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• W1995989957 abstract "Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes. Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes. β 4 expression level limits the number of functional nicotinic receptors Targeted overexpression of β4 in mice leads to strong aversion to nicotine Nicotine aversion is reversed by lentiviral expression of α5 D397N variant in vivo The medial habenula is part the circuitry controlling nicotine reinforcement Tobacco use is a major public health challenge that leads to millions of preventable deaths every year (http://www.who.int/tobacco/statistics/tobacco_atlas/en/). The principal addictive component of tobacco is the plant alkaloid nicotine, which binds and activates nicotinic acetylcholine receptors (nAChRs) (Dani and Heinemann, 1996Dani J.A. Heinemann S. Molecular and cellular aspects of nicotine abuse.Neuron. 1996; 16: 905-908Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). In the mammalian nervous system, eight alpha (α2–α7 and α9–α10) and three beta (β2–β4) subunits assemble into pentameric nAChR combinations with distinctive pharmacological and functional properties (Gotti et al., 2009Gotti C. Clementi F. Fornari A. Gaimarri A. Guiducci S. Manfredi I. Moretti M. Pedrazzi P. Pucci L. Zoli M. Structural and functional diversity of native brain neuronal nicotinic receptors.Biochem. Pharmacol. 2009; 78: 703-711Crossref PubMed Scopus (343) Google Scholar, McGehee and Role, 1995McGehee D.S. Role L.W. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons.Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (882) Google Scholar). Recently, genome-wide association studies (GWAs) have identified genetic variants in the CHRNB4/A3/A5 gene cluster as risk factors for nicotine dependence and lung cancer (Amos et al., 2010aAmos C.I. Gorlov I.P. Dong Q. Wu X. Zhang H. Lu E.Y. Scheet P. Greisinger A.J. Mills G.B. Spitz M.R. Nicotinic acetylcholine receptor region on chromosome 15q25 and lung cancer risk among African Americans: a case-control study.J. Natl. Cancer Inst. 2010; 102: 1199-1205Crossref PubMed Scopus (55) Google Scholar, Saccone et al., 2009Saccone N.L. Wang J.C. Breslau N. Johnson E.O. Hatsukami D. Saccone S.F. Grucza R.A. Sun L. Duan W. Budde J. et al.The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans.Cancer Res. 2009; 69: 6848-6856Crossref PubMed Scopus (204) Google Scholar, Thorgeirsson et al., 2008Thorgeirsson T.E. Geller F. Sulem P. Rafnar T. Wiste A. Magnusson K.P. Manolescu A. Thorleifsson G. Stefansson H. Ingason A. et al.A variant associated with nicotine dependence, lung cancer and peripheral arterial disease.Nature. 2008; 452: 638-642Crossref PubMed Scopus (1163) Google Scholar, Weiss et al., 2008Weiss R.B. Baker T.B. Cannon D.S. von Niederhausern A. Dunn D.M. Matsunami N. Singh N.A. Baird L. Coon H. McMahon W.M. et al.A candidate gene approach identifies the CHRNA5-A3-B4 region as a risk factor for age-dependent nicotine addiction.PLoS Genet. 2008; 4: e1000125Crossref PubMed Scopus (202) Google Scholar). These single nucleotide polymorphisms (SNPs) include noncoding variants across the gene cluster, as well as amino acid substitutions (http://www.ncbi.nlm.nih.gov/snp/). Given that cis-regulatory elements within the cluster coordinate transcription of these genes for assembly of α3β4-containing (α3β4∗) and α3β4α5 functional nAChRs (Scofield et al., 2010Scofield M.D. Tapper A.R. Gardner P.D. A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo.Neuroscience. 2010; 170: 1056-1064Crossref PubMed Scopus (7) Google Scholar, Xu et al., 2006Xu X. Scott M.M. Deneris E.S. Shared long-range regulatory elements coordinate expression of a gene cluster encoding nicotinic receptor heteromeric subtypes.Mol. Cell. Biol. 2006; 26: 5636-5649Crossref PubMed Scopus (32) Google Scholar), the fact that a large number of SNPs map to noncoding segments of the cluster suggests that altered regulation of these genes can contribute to the pathophysiology of tobacco use. Indeed the risk for nicotine dependence seems to stem from at least two separate mechanisms: the variability in the mRNA levels of these genes and functional changes due to nonsynonymous amino acid variants (Lu et al., 2009Lu B. Su Y. Das S. Wang H. Wang Y. Liu J. Ren D. Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80.Nature. 2009; 457: 741-744Crossref PubMed Scopus (102) Google Scholar). A number of mouse models with gene deletions, point mutations, or strain-specific variants in nAChR subunits have been critical to elucidate the role of the different nAChR combinations in nicotine addiction and withdrawal. For instance, α4β2 nAChRs, accounting for 80% of the high-affinity nicotine binding sites in the brain (Whiting and Lindstrom, 1988Whiting P.J. Lindstrom J.M. Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies.J. Neurosci. 1988; 8: 3395-3404Crossref PubMed Google Scholar), are major contributors to nicotine self-administration, as shown in β2 knockout (KO) mice (Maskos et al., 2005Maskos U. Molles B.E. Pons S. Besson M. Guiard B.P. Guilloux J.P. Evrard A. Cazala P. Cormier A. Mameli-Engvall M. et al.Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.Nature. 2005; 436: 103-107Crossref PubMed Scopus (445) Google Scholar, Picciotto, 1998Picciotto M.R. Common aspects of the action of nicotine and other drugs of abuse.Drug Alcohol Depend. 1998; 51: 165-172Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and in knockin mice with a gain-of-function mutation of α4 (Tapper et al., 2004Tapper A.R. McKinney S.L. Nashmi R. Schwarz J. Deshpande P. Labarca C. Whiteaker P. Marks M.J. Collins A.C. Lester H.A. Nicotine activation of alpha4∗ receptors: sufficient for reward, tolerance, and sensitization.Science. 2004; 306: 1029-1032Crossref PubMed Scopus (564) Google Scholar). The nAChR β4 subunit is almost always coexpressed with α3, while the auxiliary α5 subunit assembles with the α3β4 combination, but can also be incorporated in α4β2 receptor complexes. The expression of the α3β4∗ nAChR combination is restricted to a few discrete brain areas, including the medial habenula (MHb) and interpeduncular nucleus (IPN), and to autonomic ganglia (Zoli et al., 1995Zoli M. Le Novère N. Hill Jr., J.A. Changeux J.P. Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems.J. Neurosci. 1995; 15: 1912-1939Crossref PubMed Google Scholar). α3β4∗ nAChRs have a lower affinity for nicotine than α4β2 receptors and are likely less desensitized at the nicotine levels found in smokers than α4β2 nAChRs are, suggesting that α3β4∗ nAChRs could play an important role in tobacco addiction, since they retain their sensitivity to fluctuating nicotine levels in smokers (Rose, 2007Rose J.E. Multiple brain pathways and receptors underlying tobacco addiction.Biochem. Pharmacol. 2007; 74: 1263-1270Crossref PubMed Scopus (42) Google Scholar). β4 and α5 KO mice show similar phenotypes, including decreased signs of nicotine withdrawal symptoms (Jackson et al., 2008Jackson K.J. Martin B.R. Changeux J.P. Damaj M.I. Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs.J. Pharmacol. Exp. Ther. 2008; 325: 302-312Crossref PubMed Scopus (163) Google Scholar, Salas et al., 2004Salas R. Pieri F. De Biasi M. Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit.J. Neurosci. 2004; 24: 10035-10039Crossref PubMed Scopus (193) Google Scholar, Salas et al., 2009Salas R. Sturm R. Boulter J. De Biasi M. Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice.J. Neurosci. 2009; 29: 3014-3018Crossref PubMed Scopus (224) Google Scholar), hypolocomotion, and resistance to nicotine-induced seizures (Kedmi et al., 2004Kedmi M. Beaudet A.L. Orr-Urtreger A. Mice lacking neuronal nicotinic acetylcholine receptor beta4-subunit and mice lacking both alpha5- and beta4-subunits are highly resistant to nicotine-induced seizures.Physiol. Genomics. 2004; 17: 221-229Crossref PubMed Scopus (66) Google Scholar, Salas et al., 2004Salas R. Pieri F. De Biasi M. Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit.J. Neurosci. 2004; 24: 10035-10039Crossref PubMed Scopus (193) Google Scholar). It has been more difficult to assess the role of α3∗ nAChRs because KO mice die within 3 weeks after birth due to severe bladder dysfunction (Xu et al., 1999Xu W. Gelber S. Orr-Urtreger A. Armstrong D. Lewis R.A. Ou C.N. Patrick J. Role L. De Biasi M. Beaudet A.L. Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 5746-5751Crossref PubMed Scopus (238) Google Scholar). Here we show that α3β4α5 nAChR activity in vitro and in vivo is limited by the level of Chrnb4 expression, and that the ability of the β4 subunit to increase α3β4α5 currents depends on a single, unique residue (S435). This residue maps to the intracellular vestibule of the nAChR complex adjacent to the rs16969968 SNP in CHRNA5 (D398N), which is linked to a high risk of nicotine dependence in humans. We present a transgenic mouse model of the Chrnb4-Chrna3-Chrna5 gene cluster, referred to as Tabac (transgenic a3b4a5 cluster) mice, in which Chrnb4 overexpression enhances α3β4∗ nAChR levels, resulting in altered nicotine consumption and nicotine-conditioned place aversion (CPA). Lentiviral-mediated transduction of the MHb of Tabac mice with the D398N Chrna5 variant reversed the nicotine aversion induced by β4 overexpression. This study provides a mouse model for nicotine dependence, demonstrates a critical role for the MHb in the circuitry controlling nicotine consumption, and elucidates molecular mechanisms contributing to these phenotypes. Recently it has been shown that α5 competes with β4 for association with α4, and that this competition does not occur if β4 is substituted with β2 (Gahring and Rogers, 2010Gahring L.C. Rogers S.W. Nicotinic receptor subunit alpha5 modifies assembly, up-regulation, and response to pro-inflammatory cytokines.J. Biol. Chem. 2010; 285: 26049-26057Crossref PubMed Scopus (11) Google Scholar). Given that the CHRNA5-A3-B4 gene cluster regulates the coexpression of α5, β4, and α3 subunits, and that SNPs in the cluster regulatory regions and nonsynonymous variants such as rs16969968 (corresponding to D398N in CHRNA5) associate with nicotine dependence (Bierut, 2010Bierut L.J. Convergence of genetic findings for nicotine dependence and smoking related diseases with chromosome 15q24-25.Trends Pharmacol. Sci. 2010; 31: 46-51Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Bierut et al., 2008Bierut L.J. Stitzel J.A. Wang J.C. Hinrichs A.L. Grucza R.A. Xuei X. Saccone N.L. Saccone S.F. Bertelsen S. Fox L. et al.Variants in nicotinic receptors and risk for nicotine dependence.Am. J. Psychiatry. 2008; 165: 1163-1171Crossref PubMed Scopus (464) Google Scholar, Saccone et al., 2009Saccone N.L. Wang J.C. Breslau N. Johnson E.O. Hatsukami D. Saccone S.F. Grucza R.A. Sun L. Duan W. Budde J. et al.The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans.Cancer Res. 2009; 69: 6848-6856Crossref PubMed Scopus (204) Google Scholar), we were first interested in determining whether variation of the proportion of α3, β4, and α5 (wild-type [WT] and D398N) subunits influences nicotine-evoked currents. To measure this, we performed electrophysiological recordings in oocytes injected with cRNA transcripts of the different mouse subunits. In these experiments (Figure 1), the cRNA concentration of α3 was held constant (1 ng/oocyte), whereas the concentration of β4 or β2 input cRNA was varied among 1, 2, 3, 4, 5, or 10 ng. These experiments showed that β4, but not β2, was able to increase current amplitudes in a dose-dependent manner (Figures 1A and 1B). β4 overexpression did not shift the dose response curves for nicotine (Figure S1A, available online). Next we held constant the concentrations of α3 and β4 at 1:10 and added the cRNA of α5 WT or α5 D397N variant (corresponding to the human α5 variant D398N) at ratios of 1:10:1, 1:10:5, and 1:10:10 (Figure 1A). We observed a significant decrease of current amplitudes at higher concentrations of α5, and this effect was significantly more pronounced with α5 D397N. These results suggest that α5 and β4 may compete for binding to α3, in line with the studies showing such competition for binding to α4 (Gahring and Rogers, 2010Gahring L.C. Rogers S.W. Nicotinic receptor subunit alpha5 modifies assembly, up-regulation, and response to pro-inflammatory cytokines.J. Biol. Chem. 2010; 285: 26049-26057Crossref PubMed Scopus (11) Google Scholar). Given that overexpression of β2 with either α3 (Figure 1A) or α4 (Figure S1B) did not increase currents, we were interested in identifying the residues differing between β4 and β2 that mediate this effect. Since the long cytoplasmic loop is the most divergent domain between nAChR subunits (Figure S1C), and since it has been implicated in cell-surface expression and trafficking of β2 subunits (Nashmi et al., 2003Nashmi R. Dickinson M.E. McKinney S. Jareb M. Labarca C. Fraser S.E. Lester H.A. Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.J. Neurosci. 2003; 23: 11554-11567Crossref PubMed Google Scholar, Ren et al., 2005Ren X.Q. Cheng S.B. Treuil M.W. Mukherjee J. Rao J. Braunewell K.H. Lindstrom J.M. Anand R. Structural determinants of alpha4beta2 nicotinic acetylcholine receptor trafficking.J. Neurosci. 2005; 25: 6676-6686Crossref PubMed Scopus (43) Google Scholar), we generated β2–β4 chimeras exchanging either this domain, or short motifs and single residues within this domain. Replacement of the cytoplasmic loop of β2 with the corresponding sequences present in β4 (β2/β4 322–496) led to strong increase of nicotinic currents (Figure 1C). Introduction of two β4-specific motifs (a serine/tyrosine rich motif [β2/+β4 382–391] and gephyrin-like-binding motif [β2/+β4 401–419] into the β2 loop) had no influence on current amplitudes (Figure 1C). We next performed bioinformatic analyses and singled out eight β4-specific residues (indicated as T-1 to T-8 in Figure S1C) present within highly conserved motifs. Six of these residues were not further considered: T-2, T-3, T-6, and T-7 residues differ between mouse and chicken β4 subunits, which are equally potent in enhancing nicotine-evoked currents (Figure S1B); T-4 residue lies within the tested motif in the β2/+β4 382–391 chimera; and residues at position T-8 have the same charge (Figure S1C). The remaining two candidates, T-1 (S324 in β4 and T327 in β2) and T-7 (S435 in β4 and R431 in β2) (Figure S1C) were tested by point mutagenesis in the β2 subunit backbone. The β2 T327S point mutant did not increase current, whereas replacement of β2 R431 with serine resulted in a 3.5-fold current increase (Figure 1C). Furthermore, point mutation of the native S435 in the β4 subunit to the arginine residue present in β2 (β4 S435R) abolished the β4-specific activity. Thus, these data demonstrate that the distinctive ability of β4 to increase currents when overexpressed maps to a single residue (S435) that is required in β4 for current increase and can confer this property to β2. Alignment of mouse, human, and Torpedo nAChR subunit sequences indicated that S435 in β4 and D397N in α5 are located in the 25 amino-acid-long amphipathic membrane-associated stretch (MA-stretch) described in the Torpedo subunits (Unwin, 2005Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution.J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1358) Google Scholar) (Figure 2A ). Electron microscopy studies of the Torpedo nAChR have proposed a 3D density map of the receptor complex. In particular, these analyses predict that the MA-stretch of each subunit forms a curved α-helix that helps create, with other α-helices, an inverted pentagonal cone vestibule. To locate the S435 (β4) and D397 (α5) residues within the receptor pentamer, we performed homology modeling with the Torpedo nAChR using one possible α3β4α5 subunit arrangement. This model predicted the formation of a very similar disposition of α helices in the α3β4α5 and mapped both residues to the intracellular vestibule (Figure 2B). Electrostatic mapping of the vestibule showed a particular disposition of charges with S435 and D397 located at the more distal and positively charged part of the vestibule (Figure 2C). These data indicate first that the critical residue in β4 that mediates the β4 effect is located in the receptor structure near the most common SNP of α5 to be associated with heavy smoking; and second, that this is a highly charged domain of the receptor where single residue changes may have a particularly strong effect on receptor activity. To test the hypothesis that β4 is rate limiting for nAChR assembly and function in vivo and that overexpression of β4 can strongly influence nicotine-evoked currents and behavioral responses to nicotine, we characterized a bacterial artificial chromosome (BAC) transgenic line spanning the Chrnb4-Chrna3-Chrna5 gene cluster (Gong et al., 2003Gong S. Zheng C. Doughty M.L. Losos K. Didkovsky N. Schambra U.B. Nowak N.J. Joyner A. Leblanc G. Hatten M.E. Heintz N. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.Nature. 2003; 425: 917-925Crossref PubMed Scopus (1498) Google Scholar). The BAC transgene included the intact coding sequences of the Chrnb4 gene, modified sequences of Chrna3, and incomplete sequences of Chrna5. Chrna3 was modified by insertion of an eGFP cassette followed by polyadenylation signals at the ATG translation initiator codon of Chrna3 (Figure 3A ). The upstream sequences of Chrna5, encoding exon 1 splice variants (Flora et al., 2000Flora A. Schulz R. Benfante R. Battaglioli E. Terzano S. Clementi F. Fornasari D. Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues.Eur. J. Pharmacol. 2000; 393: 85-95Crossref PubMed Scopus (25) Google Scholar), are missing in the BAC transgene (Figure 3A). To promote correct expression of Chrnb4, the BAC included the intergenic and 5′ flanking regions encompassing the cis-regulatory elements that coordinate cotranscriptional control of the genes in the cluster (Bigger et al., 1997Bigger C.B. Melnikova I.N. Gardner P.D. Sp1 and Sp3 regulate expression of the neuronal nicotinic acetylcholine receptor beta4 subunit gene.J. Biol. Chem. 1997; 272: 25976-25982Crossref PubMed Scopus (80) Google Scholar, Medel and Gardner, 2007Medel Y.F. Gardner P.D. Transcriptional repression by a conserved intronic sequence in the nicotinic receptor alpha3 subunit gene.J. Biol. Chem. 2007; 282: 19062-19070Crossref PubMed Scopus (7) Google Scholar, Xu et al., 2006Xu X. Scott M.M. Deneris E.S. Shared long-range regulatory elements coordinate expression of a gene cluster encoding nicotinic receptor heteromeric subtypes.Mol. Cell. Biol. 2006; 26: 5636-5649Crossref PubMed Scopus (32) Google Scholar). As a result of these modifications in the BAC transgene, these Tabac mice express high levels of β4, but not α5 (Figure 3B), and expression of α3 is replaced by expression of an eGFP reporter cassette to monitor the sites expressing the transgene (Figures 3C–3H). As shown in Figure 3, neurons expressing eGFP were evident in autonomic ganglia (Figure 3C), and in very restricted brain structures (Figures 3D–3H) known to express these genes (Zoli et al., 1995Zoli M. Le Novère N. Hill Jr., J.A. Changeux J.P. Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems.J. Neurosci. 1995; 15: 1912-1939Crossref PubMed Google Scholar). Immunostaining with cholinergic (ChAT) and dopaminergic (TH) markers indicated high expression of Chrna3/eGFP in ChAT neurons of the Hb-IPN system (Figures 3G and 3H). Intense Chrna3/eGFP expression was also detected in other brain areas (Figures 3D and 3E) involved in nicotine addiction, such as the ventral tegmental area (VTA), the caudal linear nucleus (Cli), the supramammilary nucleus (SuM) (Ikemoto et al., 2006Ikemoto S. Qin M. Liu Z.H. Primary reinforcing effects of nicotine are triggered from multiple regions both inside and outside the ventral tegmental area.J. Neurosci. 2006; 26: 723-730Crossref PubMed Scopus (116) Google Scholar), and the laterodorsal tegmental nucleus (Figure 3F), which provides modulatory input to the VTA (Maskos, 2008Maskos U. The cholinergic mesopontine tegmentum is a relatively neglected nicotinic master modulator of the dopaminergic system: relevance to drugs of abuse and pathology.Br. J. Pharmacol. 2008; 153: S438-S445PubMed Google Scholar). We performed in situ hybridization (ISH) experiments to verify that the BAC accurately directed expression of transgenic Chrnb4 transcripts to eGFP-positive brain areas. Tabac mice showed a prominent enrichment of Chrnb4 transcripts in α3β4∗-positive areas, such as MHb and IPN, and in brain areas that have been shown to express lower levels of Chrnb4, such as SuM (Dineley-Miller and Patrick, 1992Dineley-Miller K. Patrick J. Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system.Brain Res. Mol. Brain Res. 1992; 16: 339-344Crossref PubMed Scopus (169) Google Scholar) and VTA (Yang et al., 2009Yang K. Hu J. Lucero L. Liu Q. Zheng C. Zhen X. Jin G. Lukas R.J. Wu J. Distinctive nicotinic acetylcholine receptor functional phenotypes of rat ventral tegmental area dopaminergic neurons.J. Physiol. 2009; 587: 345-361Crossref PubMed Scopus (56) Google Scholar) (Figures 3I and 3J). RT-PCR studies showed that Chrna4, Chrna7, and Chrnb2 transcripts (which are not present in the BAC) are not altered in Tabac mice (Figure S2). Taken together these data show that Tabac mice express high levels of β4, but not α5, in α3/eGFP-labeled cells in CNS and PNS structures known to express the Chrnb4-Chrna3-Chrna5 nicotinic gene cluster, and are thus a useful mouse model in which to test the consequences of enhanced β4 expression at endogenous sites. Given the demonstration that the level of β4 expression is rate limiting for the function of α3β4α5 receptors in vitro (Figure 1), we were next interested in determining whether enhanced expression of Chrnb4 in Tabac neurons resulted in elevated nicotine-evoked currents in vivo. Previous studies have shown that neurons in the MHb express high levels of α3β4α5 receptors (Quick et al., 1999Quick M.W. Ceballos R.M. Kasten M. McIntosh J.M. Lester R.A. Alpha3beta4 subunit-containing nicotinic receptors dominate function in rat medial habenula neurons.Neuropharmacology. 1999; 38: 769-783Crossref PubMed Scopus (133) Google Scholar). Accordingly, we employed patch-clamp recordings to measure nicotine-evoked currents in MHb neurons of Tabac mice. A large proportion of MHb neurons in WT mice (n = 20 of n = 23 neurons recorded) responded to local fast application (50 ms) of nicotine (Figures 4A and 4B ). In Chrna3/eGFP-labeled MHb neurons of Tabac mice, nicotine elicited significantly increased peak currents in comparison to WT littermates (on average, 3.4-fold at 100 μM nicotine, two-way ANOVA, p < 0.05) (Figure 4B). Similarly increased responses were obtained using acetylcholine (ACh) (data not shown). Dose-response curves for nicotine showed no significant differences between WT and Tabac mice, indicating that the affinity of the receptors in the transgenic mice is not altered (Figure 4C). Application of mecamylamine (MEC), a nonselective potent inhibitor of α4β2∗ and α3β4∗ nAChRs (Bacher et al., 2009Bacher I. Wu B. Shytle D.R. George T.P. Mecamylamine - a nicotinic acetylcholine receptor antagonist with potential for the treatment of neuropsychiatric disorders.Expert Opin. Pharmacother. 2009; 10: 2709-2721Crossref PubMed Scopus (75) Google Scholar), resulted in a blockade of as much as 90% of the nicotine-elicited responses in Tabac mice (Figure 4D), demonstrating that the enhanced nicotine responses in Tabac neurons result directly from elevated levels of functional nAChRs. To determine whether these additional receptors cause enhanced neuronal excitability, the firing rate of habenular neurons was measured in current-clamp assays in response to nicotine. Neurons from WT and Tabac mice were silent at rest (−70 mV). Local nicotine application (1 μM for 3 s) elicited single action potentials in WT neurons, whereas nicotine induced a robust burst of action potentials with a 13-fold higher firing frequency on average in Tabac neurons (p < 0.005) (Figures 4E and 4F). Together, these results indicate that the increased sensitivity of MHb neurons to nicotine in Tabac mice results from the presence of additional functional nAChRs, rather than from changes in the nicotine affinity of existing receptors. To assay for elevated receptor expression across different brain structures, we performed autoradiographic radioligand binding assays with iodinated epibatidine, which mainly binds α4β2∗ and α3β4∗ nAChRs (Perry and Kellar, 1995Perry D.C. Kellar K.J. [3H]epibatidine labels nicotinic receptors in rat brain: an autoradiographic study.J. Pharmacol. Exp. Ther. 1995; 275: 1030-1034PubMed Google Scholar) (Figures 5A and 5B ). Competition with cold cytisine, which binds with higher affinity to α4β2∗ than to α3β4∗ receptors (Marks et al., 2010Marks M.J. Laverty D.S. Whiteaker P. Salminen O. Grady S.R. McIntosh J.M. Collins A.C. John Daly's compound, epibatidine, facilitates identification of nicotinic receptor subtypes.J. Mol. Neurosci. 2010; 40: 96-104Crossref PubMed Scopus (21) Google Scholar), was done to distinguish α3β4∗ from overlapping α4β2∗ binding sites (Zoli et al., 1998Zoli M. Léna C. Picciotto M.R. Changeux J.P. Identification of four classes of brain nicotinic receptors using beta2 mutant mice.J. Neurosci. 1998; 18: 4461-4472Crossref PubMed Google Scholar) (Figures 5C and 5D). In WT mice, discrete brain regions resistant to cytisine competition labeled well-known α3β4∗ sites such as MHb, IPN, and superior colliculus (Figure 5C). In Tabac mice, increased radioligand binding to cytisine-resistant sites was detected in these areas and in additional brain structures, including the VTA, SuM, substantia nigra, and striatum (Figure 5D). A strong correlation between radioligand signal and eGFP fluorescence was detected in all analyzed CNS structures (Figure 5E and Table S1). Densitometric analyses indicated significantly increased cytisine-resistant signals in α3β4∗-expressing regions in Tabac mice (Table S1), while α4β2∗ epibatidine binding sites such as cortex and thalamus did not differ between control and Tabac mice (Figures 5A and 5B), indicating that elevated surface receptors are present in sites corresponding to endogenous β4 expression sites. To exclude the possibility that the increased radioligand signal could reflect increased cell number, we quantified the cell density in MHb of Tabac and WT mice and observed no significant differences (Figure S3). These data show that the enhanced nicotine-evoked currents in Tabac mice result from β4-mediated recruitment of additional functional α3β4∗ nAChR complexes on the cell surface. Taken together, the anatomic mapping and ISH results presented in Figure 3 and the" @default.
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• W1995989957 doi "https://doi.org/10.1016/j.neuron.2011.04.013" @default.
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