Abstract
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.
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References
Abercrombie M, Johnson ML (1946) Quantitative histology of Wallerian degeneration; nuclear population in rabbit sciatic nerve. J Anat 80:37–50
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 65:101–112
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 33:35–51
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 5:889–899
De Olmos J, Heimer L (1977) Mapping of collateral projections with the HRP-method. Neurosci Lett 6:107–114
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:e34435
Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083
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 4:369–374
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 518:3149–3168
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 42:1–10
Johnson AC, Mc NA, Rossiter RJ (1950) Chemistry of Wallerian degeneration; a review of recent studies. Arch Neurol Psychiatry 64:105–121
Kobbert C, Apps R, Bechmann I, Lanciego JL, Mey J, Thanos S (2000) Current concepts in neuroanatomical tracing. Prog Neurobiol 62:327–351
Kotowicz Z (2005) Gottlieb Burckhardt and Egas Moniz—two beginnings of psychosurgery. Gesnerus 62:77–101
Kristensson K, Olsson Y (1971) Retrograde axonal transport of protein. Brain Res 29:363–365
LaVail JH, LaVail MM (1972) Retrograde axonal transport in the central nervous system. Science 176:1416–1417
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 14:693–702
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 36:327–334
Liu Y, Broman J, Edvinsson L (2004) Central projections of sensory innervation of the rat superior sagittal sinus. Neuroscience 129:431–437
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 7:222–234
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 23:3269–3283
Luo L, Callaway EM, Svoboda K (2008) Genetic dissection of neural circuits. Neuron 57:634–660
Martin X, Dolivo M (1983) Neuronal and transneuronal tracing in the trigeminal system of the rat using the herpes virus suis. Brain Res 273:253–276
Mesulam MM (1976) The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility. J Histochem Cytochem 24:1273–1280
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 26:106–117
Nauta WJ, Gygax PA (1954) Silver impregnation of degenerating axons in the central nervous system: a modified technic. Stain Technol 29:91–93
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 512:595–612
Rondorf-Klym LM, Colling J (2003) Quality of life after radical prostatectomy. Oncol Nurs Forum 30:E24–E32
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 476:21–28
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 61:127–138
Schoene-Bake JC, Parpaley Y, Weber B, Panksepp J, Hurwitz TA, Coenen VA (2010) Tractographic analysis of historical lesion surgery for depression. Neuropsychopharmacology 35:2553–2563
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 31:977–981
Thomas GA (1948) Quantitative histology of Wallerian degeneration; nuclear population in two nerves of different fibre spectrum. J Anat 82:135–145
van Strien NM, Cappaert NL, Witter MP (2009) The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network. Nat Rev Neurosci 10:272–282
Weiss P, Hiscoe HB (1948) Experiments on the mechanism of nerve growth. J Exp Zool 107:315–395
Wickersham IR, Finke S, Conzelmann KK, Callaway EM (2007) Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 4:47–49
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 39:55–64
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 39:301–310
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 32:641–646
Zhang J, Pendlebury WW, Luo P (2003) Synaptic organization of monosynaptic connections from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat. Synapse 49:157–169
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 135:497–505
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 1489:37–47
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Zhang, J., Xiong, H. (2014). Neuronal Tract Tracing with Light and Electron Microscopy. In: Xiong, H., Gendelman, H.E. (eds) Current Laboratory Methods in Neuroscience Research. Springer Protocols Handbooks. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-8794-4_3
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DOI: https://doi.org/10.1007/978-1-4614-8794-4_3
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