Rabies as a transneuronal tracer of circuits in the central nervous system
Introduction
Selected neurotropic viruses have proven particularly valuable as neuroanatomical tracers (e.g. Ugolini et al., 1989, Strack and Loewy, 1990, Zemanick et al., 1991, Strick and Card, 1992, Card et al., 1993, Hoover and Strick, 1993, Lynch et al., 1994, Middleton and Strick, 1994, Middleton and Strick, 1996, Sun et al., 1996, Jasmin et al., 1997, O'Donnell et al., 1997, Card, 1998, Loewy, 1998, Hoover and Strick, 1999). These viruses move from neuron to neuron at synaptic connections. Furthermore, they replicate in infected neurons and consequently produce an on-line amplification of the tracer signal. Taken together, these features make viruses especially useful for revealing chains of synaptically-linked neurons.
Recently, we and others have developed techniques for using rabies virus as a transneuronal tracer (e.g. Astic et al., 1993, Ugolini, 1995, Kelly and Strick, 1997). Rabies moves in a time-dependent manner through the central nervous system of infected animals. Electron microscopic studies suggest that rabies transport between neurons occurs primarily at synaptic junctions (Iwasaki and Clark, 1975, Charlton and Casey, 1979). Unlike herpes infections, rabies infections are largely confined to neurons, and glia infection is rarely seen (Iwasaki and Clark, 1975, Tsiang et al., 1983). There is no evidence that rabies is taken up by fibers of passage. Furthermore, rabies virus does not cause cell lysis. Indeed, one of the hallmarks of rabies infection is the relative lack of pathology seen in infected brains even at terminal stages of the disease (Murphy, 1977). The absence of cell destruction limits the possibility of random release and non-specific uptake of virus. Furthermore, during survival times that are appropriate for tracing studies, experimental animals remain largely free of symptoms. These features of rabies make it ideal for use as a transneuronal tracer in experimental neuroanatomy.
We have used transneuronal transport of viruses in nonhuman primates to examine the organization of cerebellar and basal ganglia loops with the cerebral cortex (Hoover and Strick, 1993, Hoover and Strick, 1999, Lynch et al., 1994, Middleton and Strick, 1994, Middleton and Strick, 1996, Kelly and Strick, 1997, Kelly and Strick, 1998, Kelly and Strick, 1999) and descending systems that control single muscles (Rathelot and Strick, 1999). Here we outline the methodological issues that must be considered when designing experiments in which rabies is used as a tracer. We emphasize the critical laboratory, surgical, and animal care precautions for these experiments. Finally, we describe the surgical and immunohistochemical procedures used to visualize transneuronal transport of rabies.
Section snippets
Rabies virus basics
The structural, chemical, and antigenic properties of rabies virus have been extensively studied. Readers are referred to Baer, 1975, Baer, 1991 for detailed descriptions and analyses of these topics. Briefly, rabies is a small (180×75 nm), enveloped RNA virus. It is classified as a member of the Rhabdoviridae family by virtue of its ‘bullet-shaped’ structure. The negative strand RNA genome of the virus encodes five proteins: nucleoprotein (N), phosphoprotein (NS or P), matrix protein (M),
Rabies as a tracer
There are a number of key factors involved in the design of experiments that use rabies as a tracer. These include virus strain, virus concentration, experimental species and route of inoculation, rate and direction of transport. Each of these factors will be discussed in a separate section below.
Rabies safety issues
The development and institution of appropriate biosafety and animal care protocols are primary concerns when designing experiments with rabies. In this section we will review the various procedures associated with vaccination of personnel, laboratory safety protocols and the care and housing of infected animals.
Immunohistochemical demonstration of virus
Following the perfusion, the brain and spinal cord are stored in 10% formalin with 20% glycerol (4°C) for 3–10 days. The fixatives used during perfusion inactivate live virus, and fixed tissue is no longer considered ‘infected’. Therefore, conventional biosafety techniques can be employed during the subsequent stages of tissue processing.
Summary
This brief review is intended to describe the characteristics of rabies virus that make it particularly well suited for use as a tracer in neuronanatomical experiments. In fact, several features of rabies infections (e.g. the absence of cell lysis, glia labeling and behavioral symptoms, as well as small injection sites) may make this virus more desirable for use as a transneuronal tracer than other neurotropic viruses. Experiments with rabies virus require specific immunization protocols and
Acknowledgements
We thank Dr Charles Rupprecht of the Centers for Disease Control for providing CVS-11 and CVS-26 strains of rabies virus, Dr Bernhard Dietzschold of Thomas Jefferson University for providing CVS-B2c and CVS-N2c strains of rabies virus, and Dr Alex Wandeler of the Animal Disease Research Institution, Ontario for providing a monoclonal antibody to rabies. This work was supported by funds from the Department of Veterans Affairs, Medical Research Service and by US Public Health Service grants
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