Advances in viral transneuronal tracing

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Abstract

Powerful transneuronal tracing technologies exploit the ability of some neurotropic viruses to travel across neuronal pathways and to function as self-amplifying markers. Two main classes of viral transneuronal tracers are available, derived from alpha-herpesviruses (Herpes Simplex virus type 1, Pseudorabies) and rabies virus. Depending on the virus type and strain, there are major differences with regard to host range, peripheral uptake, replication mechanisms, transport direction and specificity. While alpha-herpesviruses are the tracers of choice for studying autonomic innervation, rabies virus is the ideal tool for studying motor innervation, since its peripheral uptake occurs exclusively at motor endplates. Rabies virus is the only viral tracer that is entirely specific, as it moves exclusively across chemical synapses by strictly unidirectional (retrograde) transneuronal transfer without altering neuronal metabolism, allowing for the stepwise, time-dependent, identification of neuronal networks across an unlimited number of synapses. This review will highlight and contrast the different properties of these viral tracers, and summarize the methodological issues that are critical for the appropriate execution and interpretation of transneuronal tracing studies. Combinations of viral tracing with other methodologies will be evaluated. Emerging technologies, based on genetically modified herpes and rabies tracers, will be also discussed and put in perspective.

Introduction

A landmark event in the evolution of tract tracing techniques has been the development of transneuronal tracers, i.e., markers that are transferred specifically between synaptically connected neurons and allow for the visualization of entire functional neuronal networks (first-order neurons, second-order, third-order, etc.) (Kuypers and Ugolini, 1990).

There are a number of crucial requirements for a marker to be regarded as a reliable transneuronal tracer. First, such marker should be exchanged exclusively by transneuronal transfer between synaptically connected neurons. Second, transneuronal transfer should ideally occur only in one direction, to permit unequivocal interpretations. Third, the number of synaptic steps should be easily identifiable. Fourth, the marker should be able to label all groups of higher-order neurons that are connected to the transferring (first-order) neurons, in order to allow for a comprehensive mapping of the entire connectivity. Fifth, transneuronal labeling should be easily detectable, and should not disappear with time. Sixth, the presence of the marker should not alter neuronal metabolism (unless this is specifically sought for), in order to allow for neurotransmitter and functional studies of the identified neuronal networks.

Validation of any potential transneuronal tracer has necessarily required a thorough evaluation of its transmission in experimental models of known connectivity. The first transneuronal tracing methods were based on the use of some conventional tracers; as only a small amount of these tracers crossed synapses, transneuronal labeling was very weak and could be detected, at best, only in some second-order neurons; third-order neurons could not be visualized (Fig. 1A) (reviewed by Kuypers and Ugolini, 1990, Ugolini, 1995a, Morecraft et al., 2009). The last two decades have witnessed the introduction of much more sensitive techniques based on the use of some viruses as markers. These novel technologies exploit the natural capacity of some neurotropic viruses to be transported along axons and travel across neuronal pathways, demonstrated by classical findings (Goodpasture and Teague, 1923, Sabin, 1938, Kristensson et al., 1971, Kristensson et al., 1974, Kristensson et al., 1982, Tsiang, 1979, Dolivo et al., 1982, Martin and Dolivo, 1983, Dietzschold et al., 1985, Kucera et al., 1985), and their unique ability to function as self-amplifying markers by replicating in recipient neurons, thus overcoming the ‘dilution’ problem of conventional markers and producing intense transneuronal labeling (Kuypers and Ugolini, 1990) (Fig. 1B and C).

Two main classes of viral transneuronal tracer are currently available, which are derived from alpha-herpesviruses (Herpes Simplex virus type 1 and Pseudorabies) (see Kuypers and Ugolini, 1990, Ugolini, 1995a, Ugolini, 1996, Loewy, 1995, Enquist and Card, 1996, Aston-Jones and Card, 2000) and a rhabdovirus, i.e., rabies virus (Ugolini, 1995b, Ugolini, 2008, Tang et al., 1999, Kelly and Strick, 2000, Graf et al., 2002, Morcuende et al., 2002, Ugolini et al., 2006, Prevosto et al., 2009a, Prevosto et al., 2009b) (Fig. 1B and C and Fig. 2). Since they are fully competent viruses, experimental manipulation of these viral tracers must be carried out at the appropriate biosafety containment level (2 or 3 depending on national regulations) and requires specific training, as well as prior vaccination in the case of rabies virus (see Ugolini, 1995a, Ugolini, 1995b, Ugolini, 1996, Enquist and Card, 1996, Kelly and Strick, 2000).

As described here, there are major differences in the properties of alpha-herpesviruses and rabies virus, which make them suitable for different purposes. An important difference is that alpha-herpesviruses rapidly induce neuronal degeneration and can also produce spurious labeling, depending on the virus dose and post-inoculation time (e.g., Ugolini, 1995a, Loewy, 1995) (Fig. 1, Fig. 3, Fig. 4). Rabies virus (Ugolini, 1995b) is the only viral tracer that propagates exclusively by retrograde transneuronal transfer without altering neuronal metabolism, and makes it possible to trace neuronal connections across a virtually unlimited number of synapses (Fig. 1C, 5–8). The purpose of this review is to highlight and contrast the specific properties of these two categories of viral transneuronal tracers, and to summarize the methodological issues that are critical for the appropriate execution and interpretation of viral transneuronal tracing studies. Emerging technologies, based on genetically modified herpes and rabies tracers, will be also discussed and put in perspective.

Section snippets

Alpha-herpesviruses: Herpes Simplex virus type 1 and Pseudorabies

The introduction of the retrograde transneuronal tracing methodology based on the use of Herpes Simplex virus type 1 (HSV 1) (Ugolini et al., 1987, Ugolini et al., 1989, McLean et al., 1989, Kuypers and Ugolini, 1990) has been the first major step in the development of sensitive transneuronal tracers (Fig. 3). This technique was rapidly adopted worldwide, particularly in non-human primates (e.g., Zemanick et al., 1991, Hoover and Strick, 1993, Middleton and Strick, 1994, Ugolini, 1995a,

Rabies virus

The introduction of the transneuronal tracing technology based on use of rabies virus (the ‘fixed’ CVS-11 strain) (Ugolini, 1995b) (Fig. 5) has finally made available a transneuronal tracer that is completely reliable, moves unidirectionally (retrogradely) and allows for the identification of neuronal networks across a virtually unlimited number of synapses. Rabies virus is the only viral transneuronal tracer that is entirely specific, since it propagates exclusively between connected neurons,

Acknowledgements

In this overview of a vast and complex field, it was impossible to cite all available publications on herpes and rabies virus neuroinvasion mechanisms and transneuronal tracing implementations. Key findings have been illustrated as much as possible by resorting to selected papers and reviews. I apologize to those investigators whose contributions could not be included due to space limitations.

This work was supported by the European Union (QLRT-2001-00151, EUROKINESIS, and BIO4-CT98-0546,

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