Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 379, Issue 4, pp 389–395 | Cite as

Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice

  • Gitte Jositsch
  • Tamara Papadakis
  • Rainer V. Haberberger
  • Miriam Wolff
  • Jürgen Wess
  • Wolfgang Kummer
Short Communication

Abstract

Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). In this paper, we assessed the specificity of muscarinic receptor (MR) antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and eight of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at four antibody dilutions at minimum, so that finally, at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross-sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabelled in the pre-absorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice even when the pre-absorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localisation of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localisation in tissue sections of peripheral organs is limited to the M2R subtype utilising the most commonly used methodological approaches.

Keywords

Acetylcholine Muscarinic receptor Immunohistochemistry Specificity Receptor knockout strains Mouse 

Notes

Acknowledgements

We thank Ms K. Michael for skilful help in preparing the figures. Thus study was supported by the DFG (Excellence Cluster Cardio-Pulmonary System and KO 1398/5-1).

References

  1. Bowerfind WM, Fryer AD, Jacoby DB (2002) Double-stranded RNA causes airway hyperreactivity and neuronal M2 muscarinic receptor dysfunction. J Appl Physiol 92:1417–1422PubMedGoogle Scholar
  2. Costello RW, Jacoby DB, Fryer AD (1998) Pulmonary neuronal M2 muscarinic receptor function in asthma and animal models of hyperreactivity. Thorax 53:613–616PubMedCrossRefGoogle Scholar
  3. Deutsch EW, Ball CA, Berman JJ, Bova GS, Brazma A, Bumgarner RE, Campbell D, Causton HC, Christiansen JH, Daian F, Dauga D, Davidson DR, Gimenez G, Goo YA, Grimmond S, Henrich T, Herrmann BG, Johnson MH, Korb M, Mills JC, Oudes AJ, Parkinson HE, Pascal LE, Pollet N, Quackenbush J, Ramialison M, Ringwald M, Salgado D, Sansone SA, Sherlock G, Stoeckert CJ Jr, Swedlow J, Taylor RC, Walashek L, Warford A, Wilkinson DG, Zhou Y, Zon LI, Liu AY, True LD (2008) Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). Nat Biotechnol 26:305–312PubMedCrossRefGoogle Scholar
  4. Fayon M, De La Roque D, Berger P, Begueret H, Ousova O, Molimard M, Marthan R (2005) Increased relaxation of immature airways to β2-adrenoceptor agonists is related to attenuated expression of postjunctional smooth muscle muscarinic M2 receptors. J Appl Physiol 98:1526–1533PubMedCrossRefGoogle Scholar
  5. Fisahn A, Yamada M, Duttaroy A, Gan JW, Deng CX, McBain CJ, Wess J (2002) Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents. Neuron 33:615–624PubMedCrossRefGoogle Scholar
  6. Forssmann WG, Pickel V, Reinecke M, Hock D, Metz J (1981) Immunohistochemistry and immunocytochemistry of nervous tissue. In: Heym CH, Forssmann WG (eds) Techniques in neuroanatomical research. Springer, New York, pp 171–205Google Scholar
  7. Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng H, Wess J (1999a) Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci U S A 96:1692–1697PubMedCrossRefGoogle Scholar
  8. Gomeza J, Zhang L, Kostenis E, Felder C, Brodkin J, Shannon H, Xia B, Deng C, Wess J (1999b) Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci U S A 96:1692–1697PubMedCrossRefGoogle Scholar
  9. Gosens R, Zaagsma J, Meurs H, Halayko AJ (2006) Muscarinic receptor signalling in the pathophysiology of asthma and COPD. Respir Res 7:73PubMedCrossRefGoogle Scholar
  10. Gross NJ, Skorodin MS (1984) Role of the parasympathetic system in airway obstruction due to emphysema. N Engl J Med 311:421–425PubMedGoogle Scholar
  11. Grube D (1980) Immunoreactivities of gastrin (G-) cells. II. Non-specific binding of immunoglobulins to G-cells by ionic interactions. Histochemistry 66:149–167PubMedCrossRefGoogle Scholar
  12. Haberberger R, Henrich M, Couraud JY, Kummer W (1999) Muscarinic M2-receptors in rat thoracic dorsal root ganglia. Neurosci Lett 266:177–180PubMedCrossRefGoogle Scholar
  13. Haberberger R, Scholz R, Kummer W, Kress M (2000) M2-receptor subtype does not mediate muscarine-induced increases in [Ca2+]i in nociceptive neurons of rat dorsal root ganglia. J Neurophysiol 84:1934–1941PubMedGoogle Scholar
  14. Levey AI, Edmunds SM, Hersch SM, Wiley RG, Heilman CJ (1995) Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat. J Comp Neurol 351:339–356PubMedCrossRefGoogle Scholar
  15. Lorincz A, Nusser Z (2008) Speceficity of immunoreactions: the importance of testing specificity in each method. J Neurosci 28:9083–9086PubMedCrossRefGoogle Scholar
  16. Mansfield KJ, Liu L, Mitchelson FJ, Moore KH, Millard RJ, Burcher E (2005) Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing. Br J Pharmacol 144:1089–1099PubMedCrossRefGoogle Scholar
  17. Michel MC, Barendrecht MM (2008) Physiological and pathological regulation of the autonomic control of urinary bladder contractility. Pharmacol Ther 117:297–312PubMedCrossRefGoogle Scholar
  18. Roffel AF, Elzinga CR, Van Amsterdam RG, De Zeeuw RA, Zaagsma J (1988) Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function. Eur J Pharmacol 153:73–82PubMedCrossRefGoogle Scholar
  19. Tong YC, Cheng JT, Hsu CT (2006) Alterations of M(2)-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder. Neurosci Lett 406:216–221PubMedCrossRefGoogle Scholar
  20. True LD (2008) Quality control in molecular immunohistochemistry. Histochem Cell Biol 130:473–480PubMedCrossRefGoogle Scholar
  21. Tyagi S, Tyagi P, Van le S, Yoshimura N, Chancellor MB, de Miguel F (2006) Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor. J Urol 176:1673–1678PubMedCrossRefGoogle Scholar
  22. Watanabe M, Fukaya M, Sakimura K, Manabe T, Mishina M, Inoue Y (1998) Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur J Neurosci 10:478–487PubMedCrossRefGoogle Scholar
  23. Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Deng CX, Wess J (2001a) Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature 410:207–212PubMedCrossRefGoogle Scholar
  24. Yamada M, Lamping KG, Duttaroy A, Zhang W, Cui Y, Bymaster FP, McKinzie DL, Felder CC, Deng CX, Faraci FM, Wess J (2001b) Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci U S A 98:14096–14101PubMedCrossRefGoogle Scholar
  25. Zarghooni S, Wunsch J, Bodenbenner M, Bruggmann D, Grando SA, Schwantes U, Wess J, Kummer W, Lips KS (2007) Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium. Life Sci 80:2308–2313PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Gitte Jositsch
    • 1
  • Tamara Papadakis
    • 1
  • Rainer V. Haberberger
    • 2
  • Miriam Wolff
    • 1
  • Jürgen Wess
    • 3
  • Wolfgang Kummer
    • 1
  1. 1.Institute of Anatomy and Cell BiologyJustus-Liebig-University GiessenGiessenGermany
  2. 2.Department of Anatomy & HistologyFlinders University of South AustraliaAdelaideAustralia
  3. 3.Laboratory of Bioorganic ChemistryNational Institute of Diabetes and Digestive and Kidney DiseasesBethesdaUSA

Personalised recommendations