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Identification of the Nicotinic Receptor Subtypes Expressed on Dopaminergic Terminals in the Rat Striatum

Michele Zoli, Milena Moretti, Alessio Zanardi, J. Michael McIntosh, Francesco Clementi and Cecilia Gotti
Journal of Neuroscience 15 October 2002, 22 (20) 8785-8789; DOI: https://doi.org/10.1523/JNEUROSCI.22-20-08785.2002
Michele Zoli
1Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, 41100 Modena, Italy,
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Milena Moretti
2Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20129 Milan, Italy, and
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Alessio Zanardi
1Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, 41100 Modena, Italy,
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J. Michael McIntosh
3Department of Biology, University of Utah, Salt Lake City, Utah 84112
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Francesco Clementi
2Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20129 Milan, Italy, and
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Cecilia Gotti
2Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20129 Milan, Italy, and
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    Fig. 1.

    Immunoprecipitation analysis of the subunit content of α6β2* and α4(nonα6)β2* nAChR subtypes immunopurified through affinity column from striatal extracts and labeled with 2 nm3H-Epi. The results are expressed as percentage of total 3H-Epi binding present in the solution before immunoprecipitation. Each data point is the mean ± SEM of five determinations performed in triplicate.

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    Fig. 2.

    Inhibition of 125I-Epi binding to native immunoimmobilized α6β2* (a) and α4(nonα6)β2* (b) nAChRs by several nicotinic ligands, including nicotine (Nic), acetylcholine (ACh), cytisine (Cyt), dihydro-β-erythroidine (DHBE),d-tubocurarine (dTC) (a, b), α-CntxMII (c), and MLA (d). The curves were obtained by fitting three or four separate experiments using the LIGAND program (Munson and Rodbard, 1980).

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    Fig. 3.

    a, b,3H-WIN-35,428 (a) and3H-Epi binding (b) in rat striatal membranes obtained from control and 6-OHDA lesioned rats.c, Immunoprecipitation of nAChR subunits in 2% Triton X-100 extracts from control and 6-OHDA lesioned striata. Each value represents the mean ± SEM of three separate experiments. Statistical analysis according to Mann–Whitney U test, **p < 0.01.

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    Table 1.

    Affinity of nicotinic agonists and antagonists for immunoimmobilized nAChR subtypes

    α6β2*α4(non α6)β2*
    Kd (nm)
     125I-Epibatidine0.034 (34)0.041 (25)
    Ki (nm)
     Cytisine0.65 (18)0.19 (16)
     Nicotine2.5 (23)1.75 (23)
     Acetylcholine8.0 (34)8.6 (23)
     Dihydro-β-erythroidine524 (25)274 (21)
     d-Tubocurarine3,110 (20)16,100 (28)
     α-Conotoxin MII1.3 (45)–
    >10,000>10,000
     Methyllycaconitine40 (47)–
    20,800 (18)25,000 (23)
    • Kd and Ki values were derived from curves of 125I-Epi saturation and competition binding, respectively, to α6β2* or α4(nonα6)β2

    • ↵* immunoimmobilized receptors. Curves obtained from three or four separate experiments were fitted using a nonlinear least-squares analysis program. For both α-conotoxin MII and methyllycaconitine, a two-site model was statistically significant (F test), whereas for d-tubocurarine and cytisine the data were better fitted with a one-site model. Numbers in parentheses represent percentage of coefficient of variation.

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The Journal of Neuroscience: 22 (20)
Journal of Neuroscience
Vol. 22, Issue 20
15 Oct 2002
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Identification of the Nicotinic Receptor Subtypes Expressed on Dopaminergic Terminals in the Rat Striatum
Michele Zoli, Milena Moretti, Alessio Zanardi, J. Michael McIntosh, Francesco Clementi, Cecilia Gotti
Journal of Neuroscience 15 October 2002, 22 (20) 8785-8789; DOI: 10.1523/JNEUROSCI.22-20-08785.2002

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Identification of the Nicotinic Receptor Subtypes Expressed on Dopaminergic Terminals in the Rat Striatum
Michele Zoli, Milena Moretti, Alessio Zanardi, J. Michael McIntosh, Francesco Clementi, Cecilia Gotti
Journal of Neuroscience 15 October 2002, 22 (20) 8785-8789; DOI: 10.1523/JNEUROSCI.22-20-08785.2002
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Keywords

  • nicotinic acetylcholine receptor
  • mesostriatal dopamine pathway
  • striatum
  • immunoprecipitation
  • 6-hydroxydopamine
  • α-conotoxin MII

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