Abstract
Experimental neuroanatomical tracing techniques are fundamental to the study of the structure of the central nervous system. In the last few decades, many new methods for axonal tracing and cell labeling have been introduced. Neuroanatomical tracing applied as an isolated method produces relatively straightforward answers, for instance, whether there is connectivity from compartment Y in nucleus A to layer X in area B. However, questions that deal with the intrinsic complexity of brain circuits require the application of multiple-tracing paradigms in which two or even three different tracers are combined in single histological sections. With such paradigms we can handle questions like “are the fibers arriving in layer X of area B in contact with neurons that project to compartment Z in nucleus C,” “do these projection neurons receive as well innervation from area W,” and “what is the neurochemical signature of these connectivity-identified neurons?”We illustrate this approach with examples from our studies on pallidonigral connectivity in association with nigrostriatal efferent neurons.
Analysis of the data acquired via a multiple-tracing approach provides more insight into the organization of the brain than does the analysis of data from single tracing, especially when it comes to network circuitry. Furthermore, by virtue of the simultaneous visualization of projections in the same section, these multiple techniques enable the precise determination of the degree of convergence or divergence of particular projections to a particular terminal zone or to particular neurons (the latter to be identified via retrograde tracing or via neurotransmitter immunocytochemistry). An additional advantage of multitracer methods is that the experimental animals can be most efficiently used and the number of used animals reduced.
In this chapter we will discuss in detail several existing protocols for the simultaneous detection of three different tracers, as well as methods in which we combine two tracers and the immunocytochemical detection of a neuroactive substance. Emphasis will be placed on providing a step-by-step account of each procedure. We will be dealing with peroxidase substrates and precipitates with different colors since these precipitates are persistent without specific storage measures and because at the end of the staining procedure the ensuing slides can be studied any time under any routine microscope.
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References
Akintunde, A., and Buxton, D. F., 1992, Quadruple labeling of brainstem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization, J. Neurosci. Methods 45:15–22.
Anderson, C. R., and Edwards, S. L., 1993, Subunit b of cholera toxin labels interstitial cells of Cajal in the gut of rat and mouse, Histochemistry 100:457–464.
Anderson, K. D., Karle, E. J., and Reiner, A., 1994, A pre-embedding triple-label electron microscopic immunohistochemical method as applied to the study of multiple inputs to defined tegmental neurons, J. Histochem. Cytochem. 42:49–56.
Angelucci, A., Clascá, F., and Sur, M., 1996, Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing the fine axonal morphology in adult and neonatal brains, J. Neurosci. Methods 65:101–112.
Antal, M., Freund, T. F., Somogyi, P., and McIlhinney, R. A., 1990, Simultaneous anterograde labelling of two afferent pathways to the same target area with Phaseolus vulgaris leucoagglutinin and Phaseolus vulgaris leucoagglutinin conjugated to biotin or dinitrophenol, J. Chem. Neuroanat. 3:1–9.
Balercia, G., Cheng, S., and Bentivoglio, M., 1992, Electron microscopic analysis of fluorescent neuronal labeling after photoconversion, J. Neurosci. Methods 45:87–98.
Bentivoglio, M., and Su, H. S., 1990, Photoconversion of fluorescent retrograde tracers, Neurosci. Lett. 113:45–87.
Blackstad, T. W., 1965, Mapping of experimental axon degeneration by electron microscopy of Golgi preparations, Z. Zellforsch. 67:819–834.
Blackstad, T. W., 1981, Tract tracing by electron microscopy of Golgi preparations, In: Heimer, L., and RoBards, M. (eds.), Neuroanatomical Tract-Tracing Methods, New York: Plenum Press, pp. 407–440.
Brandt, H. M., and Apkarian, A. V., 1992, Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey, J. Neurosci. Methods 45:35–40.
Bruce, K., and Grofova, I., 1992, Notes on a light and electron microscopic double-labeling methods combining anterograde tracing with Phaseolus vulgaris leucoagglutinin and retrograde tracing with cholera toxin subunit ß, J. Neurosci. Methods 45:23–33.
Chang, H. T., Kuo, H., Whittaker, J. A., and Cooper, N. G. F., 1990, Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold, J. Neurosci. Methods 35:31–37.
Coolen L. M., Jansen H. T., Goodman R. L., Wood R. I., and Lehman M. N., 1999, A new method for simultaneous demonstration of anterograde and retrograde connections in the brain: co-injections of biotinylated dextran amine and the beta subunit of cholera toxin, J. Neurosci. Methods 91:1–8.
Coolen L. M., and Wood R. I., 1998, Reciprocal connections of the medial amygdaloid nucleus in the Syrian hamster brain: simultaneous anterograde and retrograde tract tracing, J. Comp. Neurol. 399:189–209.
Cullinan, W. E., and Zaborszky, L., 1991, Organization of ascending hypothalamic projections to the rostral forebrain with special reference to the innervation of cholinergic projection neurons, J. Comp. Neurol. 306:631–667.
de Olmos, J. S., Ebbesson, S. O. E., and Heimer, L., 1981, Silver methods for the impregnation of degenerating axoplasm, In: Heimer, L., and Robards, M. (eds.), Neuroanatomical Tract-Tracing Methods, New York: Plenum Press, pp. 117–170.
Divac, I., and Mogensen, J., 1990, Long-term retrograde labeling of neurons, Brain Res. 524:339–341.
Dolleman-Van der Weel, M. J., Wouterlood, F. G., and Witter, M. P., 1995, Multiple anterograde tracing, combining Phaseolus vulgaris leucoagglutinin with rhodamine-and biotinconjugated dextran amine, J. Neurosci. Methods 51:9–21.
Edwards, S. B., and Hendrickson, A., 1981, The autoradiographic tracing of axonal connections in the central nervous system, In: Heimer, L., and RoBards, M. (eds.), Neuroanatomical Tract-Tracing Methods, New York: Plenum Press, pp. 171–205.
Fritzsch, B., 1993, Fast axonal diffusion of 3000 molecular weight dextran amines, J. Neurosci. Methods 50:95–103.
Fritzsch, B., and Wilm, C., 1990, Dextran amines in ##neuronal tracing, Trends Neurosci. 13:14.
Gaykema, R. P., van Weeghel R., Hersh, L. B., and Luiten, P. G., 1991, Prefrontal cortical projections to the cholinergic neurons in the basal forebrain, J. Comp. Neurol. 303:563–583.
Gerfen, C. R., and Sawchenko, P. E., 1984, An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), Brain Res. 290:219–238.
Gerfen, C. R., and Sawchenko, P. E., 1985, A method for anterograde axonal tracing of chemically specified circuits in the central nervous system: combined Phaseolus vulgaris leucoagglutinin (PHA-L) tract tracing and immunohistochemistry, Brain Res. 343:144–150.
Gonatas, N. K., Harper, C., Mizutani, T., and Gonatas, J. O., 1979, Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem. 27:728–734.
Gonzalo, N., Moreno, A., Erdozain, M. A., García, P., Vázquez, A., Castle, M., and Lanciego, J. L., 2001, A sequential protocol combining dual neuroanatomical tract-tracing with the visualization of local circuit neurons within the striatum, J. Neurosci. Methods 111:59–66.
Graham, R. C., and Karnovsky, M. J., 1966, The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem. 14:291–302.
Groenewegen, H. J., and Wouterlood, F. G., 1990, Light and electron microscopic tracing of neuronal connections with Phaseolus vulgaris-leucoagglutinin (PHA-L) and combinations with other neuroanatomical techniques, In:Wouterlood, F. G., Van den Pol, A., Björklund, A., and Hökfelt, T. (eds.), Handbook of Chemical Neuroanatomy, Vol. 8, Amsterdam: Elsevier Science Publishers, pp. 47–124.
Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma, D. K., Pagano R. E., Hancock, J. F., Mayor, S., and Parton, R. G., 2005, Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles, J. Cell Biol. 168:465–476.
Köbbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., and Thanos, S., 2000, Current concepts in neuroanatomical tracing, Prog. Neurobiol. 62:327–351.
Kristensson, K., and Olson, Y., 1971, Retrograde axonal transport of protein, Brain Res. 29:363–365.
Kuypers, H. G. J. M., and Huisman, A. M., 1984, Fluorescent neuronal tracers, Adv. Cell. Neurobiol. 5:307–340.
Lanciego, J. L., and Giménez-Amaya, J. M., 1999, Notes on the combined use of V-VIP and DAB peroxidase substrates for the detection of colocalising antigens, Histochem. Cell Biol. 111:305–311.
Lanciego, J. L., Goede, P. H., Witter, M. P., and Wouterlood, F. G., 1997, Use of peroxidase substrate Vector ® VIP for multiple staining in light microscopy, J. Neurosci. Methods 74:1–7.
Lanciego, J. L., Gonzalo, N., Castle, M., Sanchez-Escobar, C., Aymerich, M. S., and Obeso, J. A., 2004, Thalamic innervation of striatal and subthalamic neurons projecting to the rat entopeduncular nucleus, Eur. J. Neurosci. 19:1267–1277.
Lanciego, J. L., Luquin, M. R., Guillén, J., and Giménez-Amaya, J. M., 1998b, Multiple neuroanatomical tracing in primates, Brain Res. Protoc. 2:323–332.
Lanciego, J. L., and Wouterlood, F. G., 1994, Dual anterograde axonal tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), Neurosci. Protoc. 94-050-06.
Lanciego, J. L., Wouterlood, F. G., Erro, E., Arribas, J., Gonzalo, N., Urra, X., Cervantes, S., and Giménez-Amaya, J. M., 2000, Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section, J. Neurosci. Methods 103:127–135.
Lanciego, J. L., Wouterlood, F. G., Erro, E., and Giménez-Amaya, J. M., 1998a, Multiple axonal tracing: simultaneous detection of three tracers in the same histological section, Histochem. Cell Biol. 110:509–515.
LaVail, J. H. 1975, The retrograde transport method, Fed. Proc. 34:1618–1624.
LaVail, J. H., and LaVail, M. M., 1972, Retrograde axonal transport in the central nervous system, Science 176:1416–1417.
Luppi, P. H., Fort, P., and Jouvet, M., 1990, Iontophoretic application of unconjugated cholera toxin ß subunit (CTB) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons, Brain. Res. 534:209–224.
Luppi, P. H., Sakai, K., Salvert, D., Fort, P., and Jouvet, M., 1987, Peptidergic hypothalamic afferents to the cat nucleus raphe pallidus as revealed by double immunostaining technique using unconjugated cholera toxin as a retrograde tracer, Brain Res. 402:339–345.
Mauro, A., Germano, I., Giaccone, G., Giordana, M. T., and Schiffer, D., 1985, 1-Naphthol basic dye (1-NBD), an alternative to diaminobenzidine (DAB) in immunoperoxidase techniques, Histochemistry 83:97–102.
Mesulam, M. M., 1976, The blue reaction product in horseradish peroxidase neurohistochemistry, J. Histochem. Cytochem. 24:1273–1280.
Mesulam, M. M., 1978, Tetramethylbenzidine 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.
Mesulam, M. M., 1982, Principles of horseradish peroxidase neurochemistry and their applications for tracing neural pathways-axonal transport, enzyme histochemistry and light microscopic analysis, In: Mesulam, M. M. (ed.), Tracing Neural Connections with Horseradish Peroxidase, New York: Wiley: IBRO Handbook Series: Methods in the Neurosciences, pp. 1–551.
Millhouse, O. E., 1981, The Golgi methods, In: Heimer, L., and RoBards, M. (eds.), Neuroanatomical Tract-Tracing Methods, New York: Plenum Press, pp. 311–344.
Nance, D. M., and Burns, J., 1990, Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls, Brain Res. Bull. 25:139–145.
Novikova, L., Novikov, L., and Kellerth, J.-O., 1997, Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates, J. Neurosci. Methods 74:9–15.
Pieribone, V. A., and Aston-Jones, G., 1988, The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei, Brain Res. 475:259–271.
Rajakumar, N., Elisevich, K., and Flumerfelt, B. A., 1993, Biotinylated dextran: a versatile anterograde and retrograde neuronal tracer, Brain Res. 607:47–53.
Reiner, A., Veenman, C. L., Medina, L., Jiao, Y., Del Mar, N., and Honig, M. G., 2000, Pathway tracing using biotinylated dextran amines, J. Neurosci. Methods 103:23–37.
Risold P. Y., and Swanson L. W., 1995, Evidence for a hypothalamothalamocortical circuit mediating pheromonal influences on eye and head movements, Proc. Natl. Acad. Sci. U. S. A. 92:3902–3989.
Rosene, D. L., Roy, N. J., and Davis, B. J., 1986, A cryoprotection method that facilitates cutting frozen sections of whole monkey brain for histological and histochemical processing without freezing artifact, J. Histochem. Cytochem. 34:1301–1316.
Schmued, L. C., 1994, Anterograde and retrograde neuroanatomical tract-tracing with fluorescent compounds, Neurosci. Protoc. 94-050-02.
Schmued, L. C., and Fallon, J. H., 1986, Fluoro-Gold: a new fluorescent tracer with numerous unique properties, Brain Res. 377:147–154.
Schmued, L. C., and Heimer, L., 1990, Iontophoretic injection of fluorogold and other fluorescent tracers, J. Histochem. Cytochem. 38:721–723.
Schmued, L. C., Kyriakidis, K., and Heimer, L., 1990, In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS, Brain Res. 526:127–134.
Skirboll, L. R., Thor, K., Helke, C., Hökfelt, T., Robertson, B., and Long, R., 1989, Use of retrograde fluorescent tracers in combination with immunohistochemical methods, In: Zaborszki, L., and Heimer, L., (eds.), Neuroanatomical Tract-Tracing Methods 2, New York: Plenum Press, pp. 5–18.
Smith, Y., and Bolam, J. P., 1991, Convergence of synaptic inputs from the striatum and the globus pallidus onto identified nigrocollicular cells in the rat: a double anterograde labeling study, Neuroscience 44:45–73.
Smith, Y., and Bolam, J. P., 1992, Combined approaches to experimental neuroanatomy: combined tracing and immunocytochemical techniques for the study of neuronal microcircuits, In: Bolam, J. P. (ed.), Experimental Neuroanatomy, A Practical Approach, Oxford: Oxford University Press, pp. 239–266.
Somogyi, P., Hodgson, A. J., and Smith, A. D., 1979, An approach to tracing neuron networks in the cerebral cortex and basal ganglia. Combination of Golgi staining, retrograde transport of horseradish peroxidase and anterograde degeneration of synaptic boutons in the same material, Neuroscience 4:1805–1852.
Stoeckel, K., Schwab, M. E., and Thoenen, H., 1977, Role of gangliosides in the uptake and retrograde transport of cholera and tetanus toxin as compared to nerve growth factor and wheat germ agglutinin, Brain Res. 132:273–285.
Ter Horst, G. J., Groenewegen, H. J., Karst, H., and Luiten, P. G. M., 1984, Phaseolus vulgaris leucoagglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents, Brain Res. 307:379–383.
Thomas M. A., and Lemmer B., 2005, HistoGreen: a new alternative to 3.3′-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry? Brain Res. Protoc. 14:107–118.
Trojanowski, J. Q., Gonatas, J. O., and Gonatas, N. K., 1981, Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free HRP as orthograde transported markers, Brain Res. 223:381–385.
Trojanowski, J. Q., Gonatas, J. O., Steiber, A., and Gonatas, N. K., 1982, Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrograde transported markers than free HRP, Brain Res. 231:33–50.
Trojanowski, J. Q., and Schmidt, M. L. 1984, Interneuronal transfer of axonally transported proteins: studies with HRP and HRP conjugates of wheat germ agglutinin, cholera toxin and the B subunit of cholera toxin, Brain Res. 311:366–369.
Van Bockstaele, E. J., Wright, A. M., Cesari, D. M., and Pickel, V., 1994, Immunolabeling of retrogradely transported fluorogold. Sensitivity and application of ultrastructural analysis of transmitter-specific mesolimbic circuitry, J. Neurosci. Methods 55:65–78.
Veenman, C. L., Reiner, A., and Honig, M. G., 1992, Biotinylated dextran amine as an anterograde tracer for single-and double-label studies, J. Neurosci. Methods 41:239–254.
Vetter, D. E., Saldaña, E., and Mugnaini, E., 1993, Input from the inferior colliculus to medial olivocochlear neurons in the rat: a double label study with PHA-L and cholera toxin, Hear. Res. 70:173–186.
Wan, X. C. S., Trojanowski, J. Q., and Gonatas, J. O., 1982, Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: their uptake and clearance, transganglionic and retrograde transport and sensitivity, Brain Res. 243:215–224.
Warr, W. B., de Olmos, J. S., and Heimer, L., 1981, Horseradish peroxidase: the basic procedure, In: Heimer, L., and RoBards, M. (eds.), Neuroanatomical Tract-Tracing Methods, New York: Plenum Press, pp. 207–262.
Weiss, P., and Hiscoe, H. B., 1948, Experiments on the mechanism of nerve growth, J. Exp. Zool. 107:315–396.
Wessendorf, M. W., 1991, Fluoro-Gold: composition and mechanism of uptake, Brain Res. 553:135–148.
Woolf, N. J., Hernit, M. C., and Butcher, L. L., 1986, Cholinergic and noncholinergic projections from the rat basal forebrain revealed by combined choline acetyltransferase and Phaseolus vulgaris-leucoagglutinin immunohistochemistry, Neurosci. Lett. 66:281–286.
Wouterlood, F. G., Bol, J. G. J. M., and Steinbusch, H. W. M., 1987, Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgarisleucoagglutinin and enzyme immunocytochemistry of target neurons, J. Histochem. Cytochem. 35:817–823.
Wouterlood, F. G., Goede, P. H., Arts, M. P. M., and Groenewegen, H. J., 1992, Simultaneous characterization of efferent and afferent connectivity, neuroactive substances and morphology of neurons, J. Histochem. Cytochem. 40:457–465.
Wouterlood, F. G., Goede, P. H., and Groenewegen, H. J., 1990, The in situ detectability of the neuroanatomical tracer Phaseolus vulgaris-leucoagglutinin, J. Chem. Neuroanat. 3:11–18.
Wouterlood, F. G., and Groenewegen, H. J., 1985, Neuroanatomical tracing by use of Phaseolus vulgaris-leucoagglutinin (PHA-L): electron microscopy of PHA-L filled neuronal somata, dendrites, axons and axon terminals, Brain Res. 326:188–191.
Wouterlood, F. G., and Groenewegen, H. J., 1991, The Phaseolus vulgaris-leucoagglutinin tracing technique for the study of neuronal connections, Prog. Histochem. Cytochem. 22:1–78.
Wouterlood, F. G., and Jorritsma-Byham, B., 1993, The anterograde neuroanatomical tracer biotinylated dextran amine: comparison with the tracer PHA-L in preparations for electron microscopy, J. Neurosci. Methods 48:75–87.
Wouterlood, F. G., Mugnaini, E., and Nederlof, J., 1985, Projection of olfactory bulb efferents to layer I GABA-ergic neurons in the entorhinal area. Combination of anterograde degeneration and immunoelectron microscopy in rat, Brain Res. 343:283–296.
Zaborszky, L., and Cullinan, W. E. 1989, Hypothalamic axons terminate on forebrain cholinergic neurons: an ultrastructural double-labeling study using PHA-L tracing and ChAT immunocytochemistry, Brain Res. 479:177–184.
Zaborszky, L., Leranth, C., and Heimer, L., 1984, Ultrastructural evidence of amygdalofugal axons terminating on cholinergic cells of the rostral forebrain, Neurosci. Lett. 21:219–225.
Zhou, M., and Grofova, I., 1995, The use of peroxidase substrate Vector VIP in electron microscopic single and double antigen localization, J. Neurosci. Methods 62:149–158.
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Lanciego, J.L., Wouterlood, F.G. (2006). Multiple Neuroanatomical Tract-Tracing: Approaches for Multiple Tract-Tracing. In: Zaborszky, L., Wouterlood, F.G., Lanciego, J.L. (eds) Neuroanatomical Tract-Tracing 3. Springer, Boston, MA . https://doi.org/10.1007/0-387-28942-9_11
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