Techniques for the Localization of Parasympathetic Nerves to Smooth Muscle

  • F. E. Bloom
  • G. R. Siggins


In this chapter we shall attempt to describe the most useful of the currently available cytochemical techniques for the localization of parasympathetic axons. Our emphasis is placed upon electron microscopic methods or upon those light microscopic approaches that can be usefully combined with electrophysiologic and pharmacologic analysis. The ideal method—as yet unavailable—would be one which would demonstrate, with high selectivity, small amounts of acetylcholine (or other putative parasympathetic neurotransmitters) in situ in nerve terminals. However, few methods used in electron microscopy can selectively demonstrate molecules as small as acetylcholine. At best, the present methods may permit us some insight as to where the parasympathetic nerves to a given smooth muscle system are located, rather than from what region within the nerve terminal the neurotransmitter has been released from its storage vesicle.


Methylene Blue Parasympathetic Nerve Cholinergic Nerve Granular Vesicle Electron Microscopic Localization 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akert, K. and Sandri, S. 1968. An electron microscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. Brain Res., 7:286–295.PubMedCrossRefGoogle Scholar
  2. Bell, C. and McLean, J. R. 1967. Localization of norepinephrine and acetylcholinesterase in separate neurons supplying the guinea pig vas deferens. J. Pharmacol. Exp. Ther., 757:69–73.Google Scholar
  3. Berman, H. J. and Siggins, G. R. 1968. Neurogenic factors in the microvascular system. Fed. Proc., 27:1384–1390.PubMedGoogle Scholar
  4. Bloom, F. E. 1972. Localization of neurotransmitters by electron microscopy. Proc. Assoc. Res. Nerv. Ment.Dis., 50:25–51.Google Scholar
  5. Bloom, F. E. and Barrnett, R. J. 1967. The fine structural localization of cholinesterases in nervous tissues. Am. N.Y. Acad. Sci., 144:626–645.CrossRefGoogle Scholar
  6. Bondareff, W. 1966. Localization of alpha-methyl norepinephrine in sympathetic nerve fibers of the pineal body. Exp. Neurol., 76:131–135.CrossRefGoogle Scholar
  7. Burn, H. J. 1971. The Autonomic Nervous System. Blackwell Scientific Publications, Oxford and Edinburgh.Google Scholar
  8. Burnstock, G. and Robinson, P. M. 1967. LocaHzation of catecholamines and acetylcholinesterase in autonomic nerves. Circ. Res., 21 (Suppl:111):3-A3.Google Scholar
  9. Burt, A. M. 1970. A histochemical procedure for the localization of choline acetyltransferase activity. J. Histochem. Cytochem., 75:408–415.CrossRefGoogle Scholar
  10. Davis, D. A., Wasserkrug, H. L., Heyman, L A., Padmanabhan, K. C., Seligman, G. A., Plapinger, R. E., and Seligman, A. M. 1972. Comparisons of ultrastructural Cholinesterase demonstration in the motor end plate with alpha acetylthiol-m-toluenediazonium ion and 3-acetoxy-5-indolediazoniumion. J. Histochem. Cytochem., 20: 161–172.PubMedCrossRefGoogle Scholar
  11. Ehinger, G., Sporrong, B., and Stenevi, U. 1967. Combining the catecholamine fluorescence and methylene blue staining methods for demonstrating nerve fibers. Life Sci., 6:1973–1974.PubMedCrossRefGoogle Scholar
  12. El-Bedawi, A. and Schenk, E. A. 1967. Histochemical method for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections. J. Histochem. Cytochem., 75:580–588.CrossRefGoogle Scholar
  13. Eränkö, O. and Eränkö, L. 1971. Loss of histochemically demonstrable catecholamines and acetyl¬cholinesterase from sympathetic nerve fibers of the pineal body of the rat after chemical sympathectomy with 6-hydroxydopamine. Histochem. J., 3: 357–363.PubMedCrossRefGoogle Scholar
  14. Eränkö, O., Koelle, G. B., and Raiasanen, L. 1967. A thiocholine-lead ferrocyanide method for acetyl¬cholinesterase. J. Histochem. Cytochem., 75:674–679.Google Scholar
  15. Eränkö, O., Rechardt, L., Eränkö, L., and Cunningham, A. 1970. Light and electron microscopic histo- chemical observations on Cholinesterase containing sympathetic fibers in the pineal body of the rat. Histochem. J., 2:479–489.PubMedCrossRefGoogle Scholar
  16. Esterhuizen, A. C., Graham, J. D. P., Lever, F. D., and Spriggs, T.L.B. 1968a. Catecholamine and acetyl-cholinesterase distribution in relation to noradrenaline release. An enzyme histochemical and auto-radiographic study on the innervation of the cat nictitating muscle. Br. J. Pharmacol., 32:46–56.Google Scholar
  17. Esterhuizen, A., Spriggs, T. L. B., and Lever, J. D. 1968b. Nature. of islet-cell innervation in the cat pancreas. Diabetes, 17:33–36.PubMedGoogle Scholar
  18. Fillenz, M. 1970. Innervation of pulmonary and bronchial blood vessels of the dog. J. Anat., 106:449–461.PubMedGoogle Scholar
  19. Friesen, A. J. D., Kemp, J. W., and Woodbury, D. W. 1964. Identification of acetylcholine in sympathetic ganglia by chemical and physical methods. Science, 145:157–159.PubMedCrossRefGoogle Scholar
  20. Graf, J. 1967. Elektronmikdroskopischer nachweis der acetylcholinesterase in den postganglionären sympathetischen nerve des herzens. J. Neurochem., 14:893–897.PubMedCrossRefGoogle Scholar
  21. Graham, J. D. P., Lever, J. D., and Spriggs, T. L. B. 1968. An examination of adrenergic axons around pancreatic arterioles of the cat for the presence of acetylcholinesterase by high resolution autoradio¬graphic and histochemical methods. Br. J. Pharmacol. Chemother., 15–20.Google Scholar
  22. Hall, Z. 1971. Analysis of synaptic activity in relation to transmitter substances. In: Structure and Function of Synapses. Ed. by Pappas. Appleton-Century-Crofts, New York.Google Scholar
  23. Hanker, J. S., Anderson, W. A., and Bloom, F. E. 1972. Osmiophilic polymer generation: Catalysis by transition metal compounds in ultrastructural cytochemistry. Science, 775:991–993.CrossRefGoogle Scholar
  24. Higgins, J. A. and Barrnett, R. J. 1971. Fine structural localization of acetyl transferases. J. Cell Biol., 50:102–120.PubMedCrossRefGoogle Scholar
  25. Hökfelt, T., 1967. In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch., 97:1–74.Google Scholar
  26. Iversen, L. L. 1971. Role of transmitter uptake mechanisms in synpatic neurotransmission. Br. J. Pharmacol., 91:571–591.CrossRefGoogle Scholar
  27. Karnovsky, M.J. 1964. The localization of Cholinesterase activity in rat cardiac muscle by electron micro¬scopy. J. Cell Biol.,23:217–232.PubMedCrossRefGoogle Scholar
  28. Karnovsky, M. J. and Roots, L. 1964. A direct-coloring thiolcholine technique for cholinesterases. J. Histochem. Cytochem., 72:219–221.CrossRefGoogle Scholar
  29. Koelle, G. B. 1963. Cytological distribution and physiological functions of Cholinesterase. In: Handbuch der experimentellen Pharmakologie, Suppl. 15, pp. 287–397. Ed. by Koelle, G. B. Springer-Verlag, Berlin.Google Scholar
  30. Koelle, G. B. 1969. Concluding remarks. In: Autoradiography of Diffusible Substances, pp. 363–365. Ed. by Roth, L. J. and Stumpf, W. E. Academic Press, New York.Google Scholar
  31. Koelle, G. B. and Horn, R. S. 1968. Acetyldisulfide, a major active component in the thiolacetic acid histochemical method for acetylcholinesterase. J. Histochem. Cytochem., 76:743–753.CrossRefGoogle Scholar
  32. Mednick, M. L., Petrall, J. R., Thomas, N. C., Sternberger, L. A., Plapinger, R. E., Divis, D. A., Wasser- krug, H. L., and Seligman, A. M. 1971. Localization of acetylcholinesterase via production of osmiophilic polymers: New benzenediazonium salts with thiolacetate functions. J. Histochem. Cytochem., 79:155–160.CrossRefGoogle Scholar
  33. Nachmansohn, D. 1963. Actions on axons, and evidence for the role of acetylcholine in axonal conduction. In: Handbuch der experimentellen Pharmakologie, Vol. 15, pp. 701–740. Ed. by Koelle, G.B. Springer-Verlag, Berlin.Google Scholar
  34. Parducz, A., Halasz, N., and Joo, F. 1971. Lack of correlation between the zinc iodide-osmium positivity of cholinergic terminals and the cholinergic transmission in the sympathetic ganglia of the cat. J. Neurochem., 70:97–100.CrossRefGoogle Scholar
  35. Richardson, K. C. 1964. The fine structure of the albino rabbit iris with special reference to the identification of adrenergic and cholinergic nerves and nerve endings in its intrinsic muscles. Am. J. Anat., 114:113–206.CrossRefGoogle Scholar
  36. Richardson, K. C. 1968. Cholinergic and adrenergic axons in methylene blue-stained rat iris: An electron- microscopical study. Life Sci., 7 (Part I):599–604.CrossRefGoogle Scholar
  37. Richardson, K. C. 1969. The fine structure of autonomic nerves after vital staining with methylene blue. Anat. Ree., 164:359–311.CrossRefGoogle Scholar
  38. Robinson, P. M. 1969. A cholinergic component in the innervation of the longitudinal smooth muscle of the guinea pig vas deferens. The fine structural localization of acetylcholinesterase. J. Cell Biol., 41:462–475.PubMedCrossRefGoogle Scholar
  39. Rogers, A. W., Darnzynkiewicz, Z., Salpeter, M. M. Ostrowski, K., and Barnard, E. A. 1969. Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor end plates, measured by radioautography. J. Cell Biol., 41:665–685.PubMedCrossRefGoogle Scholar
  40. Shute, C. C. D. and Lewis, P. R. 1966. Electron microscopy of cholinergic terminals and acetylinesterase- containing neurones in the hippocampal formation of the rat. Z. Zellforseh., 69:334–343.CrossRefGoogle Scholar
  41. Siggins, G. R. and Bloom, F. E. 1970. Cytochemical and physiologic effects of 6-hydroxydopamine on periarteriolar nerves of frogs. Circ. Res., 27:23–38.PubMedCrossRefGoogle Scholar
  42. Siggins, G. R. and Weitsen, H. A. 1971. Cytochemical and physiological evidence for cholinergic vasodilation of amphibian arterioles and precapillary sphincters. I. Light microscopy. Mierovas. Res., 3:308–322.CrossRefGoogle Scholar
  43. Silver, A. 1967. Cholinesterases in CNS, Int. Rev. Neurobiol, 70:57–106.CrossRefGoogle Scholar
  44. Tranzer, J. P. and Thoenen, H. 1967. Electron microscopic localization of 5-hydroxydopamine (3,4,5- trihydroxyphenyl-ethylamine) a new “false” sympathetic transmitter. Experientia, 23:743–745.PubMedCrossRefGoogle Scholar
  45. Waterson, J. G., Hume, W. R., and de la Lande, L S. 1970. The distribution of Cholinesterase in the rabbit ear artery. J. Histochem. Cytochem., 18:211–216.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1975

Authors and Affiliations

  • F. E. Bloom
    • 1
  • G. R. Siggins
    • 1
  1. 1.Laboratory of Neuropharmacology Division of Special Mental Health ResearchNational Institute of Mental Health Saint Elizabeth’s HospitalUSA

Personalised recommendations