Neuronal Tract Tracing with Light and Electron Microscopy

  • Jingdong Zhang
  • Huangui XiongEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Neuronal tract tracing technique was developed based on two principles, Wallerian degeneration and axon flow theory, and has been enhanced by knocking in an enhancer or inhibitor to a chain of neurons in studying specific pathways with molecular biological functionality. The tract tracing, including anterograde, retrograde, and transganglion tract tracing, can be achieved through axon transportation of tracers. Based on the visibility of the tracers, they are classified as fluorescent and nonfluorescent tracers. As this chapter focuses on the methods that benefit both light microscopy (LM) and electron microscopy (EM) studies, the protocols of fluorescent dye tract tracing are not discussed. Among the nonfluorescent tracers, horseradish peroxidase (HRP) is a marvelous tracer that can be used in all anterograde, retrograde, and transganglion tract tracing; therefore, the protocol of histochemical visualization of HRP is presented herein. Cholera toxin subunit B (CTB) is also well suited for both anterograde and retrograde labeling. Biotinylated dextran amine (BDA), a predominant anterograde tracer, is broadly used in combination with other tracers for identification of pathway connections or convergent innervations. Phaseolus vulgaris Leucoagglutinin (PHA-L) is also a super anterograde tracer that can be applied together with BDA to study convergent projections or combined with any retrograde tracer to explore recipient of the convergent innervations. Fluoro-gold (FG) is a retrograde tracer that can be viewed directly with conventional fluorescent LM or by using anti-FG antibody to modify it and to make any combination as well. The protocols for double labeling either of HRP and BDA, BDA and CTB, or BDA and PHA-L, as well as different combinations of triple labeling with immunostaining of neuroactive substances are also presented. These protocols also summarize methods of histochemical and fluorescent illumination, as well as tissue processing, embedding, and immunostaining methods for EM studies. Two major double labeling combinations used in EM observation are delineated: TMB-ST (Tetramethyl benzidine–sodium tungsten) visualization of HRP combined with ABC (Avidin-biotin-complex) histochemical stain and ABC histochemical stain combined with silver-gold pre-embedding method. Each protocol is supplemented with images to illustrate the expected results by following the related protocols.


Axonal flew-based neuronal tracer Anterograde, retrograde, and transganglion tract tracing Histochemical visualization of tracer labeling Double or triple labeling Immuno-electron microscopy 


  1. Abercrombie M, Johnson ML (1946) Quantitative histology of Wallerian degeneration; nuclear population in rabbit sciatic nerve. J Anat 80:37–50PubMedCentralGoogle Scholar
  2. Angelucci A, Clasca F, Sur M (1996) Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains. J Neurosci Methods 65:101–112CrossRefPubMedGoogle Scholar
  3. Beier KT, Borghuis BG, El-Danaf RN, Huberman AD, Demb JB, Cepko CL (2013) Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry. J Neurosci 33:35–51CrossRefPubMedPubMedCentralGoogle Scholar
  4. De Mey J, Moeremans M, Geuens G, Nuydens R, De Brabander M (1981) High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol Int Rep 5:889–899CrossRefPubMedGoogle Scholar
  5. De Olmos J, Heimer L (1977) Mapping of collateral projections with the HRP-method. Neurosci Lett 6:107–114CrossRefPubMedGoogle Scholar
  6. Dong YL, Wang W, Li H, Li ZH, Zhang FX, Zhang T, Lu YC, Li JL, Wu SX, Li YQ (2012) Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes. PLoS One 7:e34435CrossRefPubMedPubMedCentralGoogle Scholar
  7. Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083CrossRefPubMedGoogle Scholar
  8. Fink RP, Heimer L (1967) Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res 4:369–374CrossRefPubMedGoogle Scholar
  9. Ge SN, Ma YF, Hioki H, Wei YY, Kaneko T, Mizuno N, Gao GD, Li JL (2010) Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat. J Comp Neurol 518:3149–3168CrossRefPubMedGoogle Scholar
  10. Gu Y, Chen Y, Ye L (1992) Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue. J Neurosci Methods 42:1–10CrossRefPubMedGoogle Scholar
  11. Johnson AC, Mc NA, Rossiter RJ (1950) Chemistry of Wallerian degeneration; a review of recent studies. Arch Neurol Psychiatry 64:105–121CrossRefPubMedGoogle Scholar
  12. Kobbert C, Apps R, Bechmann I, Lanciego JL, Mey J, Thanos S (2000) Current concepts in neuroanatomical tracing. Prog Neurobiol 62:327–351CrossRefPubMedGoogle Scholar
  13. Kotowicz Z (2005) Gottlieb Burckhardt and Egas Moniz—two beginnings of psychosurgery. Gesnerus 62:77–101PubMedGoogle Scholar
  14. Kristensson K, Olsson Y (1971) Retrograde axonal transport of protein. Brain Res 29:363–365CrossRefPubMedGoogle Scholar
  15. LaVail JH, LaVail MM (1972) Retrograde axonal transport in the central nervous system. Science 176:1416–1417CrossRefPubMedGoogle Scholar
  16. LaVail MM, Sidman M, Rausin R, Sidman RL (1974) Discrimination of light intensity by rats with inherited retinal degeneration: a behavioral and cytological study. Vision Res 14:693–702CrossRefPubMedGoogle Scholar
  17. Li JL, Wang D, Kaneko T, Shigemoto R, Nomura S, Mizuno N (2000) The relationship between neurokinin-1 receptor and substance P in the medullary dorsal horn: a light and electron microscopic immunohistochemical study in the rat. Neurosci Res 36:327–334CrossRefPubMedGoogle Scholar
  18. Liu Y, Broman J, Edvinsson L (2004) Central projections of sensory innervation of the rat superior sagittal sinus. Neuroscience 129:431–437CrossRefPubMedGoogle Scholar
  19. Luo P, Haines A, Dessem D (2001) Elucidation of neuronal circuitry: protocol(s) combining intracellular labeling, neuroanatomical tracing and immunocytochemical methodologies. Brain Res Brain Res Protoc 7:222–234CrossRefPubMedGoogle Scholar
  20. Luo P, Zhang J, Yang R, Pendlebury W (2006) Neuronal circuitry and synaptic organization of trigeminal proprioceptive afferents mediating tongue movement and jaw-tongue coordination via hypoglossal premotor neurons. Eur J Neurosci 23:3269–3283CrossRefPubMedGoogle Scholar
  21. Luo L, Callaway EM, Svoboda K (2008) Genetic dissection of neural circuits. Neuron 57:634–660CrossRefPubMedPubMedCentralGoogle Scholar
  22. Martin X, Dolivo M (1983) Neuronal and transneuronal tracing in the trigeminal system of the rat using the herpes virus suis. Brain Res 273:253–276CrossRefPubMedGoogle Scholar
  23. Mesulam MM (1976) The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility. J Histochem Cytochem 24:1273–1280CrossRefPubMedGoogle Scholar
  24. Mesulam MM (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26:106–117CrossRefPubMedGoogle Scholar
  25. Nauta WJ, Gygax PA (1954) Silver impregnation of degenerating axons in the central nervous system: a modified technic. Stain Technol 29:91–93CrossRefPubMedGoogle Scholar
  26. Pang YW, Ge SN, Nakamura KC, Li JL, Xiong KH, Kaneko T, Mizuno N (2009) Axon terminals expressing vesicular glutamate transporter VGLUT1 or VGLUT2 within the trigeminal motor nucleus of the rat: origins and distribution patterns. J Comp Neurol 512:595–612CrossRefPubMedGoogle Scholar
  27. Rondorf-Klym LM, Colling J (2003) Quality of life after radical prostatectomy. Oncol Nurs Forum 30:E24–E32CrossRefPubMedGoogle Scholar
  28. Rouiller EM, Capt M, Dolivo M, De Ribaupierre F (1989) Neuronal organization of the stapedius reflex pathways in the rat: a retrograde HRP and viral transneuronal tracing study. Brain Res 476:21–28CrossRefPubMedGoogle Scholar
  29. Ruigrok TJ, Teune TM, van der Burg J, Sabel-Goedknegt H (1995) A retrograde double-labeling technique for light microscopy. A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate. J Neurosci Methods 61:127–138CrossRefPubMedGoogle Scholar
  30. Schoene-Bake JC, Parpaley Y, Weber B, Panksepp J, Hurwitz TA, Coenen VA (2010) Tractographic analysis of historical lesion surgery for depression. Neuropsychopharmacology 35:2553–2563CrossRefPubMedPubMedCentralGoogle Scholar
  31. Tapia FJ, Varndell IM, Probert L, De Mey J, Polak JM (1983) Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides. J Histochem Cytochem 31:977–981CrossRefPubMedGoogle Scholar
  32. Thomas GA (1948) Quantitative histology of Wallerian degeneration; nuclear population in two nerves of different fibre spectrum. J Anat 82:135–145PubMedCentralGoogle Scholar
  33. van Strien NM, Cappaert NL, Witter MP (2009) The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network. Nat Rev Neurosci 10:272–282CrossRefPubMedGoogle Scholar
  34. Weiss P, Hiscoe HB (1948) Experiments on the mechanism of nerve growth. J Exp Zool 107:315–395CrossRefPubMedGoogle Scholar
  35. Wickersham IR, Finke S, Conzelmann KK, Callaway EM (2007) Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 4:47–49CrossRefPubMedGoogle Scholar
  36. Zhang JD (1998) Projections from dorsomedial part of the subnucleus oralis to the mesencephalic trigeminal neurons innervating the masseter muscle—a PHA-L and HRP double labeling study in the rat. J Hirnforsch 39:55–64PubMedGoogle Scholar
  37. Zhang JD, Yang XL (1999) Projections from subnucleus oralis of the spinal trigeminal nucleus to contralateral thalamus via the relay of juxtatrigeminal nucleus and dorsomedial part of the principal sensory trigeminal nucleus in the rat. J Hirnforsch 39:301–310PubMedGoogle Scholar
  38. Zhang JD, Wang BR, Li HM, Li JS (1991) Projections from neurons innervating the masseter muscle to the subnucleus oralis of the spinal trigeminal nucleus and adjacent lateral reticular formation in the rat. J Hirnforsch 32:641–646PubMedGoogle Scholar
  39. Zhang J, Pendlebury WW, Luo P (2003) Synaptic organization of monosynaptic connections from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat. Synapse 49:157–169CrossRefPubMedGoogle Scholar
  40. Zhang J, Yang R, Pendlebery W, Luo P (2005) Monosynaptic circuitry of trigeminal proprioceptive afferents coordinating jaw movement with visceral and laryngeal activities in rats. Neuroscience 135:497–505CrossRefPubMedGoogle Scholar
  41. Zhang J, Luo P, Ro JY, Xiong H (2012) Jaw muscle spindle afferents coordinate multiple orofacial motoneurons via common premotor neurons in rats: an electrophysiological and anatomical study. Brain Res 1489:37–47CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Pharmacology and Experimental NeuroscienceUniversity of Nebraska Medical Center, Durham Research CenterOmahaUSA

Personalised recommendations