Chapter 17 - Transneuronal Tracing with Alpha-herpesviruses: A Review of the Methodology

Abstract

This chapter focuses on transneuronal tracing with alpha-herpesviruses. For a marker to be effective as a transneuronal tracer, transfer occurs specifically between connected neurons. Transneuronal labeling is easy to detect and involve all groups of neurons that are part of a given neuronal circuit. There are two classes of transneuronal markers: conventional tracers— namely, the wheat germ agglutinin-horseradish peroxidase conjugate and nontoxic fragments of tetanus toxin and some neurotropic viruses— namely, alpha-herpesviruses such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV), and rhabdoviruses. A unique feature of viruses is their ability to replicate in recipient neurons after transneuronal transfer, thus functioning as a self-amplifying marker. As a result, second- and third-order neurons show the same intensity of labeling as first-order neurons The success of transneuronal labeling with HSV-1 and PrV is dependent upon several experimental parameters, some related to the virus and others related to the host. The chapter reviews the methodological aspects regarding the use of HSV-1 and PrV as transneuronal tracers and hence, details the structure, tropism, and replication cycle of HSV-1 and PrV. There are several important parameters considered while designing viral transneuronal tracing studies: Host variables (species, strain, and age), viral strains (HSV and PrV), and virus dose that is measured in Plaque-forming units per milliliter (PFU/ml). Excellent visualization of HSV-1 and PrV is provided by standard immunohistochemical methods—that is, immunofluorescence and immunoperoxidase in frozen sections. Immunoperoxidase staining can also be carried out in paraffin sections. The interpretation of the kinetics of viral transfer is often complicated because viral infection causes degeneration of first-order neurons. This might include virus-induced neuronal degeneration and local transfer and asynchronous labeling of different groups of second-order neurons.