McIntosh JM


Full name : McIntosh J Michael

First name : J Michael

Mail : George E. Whalen Veterans Affairs Medical Center, University of Utah, Salt Lake City, UT

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Country : USA

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References (118)

Title : SLPI Inhibits ATP-Mediated Maturation of IL-1beta in Human Monocytic Leukocytes: A Novel Function of an Old Player - Zakrzewicz_2019_Front.Immunol_10_664
Author(s) : Zakrzewicz A , Richter K , Zakrzewicz D , Siebers K , Damm J , Agne A , Hecker A , McIntosh JM , Chamulitrat W , Krasteva-Christ G , Manzini I , Tikkanen R , Padberg W , Janciauskiene S , Grau V
Ref : Front Immunol , 10 :664 , 2019
Abstract : Interleukin-1beta (IL-1beta) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1beta can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1beta maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1beta maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1beta release in human monocytic cells, without affecting the induction of pro-IL-1beta mRNA by LPS. In contrast, the ATP-independent IL-1beta release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2beta (iPLA2beta) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1beta release. Signaling involves nicotinic acetylcholine receptor subunits alpha7, alpha9, alpha10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1beta. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.
ESTHER : Zakrzewicz_2019_Front.Immunol_10_664
PubMedSearch : Zakrzewicz_2019_Front.Immunol_10_664
PubMedID: 31019507

Title : Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1beta Release by Pulmonary Epithelial Cells - Richter_2018_Molecules_23_
Author(s) : Richter K , Koch C , Perniss A , Wolf PM , Schweda EKH , Wichmann S , Wilker S , Magel I , Sander M , McIntosh JM , Padberg W , Grau V
Ref : Molecules , 23 : , 2018
Abstract : Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1beta. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1beta release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits alpha7, alpha9, and/or alpha10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.
ESTHER : Richter_2018_Molecules_23_
PubMedSearch : Richter_2018_Molecules_23_
PubMedID: 30096783

Title : Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1beta via CD36 and Nicotinic Acetylcholine Receptors - Siebers_2018_Front.Immunol_9_877
Author(s) : Siebers K , Fink B , Zakrzewicz A , Agne A , Richter K , Konzok S , Hecker A , Zukunft S , Kullmar M , Klein J , McIntosh JM , Timm T , Sewald K , Padberg W , Aggarwal N , Chamulitrat W , Santoso S , Xia W , Janciauskiene S , Grau V
Ref : Front Immunol , 9 :877 , 2018
Abstract : While interleukin (IL)-1beta is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1beta secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1beta is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1beta. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1beta regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1beta from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1beta release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1beta release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2beta, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1beta release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.
ESTHER : Siebers_2018_Front.Immunol_9_877
PubMedSearch : Siebers_2018_Front.Immunol_9_877
PubMedID: 29922281

Title : Canonical and Novel Non-Canonical Cholinergic Agonists Inhibit ATP-Induced Release of Monocytic Interleukin-1beta via Different Combinations of Nicotinic Acetylcholine Receptor Subunits alpha7, alpha9 and alpha10 - Zakrzewicz_2017_Front.Cell.Neurosci_11_189
Author(s) : Zakrzewicz A , Richter K , Agne A , Wilker S , Siebers K , Fink B , Krasteva-Christ G , Althaus M , Padberg W , Hone AJ , McIntosh JM , Grau V
Ref : Front Cell Neurosci , 11 :189 , 2017
Abstract : Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1beta (IL-1beta) by human monocytes via nicotinic acetylcholine receptors (nAChRs) composed of alpha7, alpha9 and/or alpha10 subunits. Furthermore, we identified phosphocholine (PC) and dipalmitoylphosphatidylcholine (DPPC) as novel nicotinic agonists that elicit metabotropic activity at monocytic nAChR. Interestingly, PC does not provoke ion channel responses at conventional nAChRs composed of subunits alpha9 and alpha10. The purpose of this study is to determine the composition of nAChRs necessary for nicotinic signaling in monocytic cells and to test the hypothesis that common metabolites of phosphatidylcholines, lysophosphatidylcholine (LPC) and glycerophosphocholine (G-PC), function as nAChR agonists. In peripheral blood mononuclear cells from nAChR gene-deficient mice, we demonstrated that inhibition of ATP-dependent release of IL-1beta by acetylcholine (ACh), nicotine and PC depends on subunits alpha7, alpha9 and alpha10. Using a panel of nAChR antagonists and siRNA technology, we confirmed the involvement of these subunits in the control of IL-1beta release in the human monocytic cell line U937. Furthermore, we showed that LPC (C16:0) and G-PC efficiently inhibit ATP-dependent release of IL-1beta. Of note, the inhibitory effects mediated by LPC and G-PC depend on nAChR subunits alpha9 and alpha10, but only to a small degree on alpha7. In Xenopuslaevis oocytes heterologously expressing different combinations of human alpha7, alpha9 or alpha10 subunits, ACh induced canonical ion channel activity, whereas LPC, G-PC and PC did not. In conclusion, we demonstrate that canonical nicotinic agonists and PC elicit metabotropic nAChR activity in monocytes via interaction of nAChR subunits alpha7, alpha9 and alpha10. For the metabotropic signaling of LPC and G-PC, nAChR subunits alpha9 and alpha10 are needed, whereas alpha7 is virtually dispensable. Furthermore, molecules bearing a PC group in general seem to regulate immune functions without perturbing canonical ion channel functions of nAChR.
ESTHER : Zakrzewicz_2017_Front.Cell.Neurosci_11_189
PubMedSearch : Zakrzewicz_2017_Front.Cell.Neurosci_11_189
PubMedID: 28725182

Title : Key residues in the nicotinic acetylcholine receptor beta2 subunit contribute to alpha-conotoxin LvIA binding - Zhangsun_2015_J.Biol.Chem_290_9855
Author(s) : Zhangsun D , Zhu X , Wu Y , Hu Y , Kaas Q , Craik DJ , McIntosh JM , Luo S
Ref : Journal of Biological Chemistry , 290 :9855 , 2015
Abstract : alpha-Conotoxin LvIA (alpha-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of alpha3beta2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the alpha3beta2 nAChR subtype from the alpha6beta2* (* indicates the other subunit) and alpha3beta4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the beta2 subunit versus those of the beta4 subunit on the binding of alpha-CTx LvIA. Although two beta2 mutations, alpha3beta2[F119Q] and alpha3beta2[T59K], strongly enhanced the affinity of LvIA, the beta2 mutation alpha3beta2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the beta2-T59L mutant was combined with the alpha3 subunit. There were no significant difference in inhibition of alpha3beta2[T59I], alpha3beta2[Q34A], and alpha3beta2[K79A] nAChRs when compared with wild-type alpha3beta2 nAChR. alpha-CTx LvIA displayed slower off-rate kinetics at alpha3beta2[F119Q] and alpha3beta2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the beta2 subunit contributes to alpha-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework alpha-conotoxins. Thr(59) and Phe(119) of the beta2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the beta4 subunit leads to increased affinity.
ESTHER : Zhangsun_2015_J.Biol.Chem_290_9855
PubMedSearch : Zhangsun_2015_J.Biol.Chem_290_9855
PubMedID: 25713061

Title : Evidence for a role for alpha6( *) nAChRs in l-dopa-induced dyskinesias using Parkinsonian alpha6( *) nAChR gain-of-function mice - Bordia_2015_Neurosci_295_187
Author(s) : Bordia T , McGregor M , McIntosh JM , Drenan RM , Quik M
Ref : Neuroscience , 295 :187 , 2015
Abstract : l-Dopa-induced dyskinesias (LIDs) are a serious side effect of dopamine replacement therapy for Parkinson's disease. The mechanisms that underlie LIDs are currently unclear. However, preclinical studies indicate that nicotinic acetylcholine receptors (nAChRs) play a role, suggesting that drugs targeting these receptors may be of therapeutic benefit. To further understand the involvement of alpha6beta2( *) nAChRs in LIDs, we used gain-of-function alpha6( *) nAChR (alpha6L9S) mice that exhibit a 20-fold enhanced sensitivity to nAChR agonists. Wildtype (WT) and alpha6L9S mice were lesioned by unilateral injection of 6-hydroxydopamine (6-OHDA, 3mug/ml) into the medial forebrain bundle. Three to 4wk later, they were administered l-dopa (3mg/kg) plus benserazide (15mg/kg) until stably dyskinetic. l-dopa-induced abnormal involuntary movements (AIMs) were similar in alpha6L9S and WT mice. WT mice were then given nicotine in the drinking water in gradually increasing doses to a final 300mug/ml, which resulted in a 40% decline AIMs. By contrast, there was no decrease in AIMs in alpha6L9S mice at a maximally tolerated nicotine dose of 20mug/ml. However, the nAChR antagonist mecamylamine (1mg/kg ip 30min before l-dopa) reduced l-dopa-induced AIMs in both alpha6L9S and WT mice. Thus, both a nAChR agonist and antagonist decreased AIMs in WT mice, but only the antagonist was effective in alpha6L9S mice. Since nicotine appears to reduce LIDs via desensitization, hypersensitive alpha6beta2( *) nAChRs may desensitize less readily. The present data show that alpha6beta2( *) nAChRs are key regulators of LIDs, and may be useful therapeutic targets for their management in Parkinson's disease.
ESTHER : Bordia_2015_Neurosci_295_187
PubMedSearch : Bordia_2015_Neurosci_295_187
PubMedID: 25813704

Title : The alpha7 nicotinic receptor agonist ABT-107 protects against nigrostriatal damage in rats with unilateral 6-hydroxydopamine lesions - Bordia_2015_Exp.Neurol_263_277
Author(s) : Bordia T , McGregor M , Papke RL , Decker MW , McIntosh JM , Quik M
Ref : Experimental Neurology , 263 :277 , 2015
Abstract : The finding that smoking is inversely correlated with Parkinson's disease and that nicotine attenuates nigrostriatal damage in Parkinsonian animals supports the idea that nicotine may be neuroprotective. Nicotine is thought to exert this effect by acting at nicotinic receptors (nAChRs), including the alpha7 subtype. The objective of this study was twofold: first, to test the protective potential of ABT-107, an agonist with high selectivity for alpha7 nAChRs; and second, to investigate its cellular mechanism of action. Rats were implanted with minipumps containing ABT-107 (0.25mg/kg/d). In addition, we tested the effect of nicotine (1mg/kg/d) as a positive control, and also DMXB (2mg/kg/d) which acts primarily with alpha7 but also alpha4beta2* nAChRs. Two weeks after minipump placement, the rats were lesioned by unilateral administration of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. Lesioning alone decreased contralateral forelimb use and adjusted stepping, two measures of Parkinsonism. ABT-107 and nicotine treatment significantly improved these behaviors at all weeks tested, with variable improvement with DMXB. We next investigated the cellular mechanism involved. The striatal dopamine transporter (DAT), a marker of dopaminergic integrity, was reduced ~70% with lesioning. ABT-107 or nicotine treatment significantly increased DAT levels in lesioned striatum; these drugs did not alter DAT levels in intact striatum. ABT-107 and nicotine also significantly improved basal dopamine release from lesioned striatum, as well as nicotine-stimulated dopamine release mediated via alpha4beta2* and alpha6beta2* nAChRs. These data suggest that alpha7 nAChR agonists may improve motor behaviors associated with nigrostriatal damage by enhancing striatal dopaminergic function.
ESTHER : Bordia_2015_Exp.Neurol_263_277
PubMedSearch : Bordia_2015_Exp.Neurol_263_277
PubMedID: 25261754

Title : Varenicline enhances dopamine release facilitation more than nicotine after long-term nicotine treatment and withdrawal - Perez_2015_Pharmacol.Res.Perspect_3_e00105
Author(s) : Perez XA , Khroyan TV , McIntosh JM , Quik M
Ref : Pharmacol Res Perspect , 3 :e00105 , 2015
Abstract : An important factor contributing to the high relapse rates among smokers is nicotine withdrawal symptoms. Multiple studies suggest that decreased dopamine release in nucleus accumbens plays a key role in withdrawal. However, recent reports showed that long-term nicotine exposure itself also decreases accumbal dopamine release, suggesting that additional mechanisms are involved in withdrawal. Here, we used real-time cyclic voltammetry in brain slices containing the nucleus accumbens to further elucidate the changes in dopamine release linked to nicotine withdrawal. Rats received vehicle or nicotine via the drinking water for 2-3 months. Studies assessing the expression of somatic signs in vehicle-treated, nicotine-treated, and 24-h nicotine withdrawn rats showed that nicotine withdrawal led to a significant increase in somatic signs. Subsequent voltammetry studies showed that long-term nicotine decreased single-pulse-stimulated dopamine release via an interaction at alpha6beta2* receptors. Nicotine withdrawal led to a partial recovery in alpha6beta2* receptor-mediated release. In addition, long-term nicotine treatment alone increased dopamine release paired-pulse ratios and this was partially reversed with nicotine removal. We then evaluated the effect of bath-applied nicotine and varenicline on dopamine release. Nicotine and varenicline both decreased single-pulse-stimulated release in vehicle-treated, nicotine-treated, and nicotine withdrawn rats. However, bath-applied varenicline increased paired-pulse ratios to a greater extent than nicotine during long-term nicotine treatment and after its withdrawal. Altogether these data suggest that nicotine withdrawal is associated with a partial restoration of dopamine release measures to control levels and that varenicline's differential modulation of dopamine release may contribute to its mechanism of action.
ESTHER : Perez_2015_Pharmacol.Res.Perspect_3_e00105
PubMedSearch : Perez_2015_Pharmacol.Res.Perspect_3_e00105
PubMedID: 25692023

Title : Cloning, synthesis, and characterization of alphaO-conotoxin GeXIVA, a potent alpha9alpha10 nicotinic acetylcholine receptor antagonist - Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
Author(s) : Luo S , Zhangsun D , Harvey PJ , Kaas Q , Wu Y , Zhu X , Hu Y , Li X , Tsetlin VI , Christensen S , Romero HK , McIntyre M , Dowell C , Baxter JC , Elmslie KS , Craik DJ , McIntosh JM
Ref : Proc Natl Acad Sci U S A , 112 :E4026 , 2015
Abstract : We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, alphaO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the alpha9alpha10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist alpha-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most alpha-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.
ESTHER : Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
PubMedSearch : Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
PubMedID: 26170295

Title : A novel alpha4\/7-conotoxin LvIA from Conus lividus that selectively blocks alpha3beta2 vs. alpha6\/alpha3beta2beta3 nicotinic acetylcholine receptors - Luo_2014_FASEB.J_28_1842
Author(s) : Luo S , Zhangsun D , Schroeder CI , Zhu X , Hu Y , Wu Y , Weltzin MM , Eberhard S , Kaas Q , Craik DJ , McIntosh JM , Whiteaker P
Ref : FASEB Journal , 28 :1842 , 2014
Abstract : This study was performed to discover and characterize the first potent alpha3beta2-subtype-selective nicotinic acetylcholine receptor (nAChR) ligand. A novel alpha4/7-conotoxin, alpha-CTxLvIA, was cloned from Conus lividus. Its pharmacological profile at Xenopus laevis oocyte-expressed rat nAChR subtypes was determined by 2-electrode voltage-clamp electrophysiology, and its 3-dimensional (3D) structure was determined by NMR spectroscopy. alpha-CTx LvIA is a 16-aa C-terminally-amidated peptide with 2-disulfide bridges. Using rat subunits expressed in Xenopus oocytes, we found the highest affinity of alpha-CTxLvIA was for alpha3beta2 nAChRs (IC50 8.7 nM), where blockade was reversible within 2 min. IC50 values were >100 nM at alpha6/alpha3beta2beta3, alpha6/alpha3beta4, and alpha3beta4 nAChRs, and >/=3 muM at all other subtypes tested. alpha3beta2 vs. alpha6beta2 subtype selectivity was confirmed for human-subunit nAChRs with much greater preference (300-fold) for alpha3beta2 over alpha6beta2 nAChRs. This is the first alpha-CTx reported to show high selectivity for human alpha3beta2 vs. alpha6beta2 nAChRs. alpha-CTxLvIA adopts two similarly populated conformations water: one (assumed to be bioactive) is highly structured, whereas the other is mostly random coil in nature. Selectivity differences with the similarly potent, but less selective, alpha3beta2 nAChR antagonist alpha-CTx PeIA probably reside within the three residues, which differ in loop 2, given their otherwise similar 3D structures. alpha4/7-CTx LvIA is a new, potent, selective alpha3beta2 nAChR antagonist, which will enable detailed studies of alpha3beta2 nAChR structure, function, and physiological roles.-Luo, S., Zhangsun, D., Schroeder, C. I., Zhu, X., Hu, Y., Wu, Y., Weltzin, M. M., Eberhard, S., Kaas, Q., Craik, D. J., McIntosh, J. M., Whiteaker, P. A novel alpha4/7-conotoxin LvIA from Conus lividus that selectively blocks alpha3beta2 vs. alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors.
ESTHER : Luo_2014_FASEB.J_28_1842
PubMedSearch : Luo_2014_FASEB.J_28_1842
PubMedID: 24398291

Title : Monkey adrenal chromaffin cells express alpha6beta4* nicotinic acetylcholine receptors - Hernandez-Vivanco_2014_PLoS.One_9_e94142
Author(s) : Hernandez-Vivanco A , Hone AJ , Scadden ML , Carmona-Hidalgo B , McIntosh JM , Albillos A
Ref : PLoS ONE , 9 :e94142 , 2014
Abstract : Nicotinic acetylcholine receptors (nAChRs) that contain alpha6 and beta4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, alpha6beta4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the alpha3beta4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express alpha6beta4* receptors. PCR was used to show the presence of transcripts for alpha6 and beta4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with alpha-conotoxins that target the alpha6beta4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; alpha-conotoxins that inhibit alpha6-containing nAChRs. Two additional agonists were used to probe for the expression of alpha7 and beta2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the alpha7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed alpha6beta4* nAChRs.
ESTHER : Hernandez-Vivanco_2014_PLoS.One_9_e94142
PubMedSearch : Hernandez-Vivanco_2014_PLoS.One_9_e94142
PubMedID: 24727685

Title : ABT-089 and ABT-894 reduce levodopa-induced dyskinesias in a monkey model of Parkinson's disease - Zhang_2014_Mov.Disord_29_508
Author(s) : Zhang D , Bordia T , McGregor M , McIntosh JM , Decker MW , Quik M
Ref : Movement Disorders , 29 :508 , 2014
Abstract : Levodopa-induced dyskinesias (LIDs) are a serious complication of levodopa therapy for Parkinson's disease for which there is little treatment. Accumulating evidence shows that nicotinic acetylcholine receptor (nAChR) drugs decrease LIDs in parkinsonian animals. Here, we examined the effect of two beta2 nAChR agonists, ABT-089 and ABT-894, that previously were approved for phase 2 clinical trials for other indications. Two sets of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys were administered levodopa/carbidopa (10 mg/kg and 2.5 mg/kg, respectively) twice daily 5 days a week until they were stably dyskinetic. Each set had a vehicle-treated group, an nAChR agonist-treated group, and a nicotine-treated group as a positive control. Set A monkeys had previously received other nAChR drugs (nAChR drug-primed), whereas Set B monkeys were initially nAChR drug-naive. Both sets were administered the partial agonist ABT-089 (range, 0.01-1.0 mg/kg) orally 5 days a week twice daily 30 minutes before levodopa with each dose given for 1 to 5 weeks. ABT-089 decreased LIDs by 30% to 50% compared with vehicle-treated monkeys. Nicotine reduced LIDs by 70% in a parallel group. After 4 weeks of washout, the effect of the full agonist ABT-894 (range, 0.0001-0.10 mg/kg) was assessed on LIDs in Set A and Set B. ABT-894 reduced LIDs by 70%, similar to nicotine. Both drugs acted equally well at alpha4beta2* and alpha6beta2* nAChRs; however, ABT-089 was 30 to 60 times less potent than ABT-894. Tolerance did not develop for the time periods tested (range, 3-4 months). The nAChR drugs did not worsen parkinsonism or cognitive ability. Emesis, a common problem with nAChR drugs, was not observed. ABT-894 and ABT-089 appear to be good candidate nAChR drugs for the management of LIDs in Parkinson's disease.
ESTHER : Zhang_2014_Mov.Disord_29_508
PubMedSearch : Zhang_2014_Mov.Disord_29_508
PubMedID: 24515328

Title : Presence of multiple binding sites on alpha9alpha10 nAChR receptors alludes to stoichiometric-dependent action of the alpha-conotoxin, Vc1.1 - Indurthi_2014_Biochem.Pharmacol_89_131
Author(s) : Indurthi DC , Pera E , Kim HL , Chu C , McLeod MD , McIntosh JM , Absalom NL , Chebib M
Ref : Biochemical Pharmacology , 89 :131 , 2014
Abstract : Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, alpha9alpha10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of alpha9alpha10 nAChRs, like the alpha-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of alpha9alpha10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca(2+) concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the alpha9alpha10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of alpha9 and alpha10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of alpha10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different alpha9alpha10 nAChR stoichiometries, possibly (alpha9)(3)(alpha10)(2) and (alpha9)(2)(alpha10)(3). As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the alpha9-alpha9 interface on the hypothesized (alpha9)(3)(alpha10)(2) nAChR, in addition to the alpha10-alpha9 and or alpha9-alpha10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.
ESTHER : Indurthi_2014_Biochem.Pharmacol_89_131
PubMedSearch : Indurthi_2014_Biochem.Pharmacol_89_131
PubMedID: 24548457

Title : Comparative functional expression of nAChR subtypes in rodent DRG neurons - Smith_2013_Front.Cell.Neurosci_7_225
Author(s) : Smith NJ , Hone AJ , Memon T , Bossi S , Smith TE , McIntosh JM , Olivera BM , Teichert RW
Ref : Front Cell Neurosci , 7 :225 , 2013
Abstract : We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly alpha3beta4 and alpha6beta4 nAChRs; (2) neurons that express predominantly alpha7 nAChRs; (3) neurons that express a combination of alpha3beta4/alpha6beta4 and alpha7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.
ESTHER : Smith_2013_Front.Cell.Neurosci_7_225
PubMedSearch : Smith_2013_Front.Cell.Neurosci_7_225
PubMedID: 24348328

Title : The alpha3beta4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the alpha5 subunit in the mouse - Jackson_2013_Neuropharmacol_70_228
Author(s) : Jackson KJ , Sanjakdar SS , Muldoon PP , McIntosh JM , Damaj MI
Ref : Neuropharmacology , 70 :228 , 2013
Abstract : The 15q25 gene cluster contains genes that code for the alpha5, alpha3, and beta4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the alpha5 and beta4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the alpha3beta4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the alpha3beta4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective alpha3beta4* nAChR antagonist, alpha-conotoxin AuIB, we assessed the role of alpha3beta4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because alpha5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with alpha3beta4*, we also evaluated the role of the alpha3beta4alpha5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the alpha3beta4* nAChR subtype in alpha5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in alpha5 nAChR knockout mice. This study shows that alpha3beta4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The alpha5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the alpha3beta4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome.
ESTHER : Jackson_2013_Neuropharmacol_70_228
PubMedSearch : Jackson_2013_Neuropharmacol_70_228
PubMedID: 23416040

Title : Poster: Nicotinic acetylcholine receptor regulation of striatal dopamine transmission: The different requirement for diverse subtypes in ventral versus dorsal striatum -
Author(s) : Exley R , McIntosh JM , Marks M , Maskos U , Cragg SJ
Ref : Biochemical Pharmacology , 86 :1228 , 2013

Title : Alpha4beta2 nicotinic receptors play a role in the nAChR-mediated decline in L-dopa-induced dyskinesias in parkinsonian rats - Quik_2013_Neuropharmacol_71_191
Author(s) : Quik M , Campos C , Bordia T , Strachan JP , Zhang J , McIntosh JM , Letchworth SR , Jordan K
Ref : Neuropharmacology , 71 :191 , 2013
Abstract : L-dopa-induced dyskinesias are a serious long-term side effect of dopamine replacement therapy for Parkinson's disease for which there are few treatment options. Our previous studies showed that nicotine decreased l-dopa-induced abnormal involuntary movements (AIMs). Subsequent work with knockout mice demonstrated that alpha6beta2 nicotinic receptors (nAChRs) play a key role. The present experiments were done to determine if alpha4beta2 nAChRs are also involved in l-dopa-induced dyskinesias. To approach this, we took advantage of the finding that alpha6beta2 nAChRs are predominantly present on striatal dopaminergic nerve terminals, while a significant population of alpha4beta2 nAChRs are located on other neurons. Thus, a severe dopaminergic lesion would cause a major loss in alpha6beta2, but not alpha4beta2 nAChRs. Experiments were therefore done in which rats were unilaterally lesioned with 6-hydroxydopamine, at a dose that led to severe nigrostriatal damage. The dopamine transporter, a dopamine nerve terminal marker, was decreased by >99%. This lesion also decreased striatal alpha6beta2 nAChRs by 97%, while alpha4beta2 nAChRs were reduced by only 12% compared to control. A series of beta2 nAChR compounds, including TC-2696, TI-10165, TC-8831, TC-10600 and sazetidine reduced l-dopa-induced AIMs in these rats by 23-32%. TC-2696, TI-10165, TC-8831 were also tested for parkinsonism, with no effect on this behavior. Tolerance did not develop with up to 3 months of treatment. Since alpha4alpha5beta2 nAChRs are also predominantly on striatal dopamine terminals, these data suggest that drugs targeting alpha4beta2 nAChRs may reduce l-dopa-induced dyskinesias in late stage Parkinson's disease.
ESTHER : Quik_2013_Neuropharmacol_71_191
PubMedSearch : Quik_2013_Neuropharmacol_71_191
PubMedID: 23583932

Title : Nicotine-mediated improvement in L-dopa-induced dyskinesias in MPTP-lesioned monkeys is dependent on dopamine nerve terminal function - Quik_2013_Neurobiol.Dis_50_30
Author(s) : Quik M , Mallela A , Chin M , McIntosh JM , Perez XA , Bordia T
Ref : Neurobiol Dis , 50 :30 , 2013
Abstract : L-dopa-induced dyskinesias (LIDs) are abnormal involuntary movements that develop with long term L-dopa therapy for Parkinson's disease. Studies show that nicotine administration reduced LIDs in several parkinsonian animal models. The present work was done to understand the factors that regulate the nicotine-mediated reduction in LIDs in MPTP-lesioned nonhuman primates. To approach this, we used two groups of monkeys, one with mild-moderate and the other with more severe parkinsonism rendered dyskinetic using L-dopa. In mild-moderately parkinsonian monkeys, nicotine pretreatment (300 mug/ml via drinking water) prevented the development of LIDs by ~75%. This improvement was maintained when the nicotine dose was lowered to 50 mug/ml but was lost with nicotine removal. Nicotine re-exposure again decreased LIDs. By contrast, nicotine treatment did not reduce LIDs in monkeys with more severe parkinsonism. We next determined how nicotine's ability to reduce LIDs correlated with lesion-induced changes in the striatal dopamine transporter and (3)H-dopamine release in these two groups of monkeys. The striatal dopamine transporter was reduced to 54% and 28% of control in mild-moderately and more severely parkinsonian monkeys, respectively. However, basal, K(+), alpha4beta2* and alpha6beta2* nAChR-evoked (3)H-dopamine release were near control levels in striatum of mild-moderately parkinsonian monkeys. By contrast, these same release measures were reduced to a significantly greater extent in striatum of more severely parkinsonian monkeys. Thus, nicotine best improves LIDs in lesioned monkeys in which striatal dopamine transmission is still relatively intact. These data suggest that nicotine treatment would most effectively reduce LIDs in patients with mild to moderate Parkinson's disease.
ESTHER : Quik_2013_Neurobiol.Dis_50_30
PubMedSearch : Quik_2013_Neurobiol.Dis_50_30
PubMedID: 23009753

Title : Positional scanning mutagenesis of alpha-conotoxin PeIA identifies critical residues that confer potency and selectivity for alpha6\/alpha3beta2beta3 and alpha3beta2 nicotinic acetylcholine receptors - Hone_2013_J.Biol.Chem_288_25428
Author(s) : Hone AJ , Ruiz M , Scadden M , Christensen S , Gajewiak J , Azam L , McIntosh JM
Ref : Journal of Biological Chemistry , 288 :25428 , 2013
Abstract : The nicotinic acetylcholine receptor (nAChR) subtype alpha6beta2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The alpha6 subunit is closely related to the alpha3 subunit, and this presents a problem in designing ligands that discriminate between alpha6beta2* and alpha3beta2* nAChRs. We used positional scanning mutagenesis of alpha-conotoxin PeIA, which targets both alpha6beta2* and alpha3beta2*, in combination with mutagenesis of the alpha6 and alpha3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed alpha6/alpha3beta2beta3 and alpha3beta2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the alpha6 and alpha3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for alpha3beta2 but not for alpha6/alpha3beta2beta3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on alpha6/alpha3beta2beta3 than alpha3beta2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the alpha6 subunit and Lys(152) of the alpha3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target alpha6beta2* nAChRs.
ESTHER : Hone_2013_J.Biol.Chem_288_25428
PubMedSearch : Hone_2013_J.Biol.Chem_288_25428
PubMedID: 23846688

Title : The nicotine-mediated decline in l-dopa-induced dyskinesias is associated with a decrease in striatal dopamine release - Bordia_2013_J.Neurochem_125_291
Author(s) : Bordia T , McIntosh JM , Quik M
Ref : Journal of Neurochemistry , :291 , 2013
Abstract : l-dopa-induced dyskinesias (LIDs) are a side effect of Parkinson's disease therapy that is thought to arise, at least in part, because of excessive dopaminergic activity. Thus, drugs that regulate dopaminergic tone may provide an approach to manage LIDs. Our previous studies showed that nicotine treatment reduced LIDs in Parkinsonian animal models. This study investigates whether nicotine may exert its beneficial effects by modulating pre-synaptic dopaminergic function. Rats were unilaterally lesioned by injection of 6-hydroxydopamine (6-OHDA) (2 x 3 ug per site) into the medial forebrain bundle to yield moderate Parkinsonism. They were then implanted with minipumps containing vehicle or nicotine (2.0 mg/kg/d) and rendered dyskinetic with l-dopa (8 mg/kg plus 15 mg/kg benserazide). Lesioning alone decreased the striatal dopamine transporter, nicotinic receptor (nAChR) levels, and nAChR-mediated 3 H-dopamine release, consistent with previous results. Nicotine administration reduced l-dopa-induced abnormal involuntary movements throughout the course of the study (4 months). Nicotine treatment led to declines in the striatal dopamine transporter, alpha6beta2* nAChRs and various components of alpha6beta2* and alpha4beta2* nAChR-mediated release. l-dopa treatment had no effect. These data suggest that nicotine may improve LIDs in Parkinsonian animal models by dampening striatal dopaminergic activity.
ESTHER : Bordia_2013_J.Neurochem_125_291
PubMedSearch : Bordia_2013_J.Neurochem_125_291
PubMedID: 23373725

Title : Nicotinic acetylcholine receptors containing the alpha6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons - Liu_2013_Biochem.Pharmacol_86(8)_1194
Author(s) : Liu L , Zhao-Shea R , McIntosh JM , Tapper AR
Ref : Biochemical Pharmacology , 86 :1194 , 2013
Abstract : Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the alpha6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the alpha6 nAChR subunit (alpha6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the alpha6 subtype-selective nAChR antagonist, alpha-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in alpha6 KO slices. Finally, pre-application of alpha-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that alpha6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse.
ESTHER : Liu_2013_Biochem.Pharmacol_86(8)_1194
PubMedSearch : Liu_2013_Biochem.Pharmacol_86(8)_1194
PubMedID: 23811312

Title : Striatal dopamine transmission is reduced after chronic nicotine with a decrease in alpha6-nicotinic receptor control in nucleus accumbens - Exley_2013_Eur.J.Neurosci_38_3036
Author(s) : Exley R , Clements MA , Hartung H , McIntosh JM , Franklin M , Bermudez I , Cragg SJ
Ref : European Journal of Neuroscience , 38 :3036 , 2013
Abstract : Nicotine directly regulates striatal dopamine (DA) neurotransmission via presynaptic nicotinic acetylcholine receptors (nAChRs) that are alpha6beta2 and/or alpha4beta2 subunit-containing, depending on region. Chronic nicotine exposure in smokers upregulates striatal nAChR density, with some reports suggesting differential impact on alpha6- or alpha4-containing nAChRs. Here, we explored whether chronic nicotine exposure modifies striatal DA transmission, whether the effects of acute nicotine on DA release probability persist and whether there are modifications to the regulation of DA release by alpha6-subunit-containing (*) relative to non-alpha6* nAChRs in nucleus accumbens (NAc) and in caudate-putamen (CPu). We detected electrically evoked DA release at carbon-fiber microelectrodes in striatal slices from mice exposed for 4-8 weeks to nicotine (200 mug/mL in saccharin-sweetened drinking water) or a control saccharin solution. Chronic nicotine exposure subtly reduced striatal DA release evoked by single electrical pulses, and in NAc enhanced the range of DA release evoked by different frequencies. Effects of acute nicotine (500 nm) on DA release probability and its sensitivity to activity were apparent. However, in NAc there was downregulation of the functional dominance of alpha6-nAChRs (alpha6alpha4beta2beta3), and an emergence in function of non-alpha6* nAChRs. In CPu, there was no change in the control of DA release by its alpha6 nAChRs (alpha6beta2beta3) relative to non-alpha6. These data suggest that chronic nicotine subtly modifies the regulation of DA transmission, which, in NAc, is through downregulation of function of a susceptible population of alpha6alpha4beta2beta3 nAChRs. This imbalance in function of alpha6:non-alpha6 nAChRs might contribute to DA dysregulation in nicotine addiction.
ESTHER : Exley_2013_Eur.J.Neurosci_38_3036
PubMedSearch : Exley_2013_Eur.J.Neurosci_38_3036
PubMedID: 23841846

Title : Long-term nicotine treatment down-regulates alpha6beta2* nicotinic receptor expression and function in nucleus accumbens - Perez_2013_J.Neurochem_127_762
Author(s) : Perez XA , McIntosh JM , Quik M
Ref : Journal of Neurochemistry , 127 :762 , 2013
Abstract : Long-term nicotine exposure induces alterations in dopamine transmission in nucleus accumbens that sustain the reinforcing effects of smoking. One approach to understand the adaptive changes that arise involves measurement of endogenous dopamine release using voltammetry. We therefore treated rats for 2-3 months with nicotine and examined alterations in nAChR subtype expression and electrically evoked dopamine release in rat nucleus accumbens shell, a region key in addiction. Long-term nicotine treatment selectively decreased stimulated alpha6beta2* nAChR-mediated dopamine release compared with vehicle-treated rats. It also reduced alpha6beta2* nAChRs, suggesting the receptor decline may contribute to the functional loss. This decreased response in release after chronic nicotine treatment was still partially sensitive to the agonist nicotine. Studies with an acetylcholinesterase inhibitor demonstrated that the response was also sensitive to increased endogenous acetylcholine. However, unlike the agonists, nAChR antagonists decreased dopamine release only in vehicle- but not nicotine-treated rats. As antagonists function by blocking the action of acetylcholine, their ineffectiveness suggests that reduced acetylcholine levels partly underlie the dampened alpha6beta2* nAChR-mediated function in nicotine-treated rats. As long-term nicotine modifies dopamine release by decreasing alpha6beta2* nAChRs and their function, these data suggest that interventions that target this subtype may be useful for treating nicotine dependence. Long-term nicotine treatment decreases dopamine (DA) transmission in the mesolimbic dopaminergic system. Our data suggest this may involve a decrease in alpha6beta2* nicotinic receptor expression and function. These changes may play a key role in nicotine reward and dependence.
ESTHER : Perez_2013_J.Neurochem_127_762
PubMedSearch : Perez_2013_J.Neurochem_127_762
PubMedID: 23992036

Title : Ventral tegmental area alpha6beta2 nicotinic acetylcholine receptors modulate phasic dopamine release in the nucleus accumbens core - Wickham_2013_Psychopharmacology.(Berl)_229_73
Author(s) : Wickham R , Solecki W , Rathbun L , McIntosh JM , Addy NA
Ref : Psychopharmacology (Berl) , 229 :73 , 2013
Abstract : RATIONALE: Phasic dopamine (DA) signaling underlies reward learning. Cholinergic and glutamatergic inputs into the ventral tegmental area (VTA) are crucial for modulating burst firing activity and subsequent phasic DA release in the nucleus accumbens (NAc), but the specific VTA nicotinic receptor subtypes that regulate phasic DA release have not been identified. OBJECTIVE: The goal was to determine the role of VTA N-methyl-D-aspartate receptors (NMDARs) and specific subtypes of nicotinic acetylcholine receptors (nAChRs) in regulating phasic DA release in the NAc core.
METHODS: Fast-scan cyclic voltammetry in anesthetized rats was combined with intra-VTA micro-infusion to evaluate the ability of glutamatergic and cholinergic drugs to modulate stimulated phasic DA release in the NAc core.
RESULTS: VTA NMDAR blockade with AP-5 decreased, while VTA NMDAR activation with NMDA increased NAc peak phasic DA release. Intra-VTA administration of the nonspecific nAChR antagonist mecamylamine produced a persistent decrease in phasic DA release. Infusion of the alpha6-selective antagonist alpha-conotoxin MII (alpha-ctx MII) produced a robust, but transient decrease in phasic DA, whereas infusion of selective doses of either the alpha4beta2-selective antagonist, dihydro-beta-erythroidine, or the alpha7 antagonist, methyllycaconitine, had no effect. Co-infusion of AP-5 and alpha-ctx MII produced a similar phasic DA decrease as either drug alone, with no additive effect.
CONCLUSIONS: The results suggest that VTA alpha6beta2 nAChRs, but not alpha4beta2 or alpha7 nAChRs, regulate phasic DA release in the NAc core and that VTA alpha6beta2 nAChRs and NMDA receptors act at a common site or target to regulate NAc phasic DA signaling.
ESTHER : Wickham_2013_Psychopharmacology.(Berl)_229_73
PubMedSearch : Wickham_2013_Psychopharmacology.(Berl)_229_73
PubMedID: 23624852

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 : The antinociceptive effects of nicotinic partial agonists varenicline and sazetidine-A in murine acute and tonic pain models - AlSharari_2012_J.Pharmacol.Exp.Ther_342_742
Author(s) : AlSharari SD , Carroll FI , McIntosh JM , Damaj MI
Ref : Journal of Pharmacology & Experimental Therapeutics , 342 :742 , 2012
Abstract : Nicotinic agonists display a wide-range profile of antinociceptive activity in acute, tonic, and chronic pain models. However, their effectiveness is limited by their unacceptable side effects. We investigated the antinociceptive effects of two new alpha4beta2* nicotinic partial agonists, varenicline and sazetidine-A, in acute thermal and tonic pain mouse models. Both drugs failed to induce significant effects in the tail-flick and hot-plate tests after subcutaneous administration. However, they blocked nicotine's effects in these tests at very low doses. In contrast to acute pain tests, varenicline and sazetidine-A dose-dependently induced an analgesic effect in the mouse formalin test after systemic administration. Their antinociceptive effects were mediated, however, by different nicotinic acetylcholine receptor (nAChR) subtypes. Sazetidine-A effects were mediated by beta2* nAChR subtypes, whereas varenicline actions were attributed to alpha3beta4 nAChRs. Moreover, low inactive doses of varenicline blocked nicotine's actions in phase II of the formalin test. Overall, our results suggest that the antagonistic actions of varenicline at low doses are mediated by beta2*-nAChRs and at higher doses as an agonist by alpha3beta4*-nAChRs. In contrast, both actions of sazetidine-A are mediated by beta2*-nAChR subtypes. These results suggest that nicotinic partial agonists possess analgesic effects in a rodent tonic pain model and may provide a potential treatment for the treatment of chronic pain disorders.
ESTHER : AlSharari_2012_J.Pharmacol.Exp.Ther_342_742
PubMedSearch : AlSharari_2012_J.Pharmacol.Exp.Ther_342_742
PubMedID: 22678099

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 acetylcholine receptors in dorsal root ganglion neurons include the alpha6beta4* subtype - Hone_2012_FASEB.J_26_917
Author(s) : Hone AJ , Meyer EL , McIntyre M , McIntosh JM
Ref : FASEB Journal , 26 :917 , 2012
Abstract : The alpha6-containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinson's disease and substance abuse. In contrast, little is known about the role of alpha6* nAChRs in the peripheral nervous system (where the asterisk denotes the possible presence of additional subunits). Dorsal root ganglia (DRG) neurons are known to express nAChRs with a pharmacology consistent with an alpha7, alpha3beta4*, and alpha4beta2* composition. Here we present evidence that DRG neurons also express alpha6* nAChRs. We used RT-PCR to show the presence of alpha6 subunit transcripts and patch-clamp electrophysiology together with subtype-selective alpha-conotoxins to pharmacologically characterize the nAChRs in rat DRG neurons. alpha-Conotoxin BuIA (500 nM) blocked acetylcholine-gated currents (I(ACh)) by 90.3 +/- 3.0%; the recovery from blockade was very slow, indicating a predominance of alpha(x)beta4* nAChRs. Perfusion with either 300 nM BuIA[T5A;P6O] or 200 nM MII[E11A], alpha-conotoxins that target the alpha6beta4* subtype, blocked I(ACh) by 49.3 +/- 5 and 46.7 +/- 8%, respectively. In these neurons, I(ACh) was relatively insensitive to 200 nM ArIB[V11L;V16D] (9.4+/-2.0% blockade) or 500 nM PnIA (23.0+/-4% blockade), alpha-conotoxins that target alpha7 and alpha3beta2*/alpha6beta2* nAChRs, respectively. We conclude that alpha6beta4* nAChRs are among the subtypes expressed by DRG, and to our knowledge, this is the first demonstration of alpha6beta4* in neurons outside the CNS.
ESTHER : Hone_2012_FASEB.J_26_917
PubMedSearch : Hone_2012_FASEB.J_26_917
PubMedID: 22024738

Title : alpha-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks alpha6beta2beta3 versus alpha6beta4 nicotinic acetylcholine receptors - Hone_2012_Mol.Pharmacol_82_972
Author(s) : Hone AJ , Scadden M , Gajewiak J , Christensen S , Lindstrom JM , McIntosh JM
Ref : Molecular Pharmacology , 82 :972 , 2012
Abstract : Nicotinic acetylcholine receptors (nAChRs) containing alpha6 and beta2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of beta2 and beta4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind alpha6beta2 nAChRs also potently bind the closely related alpha6beta4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described alpha-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including alpha6/alpha3beta2beta3 and alpha6/alpha3beta4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for alpha6/alpha3beta2beta3 nAChRs expressed in Xenopus oocytes (alpha6/alpha3 is a subunit chimera that contains the N-terminal ligand-binding domain of the alpha6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by alpha6/alpha3beta2beta3 and alpha6/alpha3beta4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to alpha6/alpha3beta2beta3 nAChRs yielded a k(off) of 0.096 +/- 0.001 min(-1) and a k(on) of 0.23 +/- 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between alpha6beta2 and alpha6beta4 nAChRs.
ESTHER : Hone_2012_Mol.Pharmacol_82_972
PubMedSearch : Hone_2012_Mol.Pharmacol_82_972
PubMedID: 22914547

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 : Nicotine persistently activates ventral tegmental area dopaminergic neurons via nicotinic acetylcholine receptors containing alpha4 and alpha6 subunits - Liu_2012_Mol.Pharmacol_81_541
Author(s) : Liu L , Zhao-Shea R , McIntosh JM , Gardner PD , Tapper AR
Ref : Molecular Pharmacology , 81 :541 , 2012
Abstract : Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brain's mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by alpha-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the alpha6 subunit. alpha-conotoxin MII[H9A;L15A] also significantly reduced the long-lasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either alpha4 or alpha6 nAChR subunits. Conversely, selective activation of nAChRs containing the alpha4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to alpha-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing alpha4 and alpha6 subunits.
ESTHER : Liu_2012_Mol.Pharmacol_81_541
PubMedSearch : Liu_2012_Mol.Pharmacol_81_541
PubMedID: 22222765

Title : Varenicline is a potent partial agonist at alpha6beta2* nicotinic acetylcholine receptors in rat and monkey striatum - Bordia_2012_J.Pharmacol.Exp.Ther_342_327
Author(s) : Bordia T , Hrachova M , Chin M , McIntosh JM , Quik M
Ref : Journal of Pharmacology & Experimental Therapeutics , 342 :327 , 2012
Abstract : Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that alpha6beta2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on alpha6beta2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited alpha6beta2* nAChR binding (K(i) = 0.12 nM) as potently as alpha4beta2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with approximately 20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating alpha6beta2* versus alpha4beta2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 muM, respectively. However, it acted as a partial agonist on alpha6beta2* and alpha4beta2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal alpha6beta2* (K(i) = 0.13 nM) and alpha4beta2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both alpha6beta2* (EC(50) = 0.014 muM) and alpha4beta2* (EC(50) = 0.029 muM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at alpha6beta2* nAChRs may contribute to its efficacy as a smoking cessation aid.
ESTHER : Bordia_2012_J.Pharmacol.Exp.Ther_342_327
PubMedSearch : Bordia_2012_J.Pharmacol.Exp.Ther_342_327
PubMedID: 22550286

Title : Nicotine reduces antipsychotic-induced orofacial dyskinesia in rats - Bordia_2012_J.Pharmacol.Exp.Ther_340_612
Author(s) : Bordia T , McIntosh JM , Quik M
Ref : Journal of Pharmacology & Experimental Therapeutics , 340 :612 , 2012
Abstract : Antipsychotics are an important class of drugs for the management of schizophrenia and other psychotic disorders. They act by blocking dopamine receptors; however, because these receptors are present throughout the brain, prolonged antipsychotic use also leads to serious side effects. These include tardive dyskinesia, repetitive abnormal involuntary movements of the face and limbs for which there is little treatment. In this study, we investigated whether nicotine administration could reduce tardive dyskinesia because nicotine attenuates other drug-induced abnormal movements. We used a well established model of tardive dyskinesia in which rats injected with the commonly used antipsychotic haloperidol develop vacuous chewing movements (VCMs) that resemble human orofacial dyskinesias. Rats were first administered nicotine (minipump; 2 mg/kg per day). Two weeks later, they were given haloperidol (1 mg/kg s.c.) once daily. Nicotine treatment reduced haloperidol-induced VCMs by approximately 20% after 5 weeks, with a significant approximately 60% decline after 13 weeks. There was no worsening of haloperidol-induced catalepsy. To understand the molecular basis for this improvement, we measured the striatal dopamine transporter and nicotinic acetylcholine receptors (nAChRs). Both haloperidol and nicotine treatment decreased the transporter and alpha6beta2* nAChRs (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex) when given alone, with no further decline with combined drug treatment. By contrast, nicotine alone increased, while haloperidol reduced alpha4beta2* nAChRs in both vehicle and haloperidol-treated rats. These data suggest that molecular mechanisms other than those directly linked to the transporter and nAChRs underlie the nicotine-mediated improvement in haloperidol-induced VCMs in rats. The present results are the first to suggest that nicotine may be useful for improving the tardive dyskinesia associated with antipsychotic use.
ESTHER : Bordia_2012_J.Pharmacol.Exp.Ther_340_612
PubMedSearch : Bordia_2012_J.Pharmacol.Exp.Ther_340_612
PubMedID: 22144565

Title : Striatal alpha5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum - Exley_2012_J.Neurosci_32_2352
Author(s) : Exley R , McIntosh JM , Marks MJ , Maskos U , Cragg SJ
Ref : Journal of Neuroscience , 32 :2352 , 2012
Abstract : Polymorphisms in the gene for the alpha5 nicotinic acetylcholine receptor (nAChR) subunit are associated with vulnerability to nicotine addiction. However, the underlying normal functions of alpha5-containing nAChRs in the brain are poorly understood. Striatal dopamine (DA) transmission is critical to the acquisition and maintenance of drug addiction and is modulated strongly by nicotine acting at heteromeric beta2-containing (beta2*) nAChRs. We explored whether alpha5 subunits, as well as alpha4, alpha6, and beta3 subunits, participate in the powerful regulation of DA release probability by beta2* nAChRs in nucleus accumbens (NAc) core and in dorsal striatum [caudatoputamen (CPu)]. We detected evoked dopamine release using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in striatal slices from mice with deletions of alpha4, alpha5, alpha6, or beta3 subunits. We show that the nAChR subtypes that dominantly regulate dopamine transmission depend critically upon alpha5 subunits in the dorsal CPu in alpha4alpha5(non-alpha6)beta2-nAChRs but not in NAc core, where alpha4alpha6beta2beta3-nAChRs are required. These data reveal the distinct populations of nAChRs that govern DA transmission in NAc core versus dorsal CPu. Furthermore, they indicate that alpha5 subunits are critical to the regulation of DA transmission by alpha4beta2* nAChRs in regions of striatum associated with habitual and instrumental responses (dorsal CPu) rather than pavlovian associations (NAc).
ESTHER : Exley_2012_J.Neurosci_32_2352
PubMedSearch : Exley_2012_J.Neurosci_32_2352
PubMedID: 22396410

Title : Alpha7 nicotinic acetylcholine receptors modulate motivation to self-administer nicotine: implications for smoking and schizophrenia - Brunzell_2012_Neuropsychopharmacology_37_1134
Author(s) : Brunzell DH , McIntosh JM
Ref : Neuropsychopharmacology , 37 :1134 , 2012
Abstract : Individuals diagnosed with schizophrenia have an exceptionally high risk for tobacco dependence. Postmortem studies show that these individuals have significant reductions in alpha7 nicotinic acetylcholine receptors (nAChRs) in several brain areas. Decreased alpha7-mediated function might not only be linked to schizophrenia but also to increased tobacco consumption. The purpose of this study was to determine whether pharmacological blockade of alpha7 nAChRs would increase motivation of rats to intravenously self-administer nicotine (NIC) during a progressive ratio schedule of reinforcement (PR). Before PR, rats received local infusions of 0, 10, or 20 pmol of a selective alpha7 nAChR antagonist, alpha-conotoxin ArIB [V11L,V16D] (ArIB) into the nucleus accumbens (NAc) shell or the anterior cingulate cortex, brain areas that contribute to motivation for drug reward. We additionally sought to determine whether local infusion of 0, 10, or 40 nmol of a selective alpha7 nAChR agonist, PNU 282987, into these brain areas would decrease motivation for NIC use. Infusion of ArIB into the NAc shell and anterior cingulate cortex resulted in a significant increase in active lever pressing, breakpoints, and NIC intake, suggesting that a decrease in alpha7 nAChR function increases motivation to work for NIC. In contrast, PNU 282987 infusion resulted in reductions in these measures when administered into the NAc shell, but had no effect after administration into the anterior cingulate cortex. These data identify reduction of alpha7 nAChR function as a potential mechanism for elevated tobacco use in schizophrenia and also identify activation of alpha7 nAChRs as a potential strategy for tobacco cessation therapy.
ESTHER : Brunzell_2012_Neuropsychopharmacology_37_1134
PubMedSearch : Brunzell_2012_Neuropsychopharmacology_37_1134
PubMedID: 22169946

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 : Long-term nicotine exposure depresses dopamine release in nonhuman primate nucleus accumbens - Perez_2012_J.Pharmacol.Exp.Ther_342_335
Author(s) : Perez XA , Ly J , McIntosh JM , Quik M
Ref : Journal of Pharmacology & Experimental Therapeutics , 342 :335 , 2012
Abstract : Tobacco use is a leading cause of preventable deaths worldwide. However, current smoking cessation therapies have very limited long-term success rates. Considerable research effort is therefore focused on identification of central nervous system changes with nicotine exposure because this may lead to more successful treatment options. Although recent work suggests that alpha6beta2* nicotinic acetylcholine receptors (nAChRs) play a dominant role in dopaminergic function in rodent nucleus accumbens, the effects of long-term nicotine exposure remain to be determined. Here, we used cyclic voltammetry to investigate alpha6beta2* nAChR-mediated release with long-term nicotine treatment in nonhuman primate nucleus accumbens shell. Control studies showed that nAChR-mediated dopamine release occurs predominantly through the alpha6beta2* receptor subtype. Unexpectedly, there was a complete loss of alpha6beta2* nAChR-mediated activity after several months of nicotine treatment. This decline in function was observed with both single- and multiple-pulse-stimulated dopamine release. Paired-pulse studies showed that the facilitation of dopamine release with multiple pulsing observed in controls in the presence of nAChR antagonist was lost with long-term nicotine treatment. Nicotine-evoked [(3)H]dopamine release from nucleus accumbens synaptosomes was similar in nicotine- and vehicle-treated monkeys, indicating that long-term nicotine administration does not directly modify alpha6beta2* nAChR-mediated dopamine release. Dopamine uptake rates, as well as dopamine transporter and alpha6beta2* nAChRs levels, were also not changed with nicotine administration. These data indicate that nicotine exposure, as occurs with smoking, has major effects on cellular mechanisms linked to alpha6beta2* nAChR-mediated dopamine release and that this receptor subtype may represent a novel therapeutic target for smoking cessation.
ESTHER : Perez_2012_J.Pharmacol.Exp.Ther_342_335
PubMedSearch : Perez_2012_J.Pharmacol.Exp.Ther_342_335
PubMedID: 22562772

Title : alpha4beta2 nicotinic acetylcholine receptors in the early postnatal mouse superior cervical ganglion - Scholze_2011_Dev.Neurobiol_71_390
Author(s) : Scholze P , Ciuraszkiewicz A , Groessl F , Orr-Urtreger A , McIntosh JM , Huck S
Ref : Dev Neurobiol , 71 :390 , 2011
Abstract : Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that alpha3 and beta4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain alpha4 have been controversial. Here, we have searched for the presence of alpha4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that alpha4-containing receptors constitute about 20% of hetero-oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of alpha4 approaches zero. In contrast, the number of alpha4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the beta2 subunit by using alpha5beta2-double knockout (KO) mice removes all alpha4-containing receptors, suggesting that in the postnatal mouse SCG, alpha4 co-assembles only with beta2 but not with beta4. alpha4beta2 receptors are, on the other hand, up-regulated in the SCG of P3 alpha5beta4-double KO mice, where they make up about 50% of receptors that bind [(3) H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from alpha5beta4-double KO mice maintained for one to two days in culture comprise <10% of alpha4beta2 and >90% of alpha3beta2, as determined by patch clamp recordings with alpha4beta2- and alpha3beta2-specific ligands. We propose that in the P3 SCG of wild type mice, alpha3beta4 (+/-alpha5) represent about 62% of receptors, whereas 17% are alpha3beta2beta4, and 21% are alpha4beta2 (+/-alpha5) receptors.
ESTHER : Scholze_2011_Dev.Neurobiol_71_390
PubMedSearch : Scholze_2011_Dev.Neurobiol_71_390
PubMedID: 21485013

Title : The novel small molecule alpha9alpha10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic - Holtman_2011_Eur.J.Pharmacol_670_500
Author(s) : Holtman JR , Dwoskin LP , Dowell C , Wala EP , Zhang Z , Crooks PA , McIntosh JM
Ref : European Journal of Pharmacology , 670 :500 , 2011
Abstract : Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the alpha9alpha10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the alpha9alpha10 subtype with an IC(5)(0) of 0.51 (0.35-0.72) nM. Antagonist activity at other nicotinic subtypes (alpha1beta1deltaepsilon, alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, alpha6/alpha3beta2beta3, alpha6/alpha3beta4 and alpha7) was 10-1000-fold lower than at the alpha9alpha10 subtype. In competition binding assays, the k(i) of ZZ-204G at gamma-aminobutyric acid(A), serotonin(3), gamma-aminobutyric acid(B), kappa- and mu-opioid receptors was 1000- to >10,000-fold lower than at alpha9alpha10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for alpha9alpha10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.
ESTHER : Holtman_2011_Eur.J.Pharmacol_670_500
PubMedSearch : Holtman_2011_Eur.J.Pharmacol_670_500
PubMedID: 21944926

Title : Functional nicotinic acetylcholine receptors containing alpha6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons - Yang_2011_J.Neurosci_31_2537
Author(s) : Yang K , Buhlman L , Khan GM , Nichols RA , Jin G , McIntosh JM , Whiteaker P , Lukas RJ , Wu J
Ref : Journal of Neuroscience , 31 :2537 , 2011
Abstract : Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR alpha6 subunit message is abundant in the VTA, but alpha6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for alpha6beta2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. alpha-Conotoxin (alpha-Ctx) MII labeling coupled with immunocytochemical staining localizes putative alpha6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by alpha6*-nAChR-selective alpha-Ctx MII or alpha-Ctx PIA (1 nm) but not by alpha7 (10 nm methyllycaconitine) or alpha4* (1 mum dihydro-beta-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR beta2 knock-out mice. Moreover, ACh induces an alpha-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that alpha6beta2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic alpha6beta2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.
ESTHER : Yang_2011_J.Neurosci_31_2537
PubMedSearch : Yang_2011_J.Neurosci_31_2537
PubMedID: 21325521

Title : bPiDI: a novel selective alpha6beta2* nicotinic receptor antagonist and preclinical candidate treatment for nicotine abuse - Wooters_2011_Br.J.Pharmacol_163_346
Author(s) : Wooters TE , Smith AM , Pivavarchyk M , Siripurapu KB , McIntosh JM , Zhang Z , Crooks PA , Bardo MT , Dwoskin LP
Ref : British Journal of Pharmacology , 163 :346 , 2011
Abstract : BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) containing alpha6beta2 subunits expressed by dopamine neurons regulate nicotine-evoked dopamine release. Previous results show that the alpha6beta2* nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) inhibits nicotine-evoked dopamine release from dorsal striatum and decreases nicotine self-administration in rats. However, overt toxicity emerged with repeated bPiDDB treatment. The current study evaluated the preclinical pharmacology of a bPiDDB analogue. EXPERIMENTAL APPROACH: The C(1)(0) analogue of bPiDDB, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), was evaluated preclinically for nAChR antagonist activity. KEY
RESULTS: bPiDI inhibits nicotine-evoked [(3)H]dopamine overflow (IC(5)(0)= 150 nM, I(max)=58%) from rat striatal slices. Schild analysis revealed a rightward shift in the nicotine concentration-response curve and surmountability with increasing nicotine concentration; however, the Schild regression slope differed significantly from 1.0, indicating surmountable allosteric inhibition. Co-exposure of maximally inhibitory concentrations of bPiDI (1 microM) and the alpha6beta2* nAChR antagonist alpha-conotoxin MII (1 nM) produced inhibition not different from either antagonist alone, indicating that bPiDI acts at alpha6beta2* nAChRs. Nicotine treatment (0.4, 10 days) increased more than 100-fold the potency of bPiDI (IC(5)(0)=1.45 nM) to inhibit nicotine-evoked dopamine release. Acute treatment with bPiDI (1.94-5.83, s.c.) specifically reduced nicotine self-administration relative to responding for food. Across seven daily treatments, bPiDI decreased nicotine self-administration; however, tolerance developed to the acute decrease in food-maintained responding. No observable body weight loss or lethargy was observed with repeated bPiDI. CONCLUSIONS AND IMPLICATIONS: These results are consistent with the hypothesis that alpha6beta2* nAChR antagonists have potential for development as pharmacotherapies for tobacco smoking cessation.
ESTHER : Wooters_2011_Br.J.Pharmacol_163_346
PubMedSearch : Wooters_2011_Br.J.Pharmacol_163_346
PubMedID: 21232049

Title : Indolizidine (-)-235B' and related structural analogs: discovery of nicotinic receptor antagonists that inhibit nicotine-evoked [3H]dopamine release - Pivavarchyk_2011_Eur.J.Pharmacol_658_132
Author(s) : Pivavarchyk M , Smith AM , Zhang Z , Zhou D , Wang X , Toyooka N , Tsuneki H , Sasaoka T , McIntosh JM , Crooks PA , Dwoskin LP
Ref : European Journal of Pharmacology , 658 :132 , 2011
Abstract : Although several therapeutic agents are available to aid in tobacco smoking cessation, relapse rates continue to be high, warranting the development of alternative pharmacotherapies. Nicotine-evoked dopamine release from its presynaptic terminals in the central nervous system leads to reward which maintains continued tobacco use. The ability of indolizidine (-)-235B' and a sub-library of structurally related analogs to inhibit nicotine-evoked [(3)H]dopamine release from rat striatal slices was determined in the current study. Indolizidine (-)-235B' inhibited nicotine-evoked [(3)H]dopamine release in a concentration-dependent manner (IC(50)=42 nM, I(max)=55%). Compound (-)-237D, the double bond-reduced analog, afforded the greatest inhibitory potency (IC(50)=0.18 nM, I(max)=76%), and was 233-fold more potent than indolizidine (-)-235B'. The des-8-methyl aza-analog of indolizidine (-)-235B', ZZ-272, also inhibited nicotine-evoked [(3)H]dopamine release (IC(50)=413 nM, I(max)=59%). Concomitant exposure to maximally effective concentrations of indolizidine (-)-235B', ZZ-272 or (-)-237D with a maximally effective concentration of alpha-conotoxin MII, a selective antagonist for alpha6beta2-containing nicotinic receptors, resulted in inhibition of nicotine-evoked [(3)H]dopamine release no greater than that produced by each compound alone. The latter results suggest that indolizidine (-)-235B', (-)-237D, ZZ-272 and alpha-conotoxin MII inhibit the same alpha-conotoxin MII-sensitive nicotinic receptor subtypes. Thus, indolizidine (-)-235B' and its analogs act as antagonists of alpha6beta2-nicotinic receptors and constitute a novel structural scaffold for the discovery of pharmacotherapies for smoking cessation.
ESTHER : Pivavarchyk_2011_Eur.J.Pharmacol_658_132
PubMedSearch : Pivavarchyk_2011_Eur.J.Pharmacol_658_132
PubMedID: 21371454

Title : Neurotoxicity induced by okadaic acid in the human neuroblastoma SH-SY5Y line can be differentially prevented by alpha7 and beta2* nicotinic stimulation - Del Barrio_2011_Toxicol.Sci_123_193
Author(s) : Del Barrio L , Martin-de-Saavedra MD , Romero A , Parada E , Egea J , Avila J , McIntosh JM , Wonnacott S , Lopez MG
Ref : Toxicol Sci , 123 :193 , 2011
Abstract : A good model of neuronal death that reproduces the characteristic tau (tau) hyperphosphorylation of Alzheimers disease is the use of okadaic acid (OA). The aim of this study was to determine the contribution of alpha7 and beta2* nicotinic acetylcholine receptor (nAChR) subtypes to neuroprotection against OA in the SH-SY5Y cell line by using the selective alpha7 and beta2* nAChR agonists PNU 282987 and 5-Iodo-A85380, respectively. The results of this study show that both alpha7 and beta2* nAChR can afford neuroprotection against OA-induced neurotoxicity. Protection mediated by alpha7 nAChRs was independent of Ca(2+) and involved the intracellular signaling pathway Janus Kinase-2/Phosphatidylinositol-3-kinase/Akt. When Ca(2+) entry was promoted through the alpha7 nAChR by using the alpha7-selective positive allosteric modulator PNU 120596, protection was lost. By contrast, protection mediated by beta2* nAChRs was Ca(2+) dependent and implicated the signaling pathways PI3K/Akt and extracellular regulated kinase 1/2. Both alpha7 and beta2* nAChR activation converged on downregulation of GSK-3beta and reduction of tau phosphorylation in cells undergoing cell death induced by OA. Therefore, targeting nAChR could offer a strategy for reducing neurodegeneration secondary to hyperphosphorylation of protein tau.
ESTHER : Del Barrio_2011_Toxicol.Sci_123_193
PubMedSearch : Del Barrio_2011_Toxicol.Sci_123_193
PubMedID: 21715663

Title : Characterizing functional alpha6beta2 nicotinic acetylcholine receptors in vitro: mutant beta2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells - Xiao_2011_Biochem.Pharmacol_82(8)_852
Author(s) : Xiao C , Srinivasan R , Drenan RM , Mackey ED , McIntosh JM , Lester HA
Ref : Biochemical Pharmacology , 82 :852 , 2011
Abstract : alpha6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of alpha6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-alpha6 and beta2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 muM ACh in 26% (5/19) of cells with evenly expressed alpha6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional alpha6-eGFPbeta2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into beta2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased alpha6-eGFPbeta2 nAChR current amplitude. alpha6-eGFPbeta2 nAChRs were also activated by nicotine and by TC-2403. The alpha6-eGFPbeta2 currents were desensitized by 1muM nicotine, blocked by alpha-conotoxin MII, partially inhibited by dihydro-beta-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that alpha6-eGFPbeta2 nAChRs had similar single-channel conductance to, but longer open time than, alpha4-eGFPbeta2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of alpha6* nAChRs for both pharmacological study and drug screening.
ESTHER : Xiao_2011_Biochem.Pharmacol_82(8)_852
PubMedSearch : Xiao_2011_Biochem.Pharmacol_82(8)_852
PubMedID: 21609715

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 : Discovery of non-peptide, small molecule antagonists of alpha9alpha10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain - Zheng_2011_Bioorg.Med.Chem.Lett_21_2476
Author(s) : Zheng G , Zhang Z , Dowell C , Wala E , Dwoskin LP , Holtman JR , McIntosh JM , Crooks PA
Ref : Bioorganic & Medicinal Chemistry Lett , 21 :2476 , 2011
Abstract : A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective alpha9alpha10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of alpha9alpha10 nAChRs and greater selectivity over alpha7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.
ESTHER : Zheng_2011_Bioorg.Med.Chem.Lett_21_2476
PubMedSearch : Zheng_2011_Bioorg.Med.Chem.Lett_21_2476
PubMedID: 21397497

Title : Distinct contributions of nicotinic acetylcholine receptor subunit alpha4 and subunit alpha6 to the reinforcing effects of nicotine - Exley_2011_Proc.Natl.Acad.Sci.U.S.A_108_7577
Author(s) : Exley R , Maubourguet N , David V , Eddine R , Evrard A , Pons S , Marti F , Threlfell S , Cazala P , McIntosh JM , Changeux JP , Maskos U , Cragg SJ , Faure P
Ref : Proc Natl Acad Sci U S A , 108 :7577 , 2011
Abstract : Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit alpha4- and subunit alpha6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the alpha4 subunit, but not the alpha6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits alpha4 and alpha6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that alpha4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing alpha4 and alpha6 subunits, underlies nicotine self-administration and its long-term maintenance.
ESTHER : Exley_2011_Proc.Natl.Acad.Sci.U.S.A_108_7577
PubMedSearch : Exley_2011_Proc.Natl.Acad.Sci.U.S.A_108_7577
PubMedID: 21502501

Title : Poster: Nicotinic cholinergic receptors in dorsal root ganglion neurons include the alpha6\/beta4 subtype -
Author(s) : Hone AJ , Meyer EL , McIntyre M , McIntosh JM
Ref : Biochemical Pharmacology , 82 :1033 , 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 : Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea - Roux_2011_J.Neurosci_31_15092
Author(s) : Roux I , Wersinger E , McIntosh JM , Fuchs PA , Glowatzki E
Ref : Journal of Neuroscience , 31 :15092 , 2011
Abstract : In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by alpha9/alpha10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mm acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as alpha9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated alpha-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6-P8) on outer hair cells in the basal cochlea.
ESTHER : Roux_2011_J.Neurosci_31_15092
PubMedSearch : Roux_2011_J.Neurosci_31_15092
PubMedID: 22016543

Title : alpha-Conotoxin BuIA[T5A\;P6O]: a novel ligand that discriminates between alpha6ss4 and alpha6ss2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release - Azam_2010_FASEB.J_24_5113
Author(s) : Azam L , Maskos U , Changeux JP , Dowell CD , Christensen S , De Biasi M , McIntosh JM
Ref : FASEB Journal , 24 :5113 , 2010
Abstract : alpha6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of alpha6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. alpha-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of alpha-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at alpha6/alpha3beta2beta3 but only by 6-fold at alpha6/alpha3beta4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in alpha6-null mutant mice. alpha-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in beta4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain alpha6beta2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related alpha6* nAChRs; alpha6beta4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.
ESTHER : Azam_2010_FASEB.J_24_5113
PubMedSearch : Azam_2010_FASEB.J_24_5113
PubMedID: 20739611

Title : Alexa Fluor 546-ArIB[V11L\;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors - Hone_2010_J.Neurochem_114_994
Author(s) : Hone AJ , Whiteaker P , Mohn JL , Jacob MH , McIntosh JM
Ref : Journal of Neurochemistry , 114 :994 , 2010
Abstract : The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.
ESTHER : Hone_2010_J.Neurochem_114_994
PubMedSearch : Hone_2010_J.Neurochem_114_994
PubMedID: 20492354

Title : Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes - David_2010_Eur.J.Neurosci_31_978
Author(s) : David R , Ciuraszkiewicz A , Simeone X , Orr-Urtreger A , Papke RL , McIntosh JM , Huck S , Scholze P
Ref : European Journal of Neuroscience , 31 :978 , 2010
Abstract : Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild-type (WT) mice expressed: alpha 3 beta 4 (55%), alpha 3 beta 4 alpha 5 (24%) and alpha 3 beta 4 beta 2 (21%) nAChRs. nAChRs in beta 4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the alpha 5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between alpha 5 beta 4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of alpha 5 or beta2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct alpha 3 beta4 and alpha 3 beta 2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the alpha-conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG.
ESTHER : David_2010_Eur.J.Neurosci_31_978
PubMedSearch : David_2010_Eur.J.Neurosci_31_978
PubMedID: 20377613

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 : Nicotinic receptor-mediated reduction in L-DOPA-induced dyskinesias may occur via desensitization - Bordia_2010_J.Pharmacol.Exp.Ther_333_929
Author(s) : Bordia T , Campos C , McIntosh JM , Quik M
Ref : Journal of Pharmacology & Experimental Therapeutics , 333 :929 , 2010
Abstract : L-DOPA-induced dyskinesias in Parkinson's disease are a significant clinical problem for which few therapies are available. We recently showed that nicotine reduces L-DOPA-induced abnormal involuntary movements (AIMs) in parkinsonian animals, suggesting it may be useful for the treatment of L-DOPA-induced dyskinesias. The present experiments were performed to understand the mechanisms whereby nicotine reduces L-DOPA-induced AIMs. We used a well established model of dyskinesias, L-DOPA-treated unilateral 6-hydroxydopamine-lesioned rats. Dose-ranging studies showed that injection of 0.1 mg/kg nicotine once or twice daily for 4 or 10 days most effectively reduced AIMs, with no worsening of parkinsonism. Importantly, a single nicotine injection did not reduce AIMs, indicating that nicotine's effect is caused by long-term rather than short-term molecular changes. Administration of the metabolite cotinine did not reduce AIMs, suggesting a direct effect of nicotine. Experiments with the nicotinic receptor (nAChR) antagonist mecamylamine were done to determine whether nicotine acted via a receptor-mediated mechanism. Unexpectedly, several days of mecamylamine injection (1.0 mg/kg) alone significantly ameliorated dyskinesias to a comparable extent as nicotine. The decline in AIMs with combined nicotine and mecamylamine treatment was not additive, suggesting that nicotine exerts its effects via a nAChR interaction. This latter finding, combined with data showing that mecamylamine reduced AIMs to a similar extent as nicotine, and that nicotine or mecamylamine treatment both decreased alpha6beta2* and increased alpha4beta2* nAChR expression, suggests that the nicotine-mediated improvement in L-DOPA-induced AIMs may involve a desensitization block. These data have important implications for the treatment of L-DOPA-induced dyskinesias in Parkinson's disease.
ESTHER : Bordia_2010_J.Pharmacol.Exp.Ther_333_929
PubMedSearch : Bordia_2010_J.Pharmacol.Exp.Ther_333_929
PubMedID: 20200117

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 : Chronic nicotine treatment increases nAChRs and microglial expression in monkey substantia nigra after nigrostriatal damage - Quik_2010_J.Mol.Neurosci_40_105
Author(s) : Quik M , Campos C , Parameswaran N , Langston JW , McIntosh JM , Yeluashvili M
Ref : Journal of Molecular Neuroscience , 40 :105 , 2010
Abstract : Our previous work had shown that long-term nicotine administration improved dopaminergic markers and nicotinic receptors (nAChRs) in the striatum of monkeys with nigrostriatal damage. The present experiments were done to determine whether nicotine treatment also led to changes in the substantia nigra, the region containing dopaminergic cell bodies. Monkeys were chronically treated with nicotine in the drinking water for 6 months after which they were injected with low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine (MPTP) for a further 6-month period. Nicotine was administered until the monkeys were euthanized 2 months after the last MPTP injection. Nicotine treatment did not affect the dopamine transporter or the number of tyrosine hydroxylase positive cells in the substantia nigra of lesioned monkeys. However, nicotine administration did lead to a greater increase in alpha3/alpha6beta2* and alpha4beta2* nAChRs in lesioned monkeys compared to controls. Nicotine also significantly elevated microglia and reduced the number of extracellular neuromelanin deposits in the substantia nigra of MPTP-lesioned monkeys. These findings indicate that long-term nicotine treatment modulates expression of several molecular measures in monkey substantia nigra that may result in an improvement in nigral integrity and/or function. These observations may have therapeutic implications for Parkinson's disease.
ESTHER : Quik_2010_J.Mol.Neurosci_40_105
PubMedSearch : Quik_2010_J.Mol.Neurosci_40_105
PubMedID: 19685015

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 : Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine - Brunzell_2010_Neuropsychopharmacology_35_665
Author(s) : Brunzell DH , Boschen KE , Hendrick ES , Beardsley PM , McIntosh JM
Ref : Neuropsychopharmacology , 35 :665 , 2010
Abstract : Beta2 subunit containing nicotinic acetylcholine receptors (beta2(*)nAChRs; asterisk ((*)) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The alpha6 subunit assembles with beta2 on DA neurons where alpha6beta2(*)nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of alpha-conotoxin MII (alpha-CTX MII), an antagonist with selectivity for alpha6beta2(*)nAChRs, the purpose of these experiments was to determine if alpha6beta2(*)nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light+tone cues (NIC) or for light+tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of alpha-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. alpha-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate cortex. Concentrations of alpha-CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion alpha-CTX MII did not affect locomotor activity in an open field. These data suggest that alpha6beta2(*)nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation.
ESTHER : Brunzell_2010_Neuropsychopharmacology_35_665
PubMedSearch : Brunzell_2010_Neuropsychopharmacology_35_665
PubMedID: 19890263

Title : Repeated nicotine administration robustly increases bPiDDB inhibitory potency at alpha6beta2-containing nicotinic receptors mediating nicotine-evoked dopamine release - Smith_2010_Biochem.Pharmacol_80_402
Author(s) : Smith AM , Pivavarchyk M , Wooters TE , Zhang Z , Zheng G , McIntosh JM , Crooks PA , Bardo MT , Dwoskin LP
Ref : Biochemical Pharmacology , 80 :402 , 2010
Abstract : The novel nicotinic receptor (nAChR) antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and alpha-conotoxin MII (alpha-CtxMII), a peptide antagonist selective for alpha6beta2-containing nAChRs, to inhibit nicotine-evoked [(3)H]DA release from striatal slices from rats repeatedly administered nicotine (0.4mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1nM) and alpha-CtxMII (1nM) resulted in inhibition of nicotine-evoked [(3)H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits alpha6beta2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [(3)H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for alpha-CtxMII was greater following repeated nicotine compared to control (I(max)=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [(3)H]DA release in nicotine-treated rats compared to control rats (IC(50)=5pM vs. 6nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of alpha6beta2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different alpha6beta2-containing nAChR subtypes.
ESTHER : Smith_2010_Biochem.Pharmacol_80_402
PubMedSearch : Smith_2010_Biochem.Pharmacol_80_402
PubMedID: 20346923

Title : alpha6ss2* and alpha4ss2* nicotinic receptors both regulate dopamine signaling with increased nigrostriatal damage: relevance to Parkinson's disease - Perez_2010_Mol.Pharmacol_78_971
Author(s) : Perez XA , Bordia T , McIntosh JM , Quik M
Ref : Molecular Pharmacology , 78 :971 , 2010
Abstract : Nicotinic receptors (nAChRs) are important modulators of dopaminergic transmission in striatum, a region critical to Parkinson's disease. The nAChRs mainly involved are the alpha6beta2* and alpha4beta2* subtypes. Lesion studies show that the alpha6beta2* receptor is decreased to a much greater extent with nigrostriatal damage than the alpha4beta2* subtype raising the question whether this latter nAChR population is more important with increased nigrostriatal damage. To address this, we investigated the effect of varying nigrostriatal damage on alpha6beta2* and alpha4beta2* receptor-modulated dopamine signaling using cyclic voltammetry. This approach offers the advantage that changes in dopamine release can be observed under different neuronal firing conditions. Total single-pulse-evoked dopamine release decreased in direct proportion to declines in the dopamine transporter and dopamine uptake. We next used alpha-conotoxinMII and mecamylamine to understand the role of the alpha4beta2* and alpha6beta2* subtypes in release. Single-pulse-stimulated alpha6beta2* and alpha4beta2* receptor dopamine release decreased to a similar extent with increasing nigrostriatal damage, indicating that both subtypes contribute to the control of dopaminergic transmission with lesioning. Total burst-stimulated dopamine release also decreased proportionately with nigrostriatal damage. However, the role of the alpha4beta2* and alpha6beta2* nAChRs varied with different degrees of lesioning, suggesting that the two subtypes play a unique function with burst firing, with a somewhat more prominent and possibly more selective role for the alpha6beta2* subtype. These data have important therapeutic implications because they suggest that drugs directed to both alpha4beta2* and alpha6beta2* nAChRs may be useful in the treatment of neurological disorders such as Parkinson's disease.
ESTHER : Perez_2010_Mol.Pharmacol_78_971
PubMedSearch : Perez_2010_Mol.Pharmacol_78_971
PubMedID: 20732972

Title : Prominent role of alpha3\/alpha6beta2* nAChRs in regulating evoked dopamine release in primate putamen: effect of long-term nicotine treatment - Perez_2009_Mol.Pharmacol_75_938
Author(s) : Perez XA , O'Leary KT , Parameswaran N , McIntosh JM , Quik M
Ref : Molecular Pharmacology , 75 :938 , 2009
Abstract : Brain dopaminergic systems are critical in motor control as evidenced by findings that their disruption results in movement disorders such as Parkinson's disease. Nicotinic acetylcholine receptor (nAChR) activation plays an important role in regulating striatal dopaminergic function. Rodent studies show that short-term nicotine exposure influences stimulated striatal dopamine release with responsiveness dependent on neuronal activity. However, studies have not yet been done in nonhuman primates, nor has work been done to evaluate the effect of long-term nicotine exposure, which is relevant for therapies for chronic neurological disorders. Here, we used voltammetry to assess the role of nAChRs on evoked dopamine release from monkey putamen slices. In both ventral and dorsal putamen, alpha3/alpha6beta2(*) nAChRs regulated > or =80% of non-burst- (single pulse) nAChR-modulated dopamine release, and alpha4beta2(*) nAChRs regulated the remainder. Similar results were observed with burst-firing in ventral but not dorsal putamen, indicating that nAChR-modulated effects on release depend on the subregion and firing frequency. Next, we investigated the consequence of long-term nicotine exposure via the drinking water on nAChR-modulated responsiveness. Nicotine treatment altered both non-burst- and burst-stimulated dopamine release in ventral but not dorsal putamen. Altogether, these data support a predominant role for alpha3/alpha6beta2(*) nAChRs in the regulation of evoked dopamine release in nonhuman primate putamen. They also show that long-term nicotine treatment selectively modifies nAChR-modulated release in distinct striatal subregions. These findings have implications for the development of treatments for addiction and neurological disorders with nAChR dysfunction.
ESTHER : Perez_2009_Mol.Pharmacol_75_938
PubMedSearch : Perez_2009_Mol.Pharmacol_75_938
PubMedID: 19144785

Title : Poster: Labeling alpha7 nAChRs on hippocampal neurons using fluorescent analogs of alpha-conotoxin ArIB[V11L\;V16A] -
Author(s) : Hone AJ , Mohn JL , Jacob M , Whiteaker P , McIntosh JM
Ref : Biochemical Pharmacology , 78 :903 , 2009

Title : Chronic nicotine selectively enhances alpha4beta2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway - Xiao_2009_J.Neurosci_29_12428
Author(s) : Xiao C , Nashmi R , McKinney S , Cai H , McIntosh JM , Lester HA
Ref : Journal of Neuroscience , 29 :12428 , 2009
Abstract : These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional alpha4beta2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and alpha4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-beta-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3-1000 mum ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional alpha4* nAChRs were not found on the neuronal somata; however, nicotine acts via alpha4beta2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these alpha4beta2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of alpha4beta2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease.
ESTHER : Xiao_2009_J.Neurosci_29_12428
PubMedSearch : Xiao_2009_J.Neurosci_29_12428
PubMedID: 19812319

Title : The role of alpha6-containing nicotinic acetylcholine receptors in nicotine reward and withdrawal - Jackson_2009_J.Pharmacol.Exp.Ther_331_547
Author(s) : Jackson KJ , McIntosh JM , Brunzell DH , Sanjakdar SS , Damaj MI
Ref : Journal of Pharmacology & Experimental Therapeutics , 331 :547 , 2009
Abstract : The alpha6 nicotinic acetylcholine receptor (nAChR) subunit is involved in nicotine-stimulated dopamine release in the striatum. It is expressed in brain regions and coexpressed with nAChR subtypes implicated in nicotine dependence behaviors; hence, this subunit may play a role in nicotine dependence. Using the alpha6-selective antagonist alpha-conotoxin H9A;L15A (MII[H9A;L15A]), we determined the role of alpha6* nAChRs in the pharmacological and behavioral effects of nicotine. We measured effects of pretreatment with MII[H9A;L15A] on analgesia, locomotion, and body temperature after a single injection of nicotine. Effects of MII[H9A;L15A] on nicotine reward were measured using the conditioned place preference (CPP) paradigm. We further measured physical (somatic signs and hyperalgesia) and affective [anxiety-related behavior and conditioned place aversion (CPA)] nicotine withdrawal behaviors after extended nicotine exposure. Results showed that MII[H9A;L15A] did not block acute nicotine effects on the behaviors measured. Conversely, MII[H9A:l15A] blocked the expression of nicotine CPP, as well as withdrawal-associated CPA and anxiety-related behavior in the elevated plus maze, but not withdrawal-induced somatic signs or hyperalgesia. These results suggest a role for the alpha6 nAChR subunit in nicotine reward and affective nicotine withdrawal but not acute nicotine-induced or physical withdrawal behaviors.
ESTHER : Jackson_2009_J.Pharmacol.Exp.Ther_331_547
PubMedSearch : Jackson_2009_J.Pharmacol.Exp.Ther_331_547
PubMedID: 19644040

Title : Poster: GZ556A and ZZ204G are novel small molecule antagonists of alpha9alpha10 nAChRs and are analgesic in rats -
Author(s) : McIntosh JM , Dwoskin LP , Crooks PA , Holtman Jr JR
Ref : Biochemical Pharmacology , 78 :921 , 2009

Title : A novel fluorescent alpha-conotoxin for the study of alpha7 nicotinic acetylcholine receptors - Hone_2009_J.Neurochem_111_80
Author(s) : Hone AJ , Whiteaker P , Christensen S , Xiao Y , Meyer EL , McIntosh JM
Ref : Journal of Neurochemistry , 111 :80 , 2009
Abstract : Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.
ESTHER : Hone_2009_J.Neurochem_111_80
PubMedSearch : Hone_2009_J.Neurochem_111_80
PubMedID: 19650873

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 : Alpha9 nicotinic acetylcholine receptors and the treatment of pain - McIntosh_2009_Biochem.Pharmacol_78(7)_693
Author(s) : McIntosh JM , Absalom N , Chebib M , Elgoyhen AB , Vincler M
Ref : Biochemical Pharmacology , 78 :693 , 2009
Abstract : Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).
ESTHER : McIntosh_2009_Biochem.Pharmacol_78(7)_693
PubMedSearch : McIntosh_2009_Biochem.Pharmacol_78(7)_693
PubMedID: 19477168

Title : UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors - Rezvani_2009_J.Neurosci_29_6883
Author(s) : Rezvani K , Teng Y , Pan Y , Dani JA , Lindstrom JM , Garcia Gras EA , McIntosh JM , De Biasi M
Ref : Journal of Neuroscience , 29 :6883 , 2009
Abstract : Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.
ESTHER : Rezvani_2009_J.Neurosci_29_6883
PubMedSearch : Rezvani_2009_J.Neurosci_29_6883
PubMedID: 19474315

Title : Nicotine is neuroprotective when administered before but not after nigrostriatal damage in rats and monkeys - Huang_2009_J.Neurochem_109_826
Author(s) : Huang LZ , Parameswaran N , Bordia T , McIntosh JM , Quik M
Ref : Journal of Neurochemistry , 109 :826 , 2009
Abstract : Nicotine reduces dopaminergic deficits in parkinsonian animals when administered before nigrostriatal damage. Here we tested whether nicotine is also beneficial when given to rats and monkeys with pre-existing nigrostriatal damage. Rats were administered nicotine before and after a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle, and the results compared with those in which rats received nicotine only after lesioning. Nicotine pre-treatment attenuated behavioral deficits and lessened lesion-induced losses of the striatal dopamine transporter, and alpha6beta2* and alpha4beta2* nicotinic receptors (nAChRs). By contrast, nicotine administered 2 weeks after lesioning, when 6-hydroxydopamine-induced neurodegenerative effects are essentially complete, did not improve these same measures. Similar results were observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. Nicotine did not enhance striatal markers when administered to monkeys with pre-existing nigrostriatal damage, in contrast to previous data that showed improvements when nicotine was given to monkeys before lesioning. These combined findings in two animal models suggest that nicotine is neuroprotective rather than neurorestorative against nigrostriatal damage. Receptor studies with (125)I-alpha-conotoxinMII and the alpha-conotoxinMII analog E11A were next performed to determine whether nicotine treatment pre- or post-lesioning differentially affected expression of alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR subtypes in striatum. The observations suggest that protection against nigrostriatal damage may be linked to striatal alpha6alpha4beta2* nAChRs.
ESTHER : Huang_2009_J.Neurochem_109_826
PubMedSearch : Huang_2009_J.Neurochem_109_826
PubMedID: 19250334

Title : Poster: Mutation of proline enables subtype selectivity of alpha-conotoxin BuIA -
Author(s) : Filchakova Y , Godoy AB , Christensen S , McIntosh JM
Ref : Biochemical Pharmacology , 78 :906 , 2009

Title : Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras - Papke_2008_Neuropharmacol_54_1189
Author(s) : Papke RL , Dwoskin LP , Crooks PA , Zheng G , Zhang Z , McIntosh JM , Stokes C
Ref : Neuropharmacology , 54 :1189 , 2008
Abstract : Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.
ESTHER : Papke_2008_Neuropharmacol_54_1189
PubMedSearch : Papke_2008_Neuropharmacol_54_1189
PubMedID: 18448138

Title : Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration - Pons_2008_J.Neurosci_28_12318
Author(s) : Pons S , Fattore L , Cossu G , Tolu S , Porcu E , McIntosh JM , Changeux JP , Maskos U , Fratta W
Ref : Journal of Neuroscience , 28 :12318 , 2008
Abstract : The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the beta2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different alpha subunit partners of beta2 (i.e., alpha4 and alpha6), the homo-pentameric alpha7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7-52.6 microg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the beta2, alpha4, alpha6 and alpha7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, beta2-VEC mice with the beta2 subunit re-expressed exclusively in the VTA, alpha4-VEC mice with selective alpha4 re-expression in the VTA, alpha6-VEC mice with selective alpha6 re-expression in the VTA, and alpha7-KO mice promptly self-administer nicotine intravenously, whereas beta2-KO, beta2-VEC in the substantia nigra, alpha4-KO and alpha6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of alpha4beta2- and alpha6beta2-subunit containing nicotinic receptors (alpha4beta2*- and alpha6beta2*-nAChRs), but not alpha7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.
ESTHER : Pons_2008_J.Neurosci_28_12318
PubMedSearch : Pons_2008_J.Neurosci_28_12318
PubMedID: 19020025

Title : N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity - Dwoskin_2008_J.Pharmacol.Exp.Ther_326_563
Author(s) : Dwoskin LP , Wooters TE , Sumithran SP , Siripurapu KB , Joyce BM , Lockman PR , Manda VK , Ayers JT , Zhang Z , Deaciuc AG , McIntosh JM , Crooks PA , Bardo MT
Ref : Journal of Pharmacology & Experimental Therapeutics , 326 :563 , 2008
Abstract : The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.
ESTHER : Dwoskin_2008_J.Pharmacol.Exp.Ther_326_563
PubMedSearch : Dwoskin_2008_J.Pharmacol.Exp.Ther_326_563
PubMedID: 18460644

Title : Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens - Exley_2008_Neuropsychopharmacology_33_2158
Author(s) : Exley R , Clements MA , Hartung H , McIntosh JM , Cragg SJ
Ref : Neuropsychopharmacology , 33 :2158 , 2008
Abstract : Modulation of striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Nicotine, by desensitizing beta2 subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) on striatal DA axons, significantly enhances how DA is released by reward-related burst activity compared to nonreward-related tonic activity. This action provides a synaptic mechanism for nicotine to facilitate the DA-dependent reinforcement. The subfamily of beta2*-nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of alpha6beta2*-nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real-time using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in mouse striatal slices. The alpha6-specific antagonist alpha-conotoxin-MII suppressed DA release evoked by single and low-frequency action potentials and concurrently enhanced release by high-frequency bursts in a manner similar to the beta2*-selective antagonist dihydro-beta-erythroidine (DHbetaE) in NAc, but less so in CPu. The greater role for alpha6*-nAChRs in NAc was not due to any confounding regional difference in ACh tone since elevated ACh levels (after the acetylcholinesterase inhibitor ambenonium) had similar outcomes in NAc and CPu. Rather, there appear to be underlying differences in nAChR subtype function in NAc and CPu. In summary, we reveal that alpha6beta2*-nAChRs dominate the effects of nicotine on DA release in NAc, whereas in CPu their role is minor alongside other beta2*-nAChRs (eg alpha4*), These data offer new insights to suggest striatal alpha6*-nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.
ESTHER : Exley_2008_Neuropsychopharmacology_33_2158
PubMedSearch : Exley_2008_Neuropsychopharmacology_33_2158
PubMedID: 18033235

Title : Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors - Innocent_2008_J.Pharmacol.Exp.Ther_327_529
Author(s) : Innocent N , Livingstone PD , Hone A , Kimura A , Young T , Whiteaker P , McIntosh JM , Wonnacott S
Ref : Journal of Pharmacology & Experimental Therapeutics , 327 :529 , 2008
Abstract : A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.
ESTHER : Innocent_2008_J.Pharmacol.Exp.Ther_327_529
PubMedSearch : Innocent_2008_J.Pharmacol.Exp.Ther_327_529
PubMedID: 18664588

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 : Chronic nicotine differentially regulates alpha6- and beta3-containing nicotinic cholinergic receptors in rat brain - Perry_2007_J.Pharmacol.Exp.Ther_322_306
Author(s) : Perry DC , Mao D , Gold AB , McIntosh JM , Pezzullo JC , Kellar KJ
Ref : Journal of Pharmacology & Experimental Therapeutics , 322 :306 , 2007
Abstract : We investigated the effects of chronic nicotine on alpha6- and beta3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [(125)I]alpha-conotoxin MII to selectively label alpha6(*) nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [(3)H]epibatidine in these two regions showed that chronic nicotine increased alpha4- and beta2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in alpha6-containing nAChRs in striatum but no change in superior colliculus. No changes in beta3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of alpha6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to alpha-conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by alpha-conotoxin MII was decreased by 56%. These findings indicate that the alpha6(*) nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a beta3 subunit with alpha6 may serve to inhibit nicotine-induced down-regulation of these receptors.
ESTHER : Perry_2007_J.Pharmacol.Exp.Ther_322_306
PubMedSearch : Perry_2007_J.Pharmacol.Exp.Ther_322_306
PubMedID: 17446303

Title : Nicotine partially protects against paraquat-induced nigrostriatal damage in mice\; link to alpha6beta2* nAChRs - Khwaja_2007_J.Neurochem_100_180
Author(s) : Khwaja M , McCormack A , McIntosh JM , Di Monte DA , Quik M
Ref : Journal of Neurochemistry , 100 :180 , 2007
Abstract : Epidemiological studies indicate that smoking is a negative, and exposure to pesticides, a positive risk factor for Parkinson's disease (PD). The purpose of this study was to assess the interplay between these two factors in a rodent model of nigrostriatal damage. To approach this, mice were administered nicotine, the agent in smoke implicated in neuroprotection. They were then treated for 3 weeks with the pesticide, paraquat, while nicotine was continued. Paraquat treatment decreased (25%) nigral dopaminergic neurons, consistent with previous results. Chronic nicotine administration significantly protected against nigral cell damage, with only a 16% decline in mice treated with both nicotine and paraquat. Paraquat treatment also decreased (14%) the striatal dopamine transporter, an effect that was partially prevented by nicotine. These changes in the striatal dopamine transporter paralleled those in a select striatal alpha6beta2* nicotinic receptor (nAChR) subtype. In contrast, striatal alpha4beta2* nAChRs were not decreased with paraquat treatment, suggesting they are on a differential subset of dopaminergic terminals. The results show that nicotine treatment partially protects against paraquat-induced declines in nigrostriatal dopaminergic neurons to which a select population of alpha6beta2* nAChRs are localized. Moreover, these data support epidemiological findings that environmental influences can elicit opposing effects on nigrostriatal dopaminergic integrity.
ESTHER : Khwaja_2007_J.Neurochem_100_180
PubMedSearch : Khwaja_2007_J.Neurochem_100_180
PubMedID: 17227438

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 : Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation - Scholze_2007_Br.J.Pharmacol_151_414
Author(s) : Scholze P , Orr-Urtreger A , Changeux JP , McIntosh JM , Huck S
Ref : British Journal of Pharmacology , 151 :414 , 2007
Abstract : BACKGROUND AND PURPOSE: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. EXPERIMENTAL APPROACH: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. KEY
RESULTS: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. CONCLUSIONS AND IMPLICATIONS: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.
ESTHER : Scholze_2007_Br.J.Pharmacol_151_414
PubMedSearch : Scholze_2007_Br.J.Pharmacol_151_414
PubMedID: 17401441

Title : Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR - Ellison_2006_Biochemistry_45_1511
Author(s) : Ellison M , Haberlandt C , Gomez-Casati ME , Watkins M , Elgoyhen AB , McIntosh JM , Olivera BM
Ref : Biochemistry , 45 :1511 , 2006
Abstract : The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.
ESTHER : Ellison_2006_Biochemistry_45_1511
PubMedSearch : Ellison_2006_Biochemistry_45_1511
PubMedID: 16445293

Title : Increases in alpha4* but not alpha3*\/alpha6* nicotinic receptor sites and function in the primate striatum following chronic oral nicotine treatment - McCallum_2006_J.Neurochem_96_1028
Author(s) : McCallum SE , Parameswaran N , Bordia T , Fan H , Tyndale RF , Langston JW , McIntosh JM , Quik M
Ref : Journal of Neurochemistry , 96 :1028 , 2006
Abstract : Knowledge of the effects of chronic nicotine is critical considering its widespread use in tobacco products and smoking cessation therapies. Although nicotine is well known to up-regulate alpha4* nAChR sites and function in the cortex, its actions in the striatum are uncertain because of the presence of multiple subtypes with potentially opposing effects. We therefore investigated the effect of long-term nicotine treatment on nAChR sites and function in the primate striatum, which offers the advantage of similar proportions of alpha3*/alpha6* and alpha4* nAChRs. Nicotine was given in drinking water, which resembles smoking in its intermittent but chronic delivery. Plasma nicotine and cotinine levels were similar to smokers. Chronic nicotine treatment (> 6 months) enhanced alpha4* nAChR-evoked [(3)H]dopamine release in striatal subregions, with an overall pattern of increase throughout the striatum when normalized to uptake. This increase correlated with elevated striatal alpha4* nAChRs. Under the same conditions, striatal alpha3*/alpha6* nAChR sites and function were decreased or unchanged. These divergent actions of chronic nicotine treatment on alpha4* versus alpha6* nAChRs, as well as effects on dopamine uptake, allow for a complex control of striatal activity to maintain dopaminergic function. Such knowledge is important for understanding nicotine dependence and the consequences of nicotine administration for the treatment of neurological disorders.
ESTHER : McCallum_2006_J.Neurochem_96_1028
PubMedSearch : McCallum_2006_J.Neurochem_96_1028
PubMedID: 16412091

Title : Expression of nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by beta 3 subunit gene deletion - Gotti_2005_Mol.Pharmacol_67_2007
Author(s) : Gotti C , Moretti M , Clementi F , Riganti L , McIntosh JM , Collins AC , Marks MJ , Whiteaker P
Ref : Molecular Pharmacology , 67 :2007 , 2005
Abstract : mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) alpha6 and beta3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that alpha6- or beta3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of beta3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that beta3-null mutation selectively reduced striatal alpha6* nAChR expression by 76% versus beta3(+/+) control. Parallel experiments showed a smaller reduction in both midbrain alpha3* and alpha6* nAChRs (34 and 42% versus beta3(+/+) control, respectively). Sedimentation coefficient determinations indicated that residual alpha6* nAChRs in beta3(-/-) striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified beta3* nAChRs demonstrated that almost all wild-type striatal beta3* nAChRs also contain alpha4, alpha6, and beta2 subunits, although a small population of non-beta3 alpha6* nAChRs is also expressed. beta3 subunit incorporation seemed to increase alpha4 participation in alpha6beta2* complexes. (125)I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of alpha6* nAChRs from the striata of beta3(-/-) mice was similar to those isolated from beta3(+/+) animals. Together, the results of these experiments show that the beta3 subunit is important for the correct assembly, stability and/or transport of alpha6* nAChRs in dopaminergic neurons and influences their subunit composition. However, beta3 subunit expression is not essential for the expression of alpha6*, high-affinity alpha-CtxMII binding nAChRs.
ESTHER : Gotti_2005_Mol.Pharmacol_67_2007
PubMedSearch : Gotti_2005_Mol.Pharmacol_67_2007
PubMedID: 15749993

Title : Functional properties of neuronal nicotinic acetylcholine receptors in the chick retina during development - Lecchi_2005_Eur.J.Neurosci_21_3182
Author(s) : Lecchi M , McIntosh JM , Bertrand S , Safran AB , Bertrand D
Ref : European Journal of Neuroscience , 21 :3182 , 2005
Abstract : Acetylcholine (ACh) has been recognized for a long time as a major neurotransmitter in the retina, however, little is known about the contribution of acetylcholine receptors in synaptic processing. Moreover, even less information is available concerning their role during development. To address this question further, we examined the physiological and pharmacological properties of neuronal nicotinic acetylcholine receptors (nAChRs) in retinal ganglion cells from embryonic (E) 12-18-day-old Leghorn chicks. Patch-clamp recordings in whole-cell configuration revealed that at E12 approximately 21% of the ganglion cells responded to acetylcholine pulses with inward currents. The number of responsive cells progressively increased to 57% at E15 to reach up to 15 positive cells out of 15 cells tested at E18. Acetylcholine-evoked responses could be subdivided, according to their time course, into fast and slowly desensitizing. Taking advantage of the selectivity of the frog toxin epibatidine (Epi), that preferentially activates heteromeric neuronal nicotinic acetylcholine receptors, we compared the currents evoked by this toxin vs. the effects of acetylcholine. A further characterization of the receptor diversity during development was to assess their sensitivity to the alpha-conotoxin MII (alpha-CTX-MII), which has been shown to preferentially block alpha6- and alpha3beta2-containing receptors. These data demonstrate that ganglion cells of the chick retina express multiple receptor subtypes that progressively develop as a function of retina maturation.
ESTHER : Lecchi_2005_Eur.J.Neurosci_21_3182
PubMedSearch : Lecchi_2005_Eur.J.Neurosci_21_3182
PubMedID: 15978026

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 : Nicotinic cholinergic receptors in the rat retina: simple and mixed heteromeric subtypes - Marritt_2005_Mol.Pharmacol_68_1656
Author(s) : Marritt AM , Cox BC , Yasuda RP , McIntosh JM , Xiao Y , Wolfe BB , Kellar KJ
Ref : Molecular Pharmacology , 68 :1656 , 2005
Abstract : Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, alpha2-alpha4, alpha6, and beta2-beta4, with RNase protection assays. The density of heteromeric nAChR binding sites is approximately 3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is approximately 30% higher than with 125I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [3H]cytisine or [3H](-)nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the beta2 subunit and a smaller fraction containing the beta4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the beta2 and beta4 subunits indicated that approximately 80% of the receptors contained beta2 subunits and approximately 25% contained beta4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of alpha and a single type of beta subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more alpha and/or beta subunits.
ESTHER : Marritt_2005_Mol.Pharmacol_68_1656
PubMedSearch : Marritt_2005_Mol.Pharmacol_68_1656
PubMedID: 16129735

Title : Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA - Quik_2005_Mol.Pharmacol_67_32
Author(s) : Quik M , Vailati S , Bordia T , Kulak JM , Fan H , McIntosh JM , Clementi F , Gotti C
Ref : Molecular Pharmacology , 67 :32 , 2005
Abstract : Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-alpha-bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.
ESTHER : Quik_2005_Mol.Pharmacol_67_32
PubMedSearch : Quik_2005_Mol.Pharmacol_67_32
PubMedID: 15470079

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 : A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors - McIntosh_2005_J.Biol.Chem_280_30107
Author(s) : McIntosh JM , Plazas PV , Watkins M , Gomez-Casati ME , Olivera BM , Elgoyhen AB
Ref : Journal of Biological Chemistry , 280 :30107 , 2005
Abstract : The alpha9 and alpha10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of alpha9 and alpha10 overlaps with that of alpha7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin alpha-conotoxin PeIA that discriminates between alpha9alpha10 and alpha7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. Alpha-conotoxin PeIA displayed a 260-fold higher selectivity for alpha-bungarotoxin-sensitive alpha9alpha10 nAChRs compared with alpha-bungarotoxin-sensitive alpha7 receptors. The IC50 of the toxin was 6.9 +/- 0.5 nM and 4.4 +/- 0.5 nM for recombinant alpha9alpha10 and wild-type hair cell nAChRs, respectively. Alpha-conotoxin PeIA bears high resemblance to alpha-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither alpha-conotoxin MII nor alpha-conotoxin GIC at concentrations of 10 microM blocked acetylcholine responses elicited in Xenopus oocytes injected with the alpha9 and alpha10 subunits. Among neuronal non-alpha-bungarotoxin-sensitive receptors, alpha-conotoxin PeIA was also active at alpha3beta2 receptors and chimeric alpha6/alpha3beta2beta3 receptors. Alpha-conotoxin PeIA represents a novel probe to differentiate responses mediated either through alpha9alpha10 or alpha7 nAChRs in those tissues where both receptors are expressed.
ESTHER : McIntosh_2005_J.Biol.Chem_280_30107
PubMedSearch : McIntosh_2005_J.Biol.Chem_280_30107
PubMedID: 15983035

Title : Cholinergic nicotinic receptor involvement in movement disorders associated with Lewy body diseases. An autoradiography study using [(125)I]alpha-conotoxinMII in the striatum and thalamus - Bohr_2005_Exp.Neurol_191_292
Author(s) : Bohr IJ , Ray MA , McIntosh JM , Chalon S , Guilloteau D , McKeith IG , Perry RH , Clementi F , Perry EK , Court JA , Piggott MA
Ref : Experimental Neurology , 191 :292 , 2005
Abstract : The presence of alpha6 subunit containing nicotinic acetylcholine receptors on nigrostriatal dopaminergic neurons has been demonstrated in rodents and monkeys. [(125)I]alpha-conotoxinMII is a radioligand that binds to alpha6, and also alpha3 subunits of nicotinic acetylcholine receptors (nAChRs). In the present study, we have compared the distribution of [(125)I]alpha-conotoxinMII binding in post mortem human tissue from four groups of patients: individuals with dementia with Lewy bodies displaying extra-pyramidal features (DLB + EPF), DLB without extra-pyramidal features (DLB - EPF) Parkinson's disease without dementia (PD) and age-matched controls. Reduced binding was observed in the putamen and caudate in PD and both DLB groups. In DLB patients, the decline was greater in DLB + EPF compared to DLB - EPF group. The declines in nicotinic receptor binding in the striatum were in part paralleled by reductions in the striatal dopamine transporter. In the thalamus, [(125)I]alpha-conotoxinMII binding was significantly reduced in the centromedian nucleus in both DLB groups, and also in the parafascicular nucleus in the DLB - EPF group. In DLB + EPF and PD patients, there was decreased binding in the ventral lateral nucleus. This study demonstrates alterations of alpha6 and/or alpha3 nAChRs binding in DLB and PD, which are likely to relate to extra-pyramidal symptoms.
ESTHER : Bohr_2005_Exp.Neurol_191_292
PubMedSearch : Bohr_2005_Exp.Neurol_191_292
PubMedID: 15649484

Title : Selective recovery of striatal 125I-alpha-conotoxinmii nicotinic receptors after nigrostriatal damage in monkeys - Lai_2004_Neurosci_127_399
Author(s) : Lai A , Sum J , Fan H , McIntosh JM , Quik M
Ref : Neuroscience , 127 :399 , 2004
Abstract : Evidence suggests that nicotinic receptors play a role in nigrostriatal function, a finding that may be relevant to Parkinson's disease. Knowledge of the conditions that regulate nicotinic receptor expression is therefore important. Previous studies showed that several different nicotinic receptors, including alpha-conotoxinMII (alpha-CtxMII)-sensitive receptors, are decreased after nigrostriatal damage. Nigrostriatal dopaminergic terminals also demonstrate a capacity for recovery after lesioning. The present experiments were therefore done to determine whether there were changes in striatal nicotinic receptors with recovery. To address this, we used two well-characterized animal models of nigrostriatal damage produced using the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Studies in mice showed that striatal 125I-alpha-CtxMII, as well as 125I-epibatidine and 125I-A85380 binding sites significantly recovered 1 month after lesioning, suggesting that alpha6* and most likely alpha4* receptors are increased. Experiments were next done in monkeys since striatal 125I-alpha-CtxMII receptors constitute a large percentage of nicotinic receptors and are more vulnerable to nigrostriatal damage in this model that closely mirrors Parkinson's disease. In monkeys allowed to recover from the toxic effects of MPTP for a 1-2 year period, there was a significant improvement in the Parkinson disability score. There was also a reversal in lesion-induced declines in striatal alpha-CtxMII-sensitive receptors, but no significant change in 125I-epibatidine and 125I-A85380 receptors. These findings suggest that alpha3*/alpha6* sites are selectively increased in monkey striatum with recovery. The present data show that recovery of 125I-alpha-CtxMII receptors occurs in parallel with the dopamine transporter, indicating that these nicotinic receptors sites are localized to presynaptic dopamine terminals in both species.
ESTHER : Lai_2004_Neurosci_127_399
PubMedSearch : Lai_2004_Neurosci_127_399
PubMedID: 15262330

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 : Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex - Amtage_2004_Brain.Res.Bull_62_413
Author(s) : Amtage F , Neughebauer B , McIntosh JM , Freiman T , Zentner J , Feuerstein TJ , Jackisch R
Ref : Brain Research Bulletin , 62 :413 , 2004
Abstract : Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [3H]noradrenaline ([3H]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [3H]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive. Ca(2+)-dependent release of [3H]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine >> DMPP > nicotine approximately cytisine > or = acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 microM) were reduced by mecamylamine, methyllycaconitine, di-hydro-beta-erythroidine (10 microM, each) and the alpha3beta2/alpha6betax-selective alpha-conotoxin MII (100/200 nM). The alpha3beta4 selective alpha-conotoxin AuIB (1 microM), and the alpha7 selective alpha-conotoxin ImI (200 nM) as well as alpha-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 microM AP-5, 100 microM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [3H]acetylcholine from human and rat neocortical slices preincubated with [3H]choline. IN CONCLUSION: (1) alpha3beta2 and/or alpha6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.
ESTHER : Amtage_2004_Brain.Res.Bull_62_413
PubMedSearch : Amtage_2004_Brain.Res.Bull_62_413
PubMedID: 15168907

Title : Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors - McIntosh_2004_Mol.Pharmacol_65_944
Author(s) : McIntosh JM , Azam L , Staheli S , Dowell C , Lindstrom JM , Kuryatov A , Garrett JE , Marks MJ , Whiteaker P
Ref : Molecular Pharmacology , 65 :944 , 2004
Abstract : Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. Alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6(*) (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3(*) versus alpha6(*) nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]alpha-conotoxin MII binding to putative alpha6beta2(*) nAChRs in mouse brain homogenates (K(i) = 3.3 nM). Thus, structure-function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.
ESTHER : McIntosh_2004_Mol.Pharmacol_65_944
PubMedSearch : McIntosh_2004_Mol.Pharmacol_65_944
PubMedID: 15044624

Title : Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes - Ellison_2004_Biochemistry_43_16019
Author(s) : Ellison M , Gao F , Wang HL , Sine SM , McIntosh JM , Olivera BM
Ref : Biochemistry , 43 :16019 , 2004
Abstract : The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.
ESTHER : Ellison_2004_Biochemistry_43_16019
PubMedSearch : Ellison_2004_Biochemistry_43_16019
PubMedID: 15609996

Title : Up-regulation of brain nicotinic acetylcholine receptors in the rat during long-term self-administration of nicotine: disproportionate increase of the alpha6 subunit - Parker_2004_Mol.Pharmacol_65_611
Author(s) : Parker SL , Fu Y , McAllen K , Luo J , McIntosh JM , Lindstrom JM , Sharp BM
Ref : Molecular Pharmacology , 65 :611 , 2004
Abstract : In male rats continually self-administering nicotine (approximately 1.5 mg free base/kg/day), we found a significant increase of nicotinic acetylcholine receptors (nAChRs) labeled by epibatidine (Epb) in 11 brain areas. A large increase of high-affinity Epb binding sites was apparent in the ventral tegmentum/substantia nigra, nucleus tractus solitarii, nucleus accumbens, thalamus/subthalamus, parietal cortex, hypothalamus, and amygdala. A smaller but significant up-regulation of high-affinity Epb sites was seen in the piriform cortex, hippocampus, caudate/putamen, and cerebellar cortex. The up-regulation of nAChRs, shown by immunoadsorption and Western blotting, involved alpha4, alpha6, and beta2 subunits. As a consequence of long-term self-administration of nicotine, the alpha6 immunoreactive (IR) binding of either labeled Epb or 125I-alpha-conotoxin MII increased to a much greater extent than did alpha4 or beta2 IR binding of Epb. In addition, the beta2 IR binding of Epb was consistently enhanced to a greater extent than was alpha4. These findings may reflect a larger surface membrane retention of alpha6-containing and, to some degree, beta2-containing nAChRs compared with alpha4-containing nAChRs during long-term self-administration of nicotine.
ESTHER : Parker_2004_Mol.Pharmacol_65_611
PubMedSearch : Parker_2004_Mol.Pharmacol_65_611
PubMedID: 14978239

Title : Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors - Dowell_2003_J.Neurosci_23_8445
Author(s) : Dowell C , Olivera BM , Garrett JE , Staheli ST , Watkins M , Kuryatov A , Yoshikami D , Lindstrom JM , McIntosh JM
Ref : Journal of Neuroscience , 23 :8445 , 2003
Abstract : Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.
ESTHER : Dowell_2003_J.Neurosci_23_8445
PubMedSearch : Dowell_2003_J.Neurosci_23_8445
PubMedID: 13679412

Title : Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice - Champtiaux_2003_J.Neurosci_23_7820
Author(s) : Champtiaux N , Gotti C , Cordero-Erausquin M , David DJ , Przybylski C , Lena C , Clementi F , Moretti M , Rossi FM , Le Novere N , McIntosh JM , Gardier AM , Changeux JP
Ref : Journal of Neuroscience , 23 :7820 , 2003
Abstract : Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (alpha4beta2*, alpha6beta2*, and alpha4alpha6beta2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromalpha4, alpha6, alpha4alpha6, and beta2 knock-out mice. Our results establish that alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonalpha6)alpha4beta2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of alpha6beta2* and alpha4beta2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonalpha6)alpha4beta2* nAChRs most likely contribute to nicotine reinforcement.
ESTHER : Champtiaux_2003_J.Neurosci_23_7820
PubMedSearch : Champtiaux_2003_J.Neurosci_23_7820
PubMedID: 12944511

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 : Methyllycaconitine is a potent antagonist of alpha-conotoxin-MII-sensitive presynaptic nicotinic acetylcholine receptors in rat striatum - Mogg_2002_J.Pharmacol.Exp.Ther_302_197
Author(s) : Mogg AJ , Whiteaker P , McIntosh JM , Marks M , Collins AC , Wonnacott S
Ref : Journal of Pharmacology & Experimental Therapeutics , 302 :197 , 2002
Abstract : The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the alpha7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [(3)H]dopamine release from rat striatal synaptosomes stimulated with 10 microM nicotine. Other alpha7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [(3)H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 microM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to alpha-conotoxin-MII (alpha-CTx-MII). MLA was not additive with a maximally effective concentration of alpha-CTx-MII (100 nM) in inhibiting [(3)H]dopamine release elicited by 10 microM nicotine or 0.2 microM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with (125)I-alpha-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B(max) values of 9.8 and 16.5 fmol/mg of protein, and K(d) values of 0.63 and 0.83 nM, respectively. MLA fully inhibited (125)I-alpha-CTx-MII binding to striatum and nucleus accumbens with a K(i) value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and alpha-CTx-MII interact with a presynaptic nAChR of subunit composition alpha3/alpha6beta2beta3* on dopamine neurons. The use of MLA as an alpha7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.
ESTHER : Mogg_2002_J.Pharmacol.Exp.Ther_302_197
PubMedSearch : Mogg_2002_J.Pharmacol.Exp.Ther_302_197
PubMedID: 12065717

Title : Loss of nicotinic receptors in monkey striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment is due to a decline in alpha-conotoxin MII sites - Kulak_2002_Mol.Pharmacol_61_230
Author(s) : Kulak JM , McIntosh JM , Quik M
Ref : Molecular Pharmacology , 61 :230 , 2002
Abstract : Nicotinic acetylcholine receptors (nAChRs) in the basal ganglia are a potential target for new therapeutics for Parkinson's disease. As an approach to detect expression of nAChRs in monkeys, we used 125I-epibatidine, an agonist at nAChRs containing alpha2 to alpha6 subunits. 125I-Epibatidine binding sites are expressed throughout the control monkey brain, including the basal ganglia. The alpha3/alpha6-selective antagonist alpha-conotoxin MII maximally inhibited 50% of binding in the caudate-putamen and had no effect on 125I-epibatidine binding in the frontal cortex or thalamus. In contrast, inhibition experiments with nicotine, cytisine, and 3-(2(S)-azetidinylmethoxy)pyridine-2HCl (A85380) showed a complete block of 125I-epibatidine binding in all regions investigated and did not discriminate between the alpha-conotoxin MII-sensitive and -insensitive populations in the striatum. To assess the effects of nigrostriatal damage, monkeys were rendered parkinsonian with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animals with moderate striatal damage (dopamine transporter levels approximately 30% of control) had a 40 to 50% decrease in 125I-epibatidine binding. Inhibition studies showed that the decrease in epibatidine binding was due to loss of alpha-conotoxin MII-sensitive nAChRs. Monkeys with severe nigrostriatal damage (dopamine transporter levels < or = 5% of control) exhibited a 55 to 60% decrease in 125I-epibatidine binding, which seemed to be due to a complete loss of alpha-conotoxin MII nAChRs and a partial loss of other nAChR subtypes. These results show that nAChRs expressed in the primate striatum have similar affinities for nicotine, cytisine, and A85380, that alpha-conotoxin MII discriminates between nAChR populations in the caudate and putamen, and that alpha-conotoxin MII-sensitive nAChRs are selectively decreased after MPTP-induced nigrostriatal damage.
ESTHER : Kulak_2002_Mol.Pharmacol_61_230
PubMedSearch : Kulak_2002_Mol.Pharmacol_61_230
PubMedID: 11752225

Title : Differential nicotinic receptor expression in monkey basal ganglia: effects of nigrostriatal damage - Quik_2002_Neurosci_112_619
Author(s) : Quik M , Polonskaya Y , McIntosh JM , Kulak JM
Ref : Neuroscience , 112 :619 , 2002
Abstract : Our previous work showed that there were marked declines in (125)I-alpha-conotoxin MII labeled nicotinic receptors in monkey basal ganglia after nigrostriatal damage, findings that suggest alpha3/alpha6 containing nicotinic receptors sites may be of relevance to Parkinson's disease. We now investigate whether there are differential changes in the distribution pattern of nicotinic receptor subtypes in the basal ganglia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned animals compared to controls to better understand the changes occurring with nigrostriatal damage. To approach this we used (125)I-alpha-conotoxin MII, a marker for alpha3/alpha6 nicotinic receptors, and (125)I-epibatidine, a ligand that labels multiple nicotinic subtypes. The results demonstrate that there were medial to lateral gradients in nicotinic receptor distribution in control striatum, as well as ventromedial to dorsolateral gradients in the substantia nigra, which resembled those of the dopamine transporter in these same brain regions. Treatment with MPTP, a neurotoxin that selectively destroys dopaminergic nigrostriatal neurons, led to a relatively uniform decrease in nicotinic receptor sites in the striatum, but a differential effect in the substantia nigra with significantly greater declines in the ventrolateral portion. Competition analysis in the striatum showed that alpha-conotoxin MII sensitive sites were primarily affected after lesioning, whereas multiple nicotinic receptor populations were decreased in the substantia nigra. From these data we suggest that in the striatum alpha3/alpha6 nicotinic receptors are primarily localized on dopaminergic nerve terminals, while multiple nicotinic receptor subtypes are present on dopaminergic cell bodies in the substantia nigra. Thus, if activation of striatal nicotinic receptors is key in the regulation of basal ganglia function, alpha3/alpha6-directed nicotinic receptor ligands may be more relevant for Parkinson's disease therapy. However, nicotinic receptor ligands with a broader specificity may be more important if receptors in the substantia nigra play a dominant role in controlling nigrostriatal activity.
ESTHER : Quik_2002_Neurosci_112_619
PubMedSearch : Quik_2002_Neurosci_112_619
PubMedID: 12074903

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 : Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum - Zoli_2002_J.Neurosci_22_8785
Author(s) : Zoli M , Moretti M , Zanardi A , McIntosh JM , Clementi F , Gotti C
Ref : Journal of Neuroscience , 22 :8785 , 2002
Abstract : Neuronal nicotinic acetylcholine receptors (nAChRs) expressed on mesostriatal dopaminergic neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Using immunoprecipitation and ligand-binding techniques, we have shown that both alpha6beta2* and alpha4(nonalpha6)beta2* nAChRs are expressed in the caudate-putamen and that only alpha6* nAChRs can bind alpha-conotoxin MII and methyllycaconitine with affinities of 1.3 and 40 nm, respectively. Further studies performed on 6-hydroxydopamine-lesioned striatum led to the identification of nAChR subtypes selectively expressed on dopaminergic terminals [alpha4alpha5beta2, alpha4alpha6beta2(beta3), and alpha6beta2(beta3)], nondopaminergic neuronal structures (alpha2alpha4beta2), or both structures (alpha4beta2). The identification of the nAChRs expressed on striatal dopaminergic terminals opens up the possibility of developing selective nAChR ligands active on dopaminergic systems and associated diseases, such as Parkinson's disease.
ESTHER : Zoli_2002_J.Neurosci_22_8785
PubMedSearch : Zoli_2002_J.Neurosci_22_8785
PubMedID: 12388584

Title : Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey - Quik_2001_J.Neurosci_21_5494
Author(s) : Quik M , Polonskaya Y , Kulak JM , McIntosh JM
Ref : Journal of Neuroscience , 21 :5494 , 2001
Abstract : Parkinson's disease, a neurodegenerative movement disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons, affects approximately 1% of the population over 50. Because nicotinic acetylcholine receptors (nAChRs) may represent an important therapeutic target for this disorder, we performed experiments to elucidate the subtypes altered with nigrostriatal damage in parkinsonian monkeys. For this purpose we used (125)I-alpha-conotoxin MII (CtxMII), a relatively new ligand that identifies alpha3 and/or alpha6 subunits containing nAChR subtypes. In brain from untreated monkeys, there was saturable (125)I-alpha-CtxMII binding to a single population of high-affinity nicotinic sites (K(d) = 0.9 nm), primarily localized in the visual, habenula-interpeduncular, and nigrostriatal-mesolimbic pathways. Administration of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine resulted in damage to the nigrostriatal system and parkinsonism. Autoradiographic analysis showed that (125)I-alpha-CtxMII sites were selectively reduced (>/=99%) in the basal ganglia and that the lesion-induced decreases correlated well with declines in the dopamine transporter, a marker of dopaminergic neuron integrity. These findings may indicate that most or all of (125)I-alpha-CtxMII-labeled nAChR subtypes in the basal ganglia are present on nigrostriatal dopaminergic neurons, in contrast to (125)I-epibatidine sites. These data suggest that the development of ligands directed to nAChR subtypes containing alpha3 and/or alpha6 subunits may yield a novel treatment strategy for parkinsonian patients with nigrostriatal dopaminergic degeneration.
ESTHER : Quik_2001_J.Neurosci_21_5494
PubMedSearch : Quik_2001_J.Neurosci_21_5494
PubMedID: 11466420

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 : Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals - Nayak_2001_J.Neurochem_76_1860
Author(s) : Nayak SV , Dougherty JJ , McIntosh JM , Nichols RA
Ref : Journal of Neurochemistry , 76 :1860 , 2001
Abstract : Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser-scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nM) induced Ca(2+) changes in a subset (10-25%) of synaptosomes. The Ca(2+) responses were dependent on extracellular Ca(2+) and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine-induced Ca(2+) responses were dose-dependent and were completely blocked by dihydro-beta-erythroidine (5 microM), differentially affected by mecamylamine (10 microM) and alpha-conotoxin MII (100 nM), and not affected by alpha-bungarotoxin (500 nM). Immunocytochemical studies using well-characterized monoclonal antibodies revealed the presence of the alpha4 and alpha3/alpha5 nicotinic subunits. The nicotine-induced responses were unaffected by prior depolarization or by a mixture of Ca(2+) channel toxins including omega-conotoxin MVIIC (500 nM), omega-conotoxin GVIA (500 nM) and agatoxin TK (200 nM). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca(2+) entry largely without involving voltage-gated Ca(2+) channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.
ESTHER : Nayak_2001_J.Neurochem_76_1860
PubMedSearch : Nayak_2001_J.Neurochem_76_1860
PubMedID: 11259504

Title : An alpha4beta4 nicotinic receptor subtype is present in chick retina: identification, characterization and pharmacological comparison with the transfected alpha4beta4 and alpha6beta4 subtypes - Barabino_2001_Mol.Pharmacol_59_1410
Author(s) : Barabino B , Vailati S , Moretti M , McIntosh JM , Longhi R , Clementi F , Gotti C
Ref : Molecular Pharmacology , 59 :1410 , 2001
Abstract : Retina from 1-day-old chicks is a valuable tissue model for studying neuronal nicotinic receptors because it expresses a large number of the developmentally regulated high affinity [(3)H]epibatidine labeled nicotinic receptors. Most of these receptors contain the beta4 subunit associated with different alpha subunits. Using a sequential immunodepletion procedure with anti-alpha6, anti-beta3, anti-beta2, and anti-beta4 antibodies, we purified an alpha4beta4 nicotinic receptor subtype that accounts for approximately 20 to 25% of the high affinity [(3)H]epibatidine labeled receptors present in retina at that developmental time. Immunoprecipitation and Western blotting experiments confirmed that the purified subtype contains only the alpha4 and beta4 subunits. This receptor binds a number of agonists and the antagonist dihydro-beta-erythroidine with nanomolar affinity, whereas it has micromolar affinity for the alpha-conotoxin MII and methyllycaconitine toxins and other nicotinic antagonists. Comparison of the pharmacological profile of this purified native subtype with that of the same subtype transiently expressed in human BOSC23 cells showed that they have very similar rank orders and absolute Ki values for several nicotinic drugs. Finally, because chick retina expresses an alpha6beta4-containing subtype with a high affinity for the alpha-conotoxin MII, we used native and transfected alpha4beta4 and alpha6beta4 subtypes to investigate the relative contributions of the alpha and beta subunits to this binding, and found that the alpha6 subunit determines the high affinity for this toxin.
ESTHER : Barabino_2001_Mol.Pharmacol_59_1410
PubMedSearch : Barabino_2001_Mol.Pharmacol_59_1410
PubMedID: 11353800

Title : Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha-conotoxin PnIB - Quiram_2000_J.Biol.Chem_275_4889
Author(s) : Quiram PA , McIntosh JM , Sine SM
Ref : Journal of Biological Chemistry , 275 :4889 , 2000
Abstract : This work uses alpha-conotoxin PnIB to probe the agonist binding site of neuronal alpha(7) acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of alpha(7) that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of alpha(7). The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the alpha(7) binding site.
ESTHER : Quiram_2000_J.Biol.Chem_275_4889
PubMedSearch : Quiram_2000_J.Biol.Chem_275_4889
PubMedID: 10671525

Title : Toxin Antagonists of the Neuronal Nicotinic Acetylcholine Receptor -
Author(s) : McIntosh JM
Ref : Handbook of Experimental Pharmacology , 144 :455 , 2000

Title : Conus peptides: novel probes for nicotinic acetylcholine receptor structure and function - McIntosh_2000_Eur.J.Pharmacol_393_205
Author(s) : McIntosh JM , Gardner S , Luo S , Garrett JE , Yoshikami D
Ref : European Journal of Pharmacology , 393 :205 , 2000
Abstract : Conus is a genus of predatory marine snails that uses venom to capture prey. Among the neurotoxins widely utilized by the cone snails are the alpha-conotoxins which are disulfide-rich peptides that target muscle or neuronal subtypes of nicotinic acetylcholine receptors. The small size and receptor subtype specificity of these peptides make them particularly useful for characterizing both native and heterologously expressed nicotinic receptors. In this report, we demonstrate that alpha-conotoxin MII potently blocks beta3-containing neuronal nicotinic receptors. Furthermore, initial evidence suggests that subpopulations of alpha3beta2beta3-containing receptors are differentially sensitive to alpha-conotoxin MII. Thus, alpha-conotoxin MII promises to be a useful tool for studying neuronal nicotinic receptors containing the beta3 subunit.
ESTHER : McIntosh_2000_Eur.J.Pharmacol_393_205
PubMedSearch : McIntosh_2000_Eur.J.Pharmacol_393_205
PubMedID: 10771014

Title : Neuronal-type acetylcholine receptors and regulation of alpha 7 gene expression in vertebrate skeletal muscle - Romano_1997_J.Neurobiol_32_69
Author(s) : Romano SJ , Pugh PC , McIntosh JM , Berg DK
Ref : Journal of Neurobiology , 32 :69 , 1997
Abstract : Several neuronal nicotinic acetylcholine receptor (AChR) genes are expressed in chick skeletal muscle during development. One of the most abundantly expressed is alpha 7, which produces a protein capable of binding alpha-bungarotoxin and is physically distinct from muscle AChRs containing the alpha 1 gene product. We show here that the alpha 7-containing species in muscle is indistinguishable pharmacologically from alpha 7-containing AChRs in neurons. In addition, immunologic analysis with subunit-specific muscle antibodies shows that the alpha 7-containing species in muscle lacks the beta 1 and delta muscle AChR gene products as it does the alpha 1. RNase protection experiments measuring alpha 7 mRNA levels indicate that the alpha 1 and alpha 7 genes may, in part, be subject to similar kinds of regulation in the tissue. Surgical denervation of leg muscle in newly hatched chicks caused a small and transient increase in alpha 7 mRNA after 8 days, while alpha 1 transcripts underwent a large and sustained increase in number. Similarly, treating myotube cultures with tetrodotoxin caused a modest increase in alpha 7 transcript levels and a large increase in alpha 1. Calcitonin gene-related peptide (CGRP) increased both kinds of transcripts in myotube cultures equally as did treatment with 8-bromo-cyclic AMP; CGRP is thought to work via a cyclic AMP-dependent pathway in muscle. In at least one respect, however, alpha 7 expression in muscle differs qualitatively from that of alpha 1: AChR-inducing activity (ARIA) increased alpha 1 mRNA levels in culture while slightly depressing alpha 7 mRNA levels. The regulatory pattern of alpha 7 expression in muscle may combine features of both alpha 7 expression in neurons and alpha 1 expression in muscle.
ESTHER : Romano_1997_J.Neurobiol_32_69
PubMedSearch : Romano_1997_J.Neurobiol_32_69
PubMedID: 8989664

Title : Alpha-conotoxin-ImI: a competitive antagonist at alpha-bungarotoxin-sensitive neuronal nicotinic receptors in hippocampal neurons - Pereira_1996_J.Pharmacol.Exp.Ther_278_1472
Author(s) : Pereira EF , Alkondon M , McIntosh JM , Albuquerque EX
Ref : Journal of Pharmacology & Experimental Therapeutics , 278 :1472 , 1996
Abstract : In the present study, the patch-clamp technique was applied to rat hippocampal neurons or myoballs in culture to study the actions of alpha-conotoxin-ImI on the native alpha-bungarotoxin-sensitive, presumably alpha 7-bearing, neuronal nicotinic receptor and on other ligand-gated channels. Preexposure of the neurons for 5 min to alpha-conotoxin-ImI decreased the peak amplitude of alpha-BGT-sensitive currents (referred to as type IA currents) in a concentration-dependent fashion. Several lines of evidence revealed that the inhibitory effect of alpha-conotoxin-ImI was competitive with respect to the agonist (IC50 approximately 85 nM) and reversible by washing. At 300 nM, alpha-conotoxin-ImI decreased by only 15% the peak amplitude of ACh-evoked currents in rat myoballs, did not affect the activation of currents gated by gamma-aminobutyric acid, glycine, N-methyl-D-aspartate, kainate, or quisqualate in hippocampal neurons, but reduced to approximately 60% the peak amplitude and shortened the decay phase of curare-sensitive, serotonin-gated currents in these neurons. The competitive and reversible nature of the alpha-conotoxin-ImI-induced inhibition of native alpha 7-bearing neuronal nicotinic receptors makes this peptide a valuable new tool for the functional and structural characterization of these receptors in the central nervous system.
ESTHER : Pereira_1996_J.Pharmacol.Exp.Ther_278_1472
PubMedSearch : Pereira_1996_J.Pharmacol.Exp.Ther_278_1472
PubMedID: 8819535

Title : alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors - Johnson_1995_Mol.Pharmacol_48_194
Author(s) : Johnson DS , Martinez J , Elgoyhen AB , Heinemann SF , McIntosh JM
Ref : Molecular Pharmacology , 48 :194 , 1995
Abstract : Through a study of cloned nicotinic receptors expressed in Xenopus oocytes, we provide evidence that alpha-conotoxin ImI, a peptide marine snail toxin that induces seizures in rodents, selectively blocks subtypes of nicotinic acetylcholine receptors. alpha-Conotoxin ImI blocks homomeric alpha 7 nicotinic receptors with the highest apparent affinity and homomeric alpha 9 receptors with 8-fold lower affinity. This toxin has no effect on receptors composed of alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 2 beta 4, alpha 3 beta 4, or alpha 4 beta 4 subunit combinations. In contrast to alpha-bungarotoxin, which has high affinity for alpha 7, alpha 9, and alpha 1 beta 1 gamma delta receptors, alpha-conotoxin ImI has low affinity for the muscle nAChR. Related Conus peptides, alpha-conotoxins MI and GI, exhibit a distinct specificity, strictly targeting the muscle subtype receptor but not alpha 7 or alpha 9 receptors. alpha-Conotoxins thus represent selective tools for the study of neuronal nicotinic acetylcholine receptors.
ESTHER : Johnson_1995_Mol.Pharmacol_48_194
PubMedSearch : Johnson_1995_Mol.Pharmacol_48_194
PubMedID: 7651351