bertrand D , Chiappinelli VA , Clarke PB , Collins AC , Dani JA , Grady SR , Kellar KJ , Lindstrom JM , Marks MJ , Quik M , Taylor P , Wonnacott S
Ref : Pharmacol Rev , 51 :397 , 1999
PubMedID: 10353988

Title : Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit - Marks_1999_J.Pharmacol.Exp.Ther_289_1090
Author(s) : Marks MJ , Whiteaker P , Calcaterra J , Stitzel JA , Bullock AE , Grady SR , Picciotto MR , Changeux JP , Collins AC
Ref : Journal of Pharmacology & Experimental Therapeutics , 289 :1090 , 1999
Abstract : Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.
ESTHER : Marks_1999_J.Pharmacol.Exp.Ther_289_1090
PubMedSearch : Marks_1999_J.Pharmacol.Exp.Ther_289_1090
PubMedID: 10215692