Frank J. Jenkins¹ and Sharon L. Turner²

¹ Department of Pathology, School of Medicine; 1Department of Infectious Diseases and Microbiology, School of Public Health;
^1,2 Division of Behavioral Medicine and Oncology, University of Pittsburgh Cancer Institute;
^1,2 Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15213 USA

Received 08/02/96; Accepted 08/08/96; On-line 09/01/96

4. TRANSNEURONAL TRACERS

Neuroscientists have long desired to map chains of neurons in order to identify communication pathways from origin to termination. With the use of antero- and retrograde tracers such as fast blue and horseradish peroxidase it is possible to identify single neurons along with their axons and terminations. However, in order to identify synaptically linked second and third order neurons, transneuronal tracers are required.

4.1. Properties of Effective Transneuronal Tracers

In order for a transneuronal tracer to be effective, it must be specific for synaptically-linked connections, possess the ability to be transported antero- or retrogradely and be sufficiently tagged for efficient and sensitive detection. Substances such as cholera toxin, tetanus toxin and wheat germ agglutinin are known to bind specifically at neuronal membranes and have been used as transneuronal tracers (25-27). These methods, however, have limitations since only small amounts of protein are transported across synapses resulting in an absence of or relatively weak labeling of linked neurons. In addition, nonspecific labeling of adjacent neurons can occur at increased injection concentrations and extended labeling times. Neurotropic viruses, specifically herpesviruses, have an advantage over these other contemporary methods in that they are able to replicate within neuron cell bodies providing signal amplification before infecting second- and third-order neurons. These viruses have also been shown to specifically label neuronal connections in both the retrograde and anterograde direction (28, 29).

4.2. Viral Transneuronal Tracers

The most common transneuronal viral tracers are herpes simplex virus 1 and 2 (HSV-1/HSV-2) and pseudorabies virus (PRV). All three viruses belong to the alpha herpesvirinae family and therefore are neurotrophic (8). The ability of these DNA viruses to specifically infect neurons contributes to their specific transneuronal transport. The most common method used to detect the presence of these viruses in neuronal tissue is by immunohistochemical staining for viral antigen.

Experiments used to obtain transneuronal tracings are modulated by the strain of virus used, the host animal, the site of injection, the amount of virus inoculated, and the time of post-inoculation analysis. The importance of these parameters is apparent from many studies which report that uncontrolled viral tracings, especially at late survival times, may lead to nonspecific labeling (30, 31).

Electron microscopic studies using HSV and PRV have demonstrated that fusion of the viral envelope with the cellular plasma membrane of neuronal extensions is followed by retrograde axonal transport of unenveloped nucleocapsids along axonal microtubules (28). Although this is the primary mode of viral transport to the neuronal nucleus, it is not exclusive. Other studies have shown anterograde transport of virus, (29, 32) and at least one report suggests that the direction of transneuronal transport may be strain dependent (33). By analyzing labeled neurons at progressive time points, it has been determined that retrograde transport occurs much faster than anterograde transport (29). Consideration of the difference in transport rate is important in tracing analyses and can be useful in determining connections between groups of neurons. For example, in groups of neurons which are highly connected by collaterals, one must consider the fact that individual neuron labeling could be due to either antero- or retrograde transport, and in such instances, there may be no way to distinguish between originating and target cells.

Although the release of herpes virus occurs at neuronal terminals, sites of virion egress do not always occur directly into synaptic clefts. Herpes-containing vesicles have been reported to fuse at presynaptic terminals releasing enveloped virus which then fuses to postsynaptic membranes adjacent to the presynaptic terminals resulting in the entry of nucleocapsids into the neuron. Astrocytes are also susceptible to PRV and HSV infection, but infected cells are only observed subsequent to an adjacent neuronal infection. Ultrastructural analyses of PRV-infected astrocytes have revealed a defect in the cytoplasmic envelopment of viral nucleocapsids rendering the nucleocapsids incapable of plasma membrane fusion. This defect results in an absence of viral egress and an accumulation of virion particles within the cellular cytoplasm (34). The resulting abortive infection effectively prevents astrocytic PRV virions from contributing to nonspecific extracellular spread. At present, no such mechanisms are known for HSV. In fact, several studies have reported that HSV is quite capable of establishing a productive infection in astrocytes (35). The inability of PRV to establish a productive infection in astrocytes provides a great advantage to PRV in ensuring specific transneuronal transport and is a major reason why PRV is considered by many to be the virus of choice for CNS transneuronal tracing studies.

Additional host mechanisms restricting the spread of the virus to non-neuronal cells are provided by the host's immune response to both PRV and HSV infections. Resident microglia, monocytes and macrophages are activated in the nervous system during viral infection and may effectively phagocytose virus and degenerating cellular debris (36). The importance of these mechanisms is apparent considering the large viral load which may be released from necrotic cells to the extracellular space in the absence of these mechanisms. T-lymphocytes may also play a role in the delineation of viral spread (37). Factors regulating these mechanisms have yet to be elucidated, but most likely involve immune-mediated cytokine production and the induction of major histocompatibility antigen expression within the nervous system.