A Survey of Current Neuroanatomical Tracing Techniques

  • Floris G. WouterloodEmail author
Part of the Neuromethods book series (NM, volume 92)


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.

Key words

Neuroanatomical tracing Anterograde transport Retrograde transport Fluorescence Combined methods 



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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© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Anatomy and Neurosciences, University Medical CenterVrije UniversityAmsterdamThe Netherlands
  2. 2.Department of Anatomy and Neurosciences, University Medical CenterVrije University Medical CenterAmsterdamThe Netherlands

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