, Volume 184, Issue 3–4, pp 314–327 | Cite as

Deletion of the beta 2 nicotinic acetylcholine receptor subunit alters development of tolerance to nicotine and eliminates receptor upregulation

  • Sarah E. McCallum
  • Allan C. Collins
  • Richard Paylor
  • Michael J. Marks
Original Investigation



Chronic nicotine exposure induces both tolerance and upregulation of [3H]nicotine binding sites in rodent and human brain. However, the mechanism for chronic tolerance is unclear because a direct relationship between tolerance and receptor upregulation is not consistently observed.


In the present experiments, the role of β2* nicotinic acetylcholine receptors (nAChRs) on tolerance development and nAChR upregulation was examined following chronic nicotine treatment of β2 wild-type (+/+), heterozygous (+/−), and null mutant (−/−) mice.


Saline or nicotine (1, 2, or 4 mg/kg/h) was infused intravenously for 10 days. Locomotor activity and body temperature responses were measured before and after nicotine challenge injection to observe changes in nicotine sensitivity. [3H]Epibatidine binding was then measured in ten brain regions.


β2+/+ mice developed dose-dependent tolerance and upregulation of [3H]epibatidine binding sites. In contrast, β2−/− mice, initially less sensitive to acute nicotine's effects, became more sensitive following treatment with the lowest chronic dose (1 mg/kg/h). β2−/− mice treated with 4.0 mg/kg/h nicotine were no longer supersensitive, indicating that tolerance developed at this higher dose. However, these changes in nicotine sensitivity occurred in the absence of any nAChR changes in either low- or high-affinity [3H]epibatidine sites. Responses of β2+/− mice were intermediate between wild-type and mutant mice.


Upregulation of nAChRs in vivo requires the presence of the β2 subunit. Changes in nicotine sensitivity occurred both in the presence (β2+/+) and absence (β2−/−) of β2* nAChRs and suggest that mechanisms involving both β2* and non-β2* nAChR subtypes modulate adaptation to chronic nicotine exposure.


Nicotine β2 null mutant mouse Tolerance Sensitivity Receptor upregulation [3H]Epibatidine 


  1. Avila AM, Davila-Garcia MI, Ascarrunz VS, Xiao Y, Kellar KJ (2003) Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor. J Pharmacol Exp Ther 64:974–986Google Scholar
  2. Badio B, Daly JW (1994) Epibatidine: a potent analgetic and nicotinic agonist. Mol Pharmacol 45:563–569PubMedGoogle Scholar
  3. Barr JE, Holmes DB, Ryan LM, Sharpless SH (1979) Techniques for the chronic cannulation of the jugular vein in mice. Pharmacol Biochem Behav 11:115–118CrossRefPubMedGoogle Scholar
  4. Barrantes GE, Rogers AT, Lindstrom J, Wonnacott S (1995) Alpha-bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterization, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment. Brain Res 672:228–236CrossRefPubMedGoogle Scholar
  5. Benwell ME, Balfour DJ, Anderson JM (1988) Evidence that tobacco smoking increases the density of (−)-[3H]nicotine binding sites in human brain. J Neurochem 50:1243–1247PubMedCrossRefGoogle Scholar
  6. Breese CR, Marks MJ, Logel J, Adams CE, Sullivan B, Collins AC, Leonard S (1997) Effect of smoking history on [3H]nicotine binding in human postmortem brain. J Pharmacol Exp Ther 282:7–13PubMedGoogle Scholar
  7. Carlson J, Armstrong B, Switzer RC III, Ellison G (2000) Selective neurotoxic effects of nicotine on axons in fasciculus retroflexus further support evidence that this is a weak link in brain across multiple drugs of abuse. Neuropharmacology 39:2792–2798CrossRefPubMedGoogle Scholar
  8. Carlson J, Noguchi K, Ellison G (2001) Nicotine produces selective degeneration in the medial habenula and fasciculus retroflexus. Brain Res 906:127–134CrossRefPubMedGoogle Scholar
  9. Champtiaux N, Han Z-Y, Bessis A, Rossi FM, Zoli M, Marubio L, McIntosh JM, Changeux J-P (2002) Distribution and pharmacology of α6 containing nicotinic acetylcholine receptors analyzed with mutant mice. J Neurosci 22:1208–1217PubMedGoogle Scholar
  10. Collins AC, Miner LL, Marks MJ (1988) Genetic influences on acute responses to nicotine and nicotine tolerance in the mouse. Pharmacol Biochem Behav 30:269–278CrossRefPubMedGoogle Scholar
  11. Davila-Garcia MI, Musachio JL, Kellar KJ (2003) Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]-epibatidine in adrenal gland, superior cervical ganglia, pineal or retina. J Neurochem 85:1237–1246CrossRefPubMedGoogle Scholar
  12. Flores CM, Rogers SW, Pabreza LA, Wolfe BB, Kellar KJ (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha4 and beta2 subunits and is up-regulated by chronic nicotine treatment. Mol Pharmacol 41:31–37PubMedGoogle Scholar
  13. Flores CM, Davila-Garcia MI, Ulrich YM, Kellar KJ (1997) Differential regulation of neuronal nicotinic receptor binding sites following chronic nicotine administration. J Neurochem 69:2216–2219PubMedCrossRefGoogle Scholar
  14. Glick SD, Maisonneuve IM, Kitchen BA (2002) Modulation of nicotine self-administration in rats by combination therapy with agents blocking α3β4 nicotinic receptors. Eur J Pharmacol 448:185–191CrossRefPubMedGoogle Scholar
  15. Lukas RJ, Changeux JP, Le Novere N, Albuquerque EX, Balfour DJ, Berg DK, Bertrand D, Chiappinelli VA, Clarke PB, Collins AC, Dani JA, Grady SR, Kellar KJ, Lindstrom JM, Marks MJ, Quik M, Taylor PW, Wonnacott S (1999) International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 51:397–401PubMedGoogle Scholar
  16. Marks MJ, Burch LB, Collins AC (1983) Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226:817–825PubMedGoogle Scholar
  17. Marks MJ, Romm E, Bealer SM, Collins AC (1985) A test battery for measuring nicotine effects in mice. Pharmacol Biochem Behav 23:325–330CrossRefPubMedGoogle Scholar
  18. Marks MJ, Stitzel JA, Collins AC (1989) Genetic influences on nicotine responses. Pharmacol Biochem Behav 33:667–678CrossRefPubMedGoogle Scholar
  19. Marks MJ, Campbell SM, Romm E, Collins AC (1991) Genotype influences the development of tolerance to nicotine in the mouse. J Pharmacol Exp Ther 259:392–402PubMedGoogle Scholar
  20. Marks MJ, Smith KW, Collins AC (1998) Differential agonist inhibition identifies multiple epibatidine binding sites in mouse brain. J Pharmacol Exp Ther 285:377–386PubMedGoogle Scholar
  21. Marks MJ, Whiteaker P, Calcaterra J, Stitzel JA, Bullock AE, Grady SR, Picciotto MR, Changeux JP, Collins AC (1999) Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit. J Pharmacol Exp Ther 289:1090–1103PubMedGoogle Scholar
  22. Marks MJ, Stitzel JA, Grady SR, Picciotto MR, Changeux JP, Collins AC (2000) Nicotinic-agonist stimulated 86Rb+ efflux and [3H]epibatidine binding of mice differing in beta2 genotype. Neuropharmacology 39:2632–2645CrossRefPubMedGoogle Scholar
  23. Marks MJ, Rowell PP, Cao J-Z, Grady SR, McCallum SE, Collins AC (2004) Subsets of acetylcholine-stimulated 86RB+ efflux and [125I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment. Neuropharmacology 46:1141–1157CrossRefPubMedGoogle Scholar
  24. Marubio LM, Mar Arroyo-Jimenez M, Cordero-Erausquin M, Lena C, Le Novere N, de Kerchove A, Huchet M, Damaj MI, Changeux JP (1999) Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature 398:805–810CrossRefPubMedGoogle Scholar
  25. McCallum SE, Caggiula AR, Booth S, Breese CR, Lee MJ, Donny EC, Leonard S, Sved AF (2000) Mecamylamine prevents tolerance but enhances whole brain [3H]epibatidine binding in response to repeated nicotine administration in rats. Psychopharmacology 150:1–8CrossRefPubMedGoogle Scholar
  26. Nguyen HN, Rasmussen BA, Perry DC (2003) Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography. J Pharmacol Exp Ther 307:1090–1097CrossRefPubMedGoogle Scholar
  27. Olale F, Gerzanich V, Kuryatov A, Wang F, Lindstrom J (1997) Chronic nicotine exposure differentially affects the function of human α3, α4, and α7 neuronal receptor subtypes. J Pharmacol Exp Ther 283:675–683PubMedGoogle Scholar
  28. Parker MJ, Beck A, Luetje CW (1998) Neuronal nicotinic receptor β2 and β4 subunits confer large differences in agonist binding affinity. Mol Pharmacol 54:1132–1139PubMedGoogle Scholar
  29. Pauly JR, Marks MJ, Gross SD, Collins AC (1991) An autoradiographic analysis of cholinergic receptors in mouse brain after chronic nicotine treatment. J Pharmacol Exp Ther 258:1127–1136PubMedGoogle Scholar
  30. Pauly JR, Grun EU, Collins AC (1992) Tolerance to nicotine following chronic treatment by injections: a potential role for corticosterone. Psychopharmacology 108:33–39CrossRefPubMedGoogle Scholar
  31. Perkins KA (2002) Chronic tolerance to nicotine in humans and its relationship to tobacco dependence. Nicotine Tob Res 4:405–422CrossRefPubMedGoogle Scholar
  32. Perry DC, Kellar KJ (1995) [3H]Epibatidine labels nicotinic receptors in rat brain: an autoradiographic study. J Pharmacol Exp Ther 275:1030–1034PubMedGoogle Scholar
  33. Perry DC, Davila-Garcia MI, Stockmeier CA, Kellar KJ (1999) Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J Pharmacol Exp Ther 289:1545–1552PubMedGoogle Scholar
  34. Perry DC, Xiao Y, Nguyen HN, Musachio JL, Davila-Garcia MI, Kellar KJ (2002) Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography. J Neurochem 82:468–481CrossRefPubMedGoogle Scholar
  35. Picciotto MR, Zoli M, Lena C, Bessis A, Lallemand Y, LeNovere N, Vincent P, Merlo EM, Brulet P, Changeux J-P (1995) Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374:65–67CrossRefPubMedGoogle Scholar
  36. Picciotto MR, Zoli M, Rimondini R, Lena C, Marubio LM, Pich EM, Fuxe K, Changeux JP (1998) Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391:173–177CrossRefPubMedGoogle Scholar
  37. Pietila K, Ahtee L (2000) Chronic nicotine administration in the drinking water affects the striatal dopamine in mice. Pharmacol Biochem Behav 66:95–103CrossRefPubMedGoogle Scholar
  38. Pietila K, Lahde T, Attila M, Ahtee L, Nordberg A (1998) Regulation of nicotinic receptors in the brain of mice withdrawn from chronic oral nicotine treatment. Naunyn Schmiedebergs Arch Pharmacol 357:176–182PubMedCrossRefGoogle Scholar
  39. Ross SA, Wong JY, Clifford JJ, Kinsella A, Massalas JS, Horne MK, Scheffer IE, Kola I, Waddington JL, Berkovic SF, Drago J (2000) Phenotypic characterization of an alpha 4 neuronal nicotinic acetylcholine receptor subunit knock-out mouse. J Neurosci 20:6431–6441PubMedGoogle Scholar
  40. Salas R, Pieri F, DeBiasi M (2004) Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit. J Neurosci 24:10035–10039CrossRefPubMedGoogle Scholar
  41. Sallette J, Bohler S, Benoit P, Soudant M, Pons S, leNoviere N, Changeux J-P, Corringer J (2004) An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J Biol Chem 279:18767–18775CrossRefPubMedGoogle Scholar
  42. Schwartz RD, Kellar KJ (1983) Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science 220:214–216PubMedCrossRefGoogle Scholar
  43. Shoaib M, Thorndike E, Schindler CW, Goldberg SR (1997) Discriminative stimulus effects of nicotine and chronic tolerance. Pharmacol Biochem Behav 56:167–173CrossRefPubMedGoogle Scholar
  44. Sparks JA, Pauly JR (1999) Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice. Psychopharmacology 141:145–153CrossRefPubMedGoogle Scholar
  45. Stolerman IP, Fink R, Jarvik ME (1973) Acute and chronic tolerance to nicotine measured by activity in rats. Psychopharmacologia 30:329–342CrossRefPubMedGoogle Scholar
  46. Tritto T, McCallum SE, Waddle SA, Hutton SH, Paylor R, Collins AC, Marks MJ (2004) Null mutant analysis of responses to nicotine: deletion of beta2 nAChR subunit but not alpha7 subunit reduces sensitivity to nicotine-induced locomotor depression and hypothermia. Nicotine Tob Res 6:145–157CrossRefPubMedGoogle Scholar
  47. Wang F, Nelson ME, Kuryatov A, Olale F, Cooper J, Keyser K, Lindstrom J (1998) Chronic nicotine treatment up-regulates human alpha beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells. J Biol Chem 273:28721–28732CrossRefPubMedGoogle Scholar
  48. Whiteaker P, Jimenez M, McIntosh JM, Collins AC, Marks MJ (2000) Identification of a novel nicotinic binding site in mouse brain using [125I]-epibatidine. Br J Pharmacol 131:729–739CrossRefPubMedGoogle Scholar
  49. Whiting PJ, Lindstrom J (1988) Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J Neurosci 8:3395–3404PubMedGoogle Scholar
  50. Wonnacott S (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol Sci 11:216–219CrossRefPubMedGoogle Scholar
  51. Xu W, Orr-Urtreger A, Nigro F, Gelber S, Sutcliffe CB, Armstrong D, Patrick JW, Role LW, Beaudet AL, De Biasi M (1999) Multiorgan autonomic dysfunction in mice lacking the β2 and the β4 subunits of neuronal nicotinic acetylcholine receptors. J Neurosci 19:9298–9305PubMedGoogle Scholar
  52. Zoli M, Lena C, Picciotto MR, Changeux JP (1998) Identification of four classes of brain nicotinic receptors using β2 mutant mice. J Neurosci 18:4461–4472PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Sarah E. McCallum
    • 1
    • 3
  • Allan C. Collins
    • 1
  • Richard Paylor
    • 2
  • Michael J. Marks
    • 1
  1. 1.Institute for Behavioral GeneticsUniversity of ColoradoBoulderUSA
  2. 2.Department of Molecular and Human GeneticsBaylor College of MedicineHoustonUSA
  3. 3.The Parkinson's InstituteSunnyvaleUSA

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