Multiple axonal tracing: simultaneous detection of three tracers in the same section

Abstract Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector®VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.

[1]  H. Groenewegen,et al.  The Phaseolus vulgaris-leucoagglutinin tracing technique for the study of neuronal connections. , 1991, Progress in histochemistry and cytochemistry.

[2]  Mian Zhou,et al.  The use of peroxidase substrate Vector® VIP in electron microscopic single and double antigen localization , 1995, Journal of Neuroscience Methods.

[3]  J. Zimmer,et al.  Biocytin pellets: an alternative technique for massive anterograde labeling of neuronal pathways in vivo and in vitro , 1993, Brain Research.

[4]  J. Sjöstrand,et al.  Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve. , 1971, Brain research.

[5]  A. M. Wright,et al.  Immunolabeling of retrogradely transported Fluoro-Gold: sensitivity and application to ultrastructural analysis of transmitter-specific mesolimbic circuitry , 1994, Journal of Neuroscience Methods.

[6]  H. T. Chang,et al.  Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold , 1990, Journal of Neuroscience Methods.

[7]  B. Stein,et al.  The corticostriatal and corticotectal projections of the feline lateral suprasylvian cortex demonstrated with anterograde biocytin and retrograde fluorescent techniques , 1991, Neuroscience Research.

[8]  A. Reiner,et al.  Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies , 1992, Journal of Neuroscience Methods.

[9]  H. Nauta.  Tracing neural connections with horseradish peroxidase M. M. Mesulam (Ed.). John Wiley, Chichester (1982). 280 pp., Cloth, $52.00/£22.00; paper, $26.00/£11.00 , 1982, Neuroscience.

[10]  A. Vania Apkarian,et al.  Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey , 1992, Journal of Neuroscience Methods.

[11]  V. Casagrande,et al.  Morphological details of primate axons and dendrites revealed by extracellular injection of biocytin: an economic and reliable alternative to PHA-L , 1991, Brain Research.

[12]  M. King,et al.  Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative , 1989, Brain Research.

[13]  D. Schiffer,et al.  1-Naphthol basic dye (1-NBD). An alternative to diaminobenzidine (DAB) in immunoperoxidase techniques. , 1985, Histochemistry.

[14]  Y. Smith,et al.  Combined approaches to experimental neuroanatomy : combined tracing and immunocytochemical techniques for the study of neuronal microcircui ts , 2022 .

[15]  N. Rajakumar,et al.  Biotinylated dextran: a versatile anterograde and retrograde neuronal tracer , 1993, Brain Research.

[16]  Floris G Wouterlood,et al.  Use of peroxidase substrate Vector VIP® for multiple staining in light microscopy , 1997, Journal of Neuroscience Methods.

[17]  M. P. Witter,et al.  Multiple anterograde tracing, combining Phaseolus vulgaris leucoagglutinin with rhodamine- and biotin-conjugated dextran amine , 1994, Journal of Neuroscience Methods.

[18]  J. Lanciego,et al.  Multiple neuroanatomical tracing in primates. , 1998, Brain research. Brain research protocols.

[19]  Garrett E. Alexander.  Basal ganglia , 1998 .

[20]  L. Heimer,et al.  Iontophoretic injection of fluoro-gold and other fluorescent tracers. , 1990, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[21]  Stanley J. Watson,et al.  The rat brain in stereotaxic coordinates (2nd edn) by George Paxinos and Charles Watson, Academic Press, 1986. £40.00/$80.00 (264 pages) ISBN 012 547 6213 , 1987, Trends in Neurosciences.

[22]  P. Izzo.  A note on the use of biocytin in anterograde tracing studies in the central nervous system: Application at both light and electron microscopic level , 1991, Journal of Neuroscience Methods.

[23]  H. Groenewegen,et al.  Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents , 1984, Brain Research.

[24]  D. Rosene,et al.  A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact. , 1986, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[25]  Y. Smith,et al.  Convergence of synaptic inputs from the striatum and the globus pallidus onto identified nigrocollicular cells in the rat: A double anterograde labelling study , 1991, Neuroscience.