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A Survey of Current Neuroanatomical Tracing Techniques

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Neural Tracing Methods

Part of the book series: Neuromethods ((NM,volume 92))

Abstract

This chapter provides a systematic description of neuroanatomical tracing methods, with a brush of history. Tracing can be based on uptake and transport of tracer in living neurons but can also be based on physical diffusion in living neurons after intracellular injection of tracer or, in fixed tissue as is the case of Golgi silver staining, based on complex anorganic chemical reactions. Because of the special fixation status of human brain tissue, the physicochemical methods are prominent with this kind of nervous tissue. Nowadays, the transport methods enjoy popularity in animal connectivity models because they produce fast and decisive results in terms of specific connectivity of functional systems. Transport-based tracing methods are best suited to visualize long-axon projections. For the study of short projection axons and interneurons, more sophisticated methods need to be applied such as pericellular injection or intracellular filling with dye after neurophysiological recording in living slice preparations or in vivo.

The chapter discusses the current neuroanatomical tracing methods and its equipment and procedures, starting with the “mother of retrograde tracing methods”: the technique of injection, uptake, and transport of the enzyme horseradish peroxidase. It continues with fluorescent dye tracing. The fluorescent compounds have two advantages: they are extremely stable and they can easily be combined with immunofluorescence to determine the neurochemical identity of the labeled neurons.

The most commonly used anterograde tracers are Phaseolus vulgaris-Leucoagglutinin (PHA-L), which requires immunohistochemical detection, and biotinylated dextran amine (BDA), which is detected via reaction with streptavidin conjugated to a reporter molecule. Single and multiple fluorescence methods receive much attention because they so perfectly combine with modern laser scanning microscopes and digital image processing.

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Notes

  1. 1.

    Diaminobenzidine and benzidine derivates such as BDHC are considered carcinogenic and should be treated with care, that is, in a dedicated container in a fume hood. Gloves should be worn at all times to avoid contact with the skin. All material in contact with DAB and BDHC should be decontaminated with chlorine after the experiment, and care must be taken not to contaminate the microscope used to monitor at intervals the progress of the histochemical reaction.

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Acknowledgments

It is a pleasure to acknowledge the continuous support provided by technicians who skillfully assisted us: Barbara Jorritsma-Byham, Annaatje Pattiselanno, Peter Goede, Amber Boekel, John Bol, Eveline Timmermans, Luciënne Baks-te Bulte, Yvon Galis, and Angela Engel. Their professional attitude, skill, and enthusiasm made the day. I am also indebted to many colleagues who at some point gave advice and contributed ideas and improvements, to name a few, Jochen Staiger, José Lanciego, Riichi Kajiwara, and Jean-Luc Boulland. It should be mentioned that a score of graduate students assisted in various projects. Finally, computer and other digital assistance by Nico Blijleven and Jeroen Beliën were of vital support.

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  1. Department of Anatomy and Neurosciences, University Medical Center, Vrije University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

    Floris G. Wouterlood

  2. Department of Anatomy and Neurosciences, University Medical Center, Vrije University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands

    Floris G. Wouterlood

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Correspondence to Floris G. Wouterlood .

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  1. Baylor College of Medicine, Houston, Texas, USA

    Benjamin R. Arenkiel

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Wouterlood, F.G. (2015). A Survey of Current Neuroanatomical Tracing Techniques. In: Arenkiel, B. (eds) Neural Tracing Methods. Neuromethods, vol 92. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1963-5_1

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