Elsevier

Brain Research Bulletin

Volume 51, Issue 1, 1 January 2000, Pages 11-28
Brain Research Bulletin

Review Articles
Recent techniques for tracing pathways in the central nervous system of developing and adult mammals

https://doi.org/10.1016/S0361-9230(99)00229-4Get rights and content

Abstract

Over the last 20 years, the choice of neural tracers has increased manyfold, and includes newly introduced anterograde tracers that allow quantitation of single-axon morphologies, and retrograde tracers that can be combined with intracellular fills for the study of dendritic arbors of neurons which have a specific projection pattern. The combination of several different tracers now permits the comparison of multiple connections in the same animal, both quantitatively and qualitatively. Moreover, the finding of new virus strains, which infect neural cells without killing them, provides a tool for studying multisynaptic connections that participate in a circuit. In this paper, the labeling characteristics, mechanism of transport and advantages/disadvantages of use are discussed for the following recently introduced neural tracers: carbocyanine dyes, fluorescent latex microspheres, fluorescent dextrans, biocytin, dextran amines, Phaseolus vulgaris leucoagglutinin, cholera toxin and viruses. We also suggest the choice of specific tracers, depending on the experimental animal, age and type of connection to be studied, and discuss quantitative methodologies.

Introduction

Since the pioneering times of Golgi and Cajal, tracing neural connections has been a challenge in neuroanatomy, with profound implications for the study of neural function and of the developmental and adult plasticity of the nervous system. This methodological venture began with the studies on retrograde and anterograde degeneration at the end of the 19th century. The retrograde degeneration method consisted in putting in evidence deafferented cell bodies (i.e., cell bodies of neurons which had their targets ablated) 17, 110, whereas anterograde degeneration techniques were based on the Wallerian degeneration (i.e., putting in evidence axon terminals following lesion of an axon or of its parent cell body) by means of the Marchi technique [122]. On the impulse of the early debates between Golgi and followers (the “reticularists”) and Cajal (the paladin of the “neuron doctrine”), the Golgi method of silver impregnation of individual neurons 38, 39, 67, 68, 69, 122 was exploited by Cajal for his detailed descriptions of neurons and their axons [21].

Several years later, beginning from the 1940s, the degeneration methods introduced by Glees [65], Nauta et al. 11, 130, 131 and Fink and Heimer [53] allowed to study anterogradely the projections of a nucleus to other centers, and were especially suited for long connections which could not be visualized with the Golgi method.

A further technical improvement consisted much later, in the 1970s, in the introduction of methods which, taking advantage of the retrograde axonal transport for horseradish peroxidase (HRP) in vivo 102, 109, allowed to trace neural connections by a simple and reliable histochemical reaction, easier to perform and more sensitive than all the degeneration methods. Tracing with HRP, which is still in use, represented a giant step in neuroanatomy, since it allows to trace connections in vivo retrogradely, anterogradely and transsynaptically (using wheat germ agglutinin-coupled HRP [60]). At the same time, Cowan and collaborators [37] developed the technique of autoradiography to study axonal connections by anterograde transaxonal transport of radiolabeled amino acids.

Based on axonal transport, a long series of markers has been developed as anterograde or retrograde tracers according to the preferential direction of their transport in the axon. Among these tracers, effective tools were provided by fluorescent dyes which, besides their reliability and sensitivity, give the opportunity to trace multiple connections employing different colors revealed simultaneously 35, 46, 57, 177, 182, 184, 185, 194. In parallel, the introduction of intracellular injections allowed to draw single cell connectivity, eventually associated to the physiological characterization of the neuron 3, 19, 20, 33, 75, 88, 118, 134, 184.

In addition, new anterograde tracers were introduced, such as the subunit B of cholera toxin (CTB), Phaseolus vulgaris leucoagglutinin (PHA-L), biocytin and dextran that allow to reveal the efferents originating from a given cell group. Nevertheless, as it will be repeatedly mentioned below, the differentiation of anterograde vs. retrograde labeling, especially in structures which have reciprocal connections (e.g., in the cerebral cortex) still represents a problem and a stimulus for seeking new tracers, since most of the actual ones are, at least in part, both anterograde and retrograde.

The introduction of carbocyanine dyes, which are effective in fixed, embryonic and neonatal tissues in both anterograde and retrograde directions, has allowed to combine an extreme precision in the placement of the dye with a detailed labeling of dendrites and axons. The use of carbocyanine dyes has also allowed to overcome, at least for shorter connections, several problems in tracing connections in young brains.

Therefore, the choice of tracers for studying neural connections has greatly expanded over the last 20 years. Neuroscientists have been, and still are, continuously seeking for new tracers, in order to improve the efficacy of labeling, to obtain an increasing ease of use, and to avoid use of radioactive labels or carcinogenic reagents. The use of neural tracers has become a common tool not only for the neuroanatomist, but also for every neuroscientist, like immunohistochemistry, histochemistry or in situ hybridization which, however, provide different information and may be coupled with tracing. The aim of this paper is to review some of the most recent methodological advances in this field, providing some suggestions to make an appropriate choice. We will also review some of the problems of quantitation and interpretation of results. For the sake of brevity, we will not deal with HRP and retrograde fluorescent tracers, such as diamidino yellow, fast blue and others 8, 9, which have been in use since a long time, for which we refer to an exhaustive account [7].

Section snippets

Fluorescent tracers

A fluorescent tracer is one that fluoresces when exposed to light of a certain wavelength. Combinations of tracers which are excited with light of different wavelengths, and which in turn emit light of different wavelengths (i.e., exhibit different colors) may be readily used for tracing multiple pathways within the same brain. We will deal in the following chapters with the most recent ones, i.e., carbocyanine dyes, dextrans and fluorescent microspheres, but many other fluorescent tracers

Biotinylated dextran

Biotinylated dextran amine (BDA) (Figs. 4F–H, TABLE 1, TABLE 2, TABLE 3) shares most of its properties with fluorescent dextrans, excepted for the need of a long immunohistochemical reaction for detection, but the final reaction product is more stable, and the quality of dendrite filling is better, than with fluorescent dextrans 14, 44, 145. BDA is an anterograde tracer, with a postinjection survival required for an effective labeling variable between 2 and 21 days according to the length of

Viruses as neuronal tracers

A relatively new and still promising tool in tracing neural pathways is represented by neurotropic viruses 23, 24, 25, 103, such as herpes simplex (HSV-1 [178], and HSV-2 [133]) and pseudorabies (PrV, a pig herpes virus 50, 51, 52, 117, 123, 187), which require specific safety measures in handling (Biosafety level 2, http://www.orcbs.msu.edu/biological/bmbl/bmbl-1.htm). The use of viruses TABLE 1, TABLE 2, TABLE 3 as neuronal markers was first suggested by Sabin [152], but only recently they

Dyes for intracellular injections

Quite often the quality of filling of neuronal processes obtained with tracers is not adequate for performing complete reconstructions of axonal and dendritic arbors and/or identification of cell type has to be combined with the study of electrophysiological properties. To fill neurons completely, dyes may be directly injected into neurons, coupling intracellular injections to the axonal transport of tracers or electrophysiology. Intracellular injections are performed either in vivo or in fixed

Criteria for tracer selection

The choice of a tracer depends on several factors, including animal species and age, pathway (some tracers are pathway-sensitive), direction of transport needed (anterograde or retrograde), and overall design of the experiment (multiple pathway tracing, or combination with other techniques) TABLE 1, TABLE 2, TABLE 3.

The first parameter to consider is the animal species: provided that the length of the pathway depends of course also on the size of the animal. Species may differ also for density

Quantitation in retrograde tracing

The aim of tract-tracing studies is not only to reveal connections between areas in the nervous system, but also to provide quantitative data on the density of a given connection, and, in developmental or manipulation studies, on changes in the refinement of connections.

Several tracers, such as BDA, biocytin or CTB, very likely fill completely axon arbors, thus allowing serial reconstructions of axonal arborizations at the computer. Appropriate software programs are available 4, 16, 64, 73, 82,

Conclusion

Over the last decades, the family of neuronal tracers has progressively grown up, with new anterograde, axonal tracers and lipophilic dyes representing revolutionary tools in the field of neuroanatomy. They allow qualitative and quantitative morphological analysis of connectivity, both under control and experimental conditions, in vivo and in fixed material. On the grounds of the data obtained with tract tracing, it is already possible to make simulations of neural activity, to be compared to

Acknowledgements

The authors are grateful to Prof. G. C. Panzica and Dr. S. Jhaveri for critical reading of the manuscript, and to Dr. G. Bronchti and Dr. A. Angelucci for their suggestions on BDA and CTB techniques. Some of the photomicrographs shown in this paper are from work done in collaboration with Prof. G.M. Innocenti and Dr. S. Jhaveri, to whom AV is indebted for advice and suggestions.

Supported by grants from MURST, AISM, Compagnia di San Paolo and Regione Piemonte to AV. MR is a recipient of a

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