Chapter 10 - Rabies Virus as a Transneuronal Tracer of Neuronal Connections

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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. Rabies virus is the only viral tracer that is entirely specific, as it propagates exclusively between connected neurons by strictly unidirectional (retrograde) transneuronal transfer, allowing for the stepwise identification of neuronal connections of progressively higher order. Transneuronal tracing studies in primates and rodent models prior to the development of clinical disease have provided valuable information on rabies pathogenesis. We have shown that rabies virus propagation occurs at chemical synapses but not via gap junctions or cell-to-cell spread. Infected neurons remain viable, as they can express their neurotransmitters and cotransport other tracers. Axonal transport occurs at high speed, and all populations of the same synaptic order are infected simultaneously regardless of their neurotransmitters, synaptic strength, and distance, showing that rabies virus receptors are ubiquitously distributed within the CNS. Conversely, in the peripheral nervous system, rabies virus receptors are present only on motor endplates and motor axons, since uptake and transneuronal transmission to the CNS occur exclusively via the motor route, while sensory and autonomic endings are not infected. Infection of sensory and autonomic ganglia requires longer incubation times, as it reflects centrifugal propagation from the CNS to the periphery, via polysynaptic connections from sensory and autonomic neurons to the initially infected motoneurons. Virus is recovered from end organs only after the development of rabies because anterograde spread to end organs is likely mediated by passive diffusion, rather than active transport mechanisms.

Introduction

A landmark event in systems neuroscience has been the development of transneuronal tracers, that is, markers that allow for the identification of the chains of synaptically connected neurons (first-order neurons, second-order, third-order, etc.) that innervate a given organ and mediate a specific behavior (Kuypers and Ugolini, 1990, Morecraft et al., 2009, Ugolini, 1995a, Ugolini, 2010). In order to be effective as transneuronal tracers, such markers should meet several requirements. First, they should propagate exclusively by transneuronal transfer between connected neurons (and not by cell-to-cell spread among neurons that are not synaptically connected). Second, transneuronal transfer should ideally be unidirectional, to permit unequivocal interpretations. Third, the number of synaptic steps should be easily identifiable. Fourth, the marker should allow for the visualization of all groups of neurons that innervate the injection site directly (first-order neurons) and indirectly (second-order neurons, third-order, fourth-order, etc.), in order to permit a comprehensive mapping of the entire connectivity. Fifth, transneuronal labeling should be easily detectable and should not disappear with time. Sixth, the marker should not substantially alter neuronal metabolism, to allow for neurotransmitter and functional studies of the identified neuronal networks.

The first transneuronal tracing methods were based on the use of conventional tracers, and their transfer occurred only when first-order neurons were filled with great quantities of the tracer. Because only a small amount of the 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, Morecraft et al., 2009, Ugolini, 1995a, Ugolini, 2010).

Sensitive transneuronal tracing technologies are based on the use of neurotropic viruses as markers (Kuypers and Ugolini, 1990, Loewy, 1995, Ugolini, 1995a, Ugolini, 1996, Ugolini, 2010). They exploit the capacity of some viruses to travel across neuronal pathways, demonstrated by classical studies (e.g., Dietzschold et al., 1985, Dolivo, 1980, Goodpasture and Teague, 1923, Kristensson et al., 1971, Kristensson et al., 1974, Kristensson et al., 1982, Kucera et al., 1985, Martin and Dolivo, 1983, Sabin, 1938, Tsiang, 1979). Their superior sensitivity is due to the ability of viruses to function as self-amplifying markers by replicating in recipient neurons, thus overcoming the “dilution” problem of conventional tracers and producing intense transneuronal labeling, as detected immunohistochemically (Kuypers and Ugolini, 1990, Ugolini, 2010; Fig. 1B–D).

There are two main classes of viral transneuronal tracers, derived from alpha-herpesviruses (herpes simplex virus type 1, HSV 1, and pseudorabies, PrV; see Aston-Jones and Card, 2000, Kuypers and Ugolini, 1990, Loewy, 1995, Ugolini, 1995a, Ugolini, 1996, Ugolini, 2010) and a rhabdovirus, that is, rabies virus (the “fixed” CVS-11 strain; Graf et al., 2002, Kelly and Strick, 2000, Morcuende et al., 2002, Prevosto et al., 2009, Prevosto et al., 2010, Tang et al., 1999, Ugolini, 1995b, Ugolini, 2008, Ugolini, 2010, Ugolini et al., 2006; Figs. 1B, C and 2). These two classes of viral tracers have very different properties (see Section II). Importantly, only rabies virus (Ugolini, 1995b) is completely reliable as transneuronal tracer because it propagates exclusively by strictly unidirectional (retrograde) transneuronal transfer and allows for the stepwise identification of neuronal networks across a virtually unlimited number of synapses (Fig. 1).

The purpose of this chapter is to highlight the specific properties of rabies virus as a transneuronal tracer, which have been identified by studying viral propagation within the central nervous system (CNS) during the preclinical period in primate and rodent models of known connectivity. The experimental findings have valuable implications for the understanding of rabies pathogenesis, which will be discussed.

Section snippets

Differences in Properties of Alpha-Herpesviruses and Rabies Virus as Transneuronal Tracers

There are major differences in the properties of alpha-herpesviruses and rabies virus, which make them suitable for different purposes. The first important difference is in their peripheral uptake: alpha-herpesviruses can infect all categories of neurons that innervate a peripheral site (e.g., a muscle), that is, primary sensory neurons, motoneurons, sympathetic, and parasympathetic neurons (e.g., Goodpasture and Teague, 1923, Kristensson et al., 1982, Kuypers and Ugolini, 1990, Martin and

Structure of rabies virus

Rabies virus is a single-strand negative RNA virus, from the genus Lyssavirus (from lyssa, the Greek word for frenzy) of the Rhabdoviruses family (from the Greek rhabdos, i.e., “rod” because of its characteristic bullet shape; Fig. 2B). The viral genome (less than 12 kb) encodes only five proteins: a nucleoprotein (N), an RNA-dependent RNA polymerase (L), a polymerase cofactor phosphorylated protein (the phosphoprotein P), a matrix protein (M), and a single external glycoprotein (G; Dietzschold

Methodologies: Genetically modified rabies tracers

The development of reverse genetics (Conzelmann, 1996, Mebatsion et al., 1996a, Mebatsion et al., 1996b) has made it possible to manipulate directly the genome of rabies virus, providing outstanding opportunities to improve knowledge of mechanisms involved in viral functions and to design new vaccines (Faul et al., 2009, Finke and Conzelmann, 2005, Schnell et al., 2005).

Preliminary results highlight the potential of reverse genetics approaches also for engineering modified rabies virus tracers

Acknowledgments

This work was supported by the European Union (QLRT-2001-00151, EUROKINESIS, and BIO4-CT98-0546, TransVirus) and the Centre National de la Recherche Scientifique (CNRS).

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