A time-honored protocol for tracing neural connections is to inject an enzyme or dye into a small area of the brain, from which it is taken up by nerve terminals and then transported along axons. However, most of these tracers are retained within neurons and do not diffuse to connected neurons. Those that can spread to connected neurons dilute rapidly, an obvious impediment to following multisynaptic circuits. Viruses provide a noteworthy alternative to these chemical tracers. The most useful are those neuroinvasive viruses that infect the central nervous system (CNS) after peripheral inoculation. Well known examples of neuroinvasive viruses are rabies virus (an RNA virus from the family Rhabdoviridae) and the α-herpesviruses (DNA viruses from the subfamily Herpesvirinae) [1
When infection spreads between functionally connected neurons, it is said to be trans-neuronal. The actual mechanism of trans-neuronal spread is understood poorly. The important point is that, unlike chemical tracers, as the infection spreads, viral-encoded proteins mark each infected neuron. Consequently, neuroinvasive viruses can be self-amplifying tracers of neuronal circuitry.
In recent years, pseudorabies virus (PRV) has become the most popular α-herpesvirus for circuit-tracing studies. Despite its name, PRV has absolutely no relationship to rabies virus. The natural hosts of PRV are swine, although the virus has a broad host range; it is neuroinvasive in essentially all mammals, except higher primates. PRV can be used in a wide range of animal models but remains safe for laboratory workers. It is amenable to genetic manipulation and the insertion of large segments of foreign DNA. The 142 kb double-stranded PRV genome has been cloned into a bacterial artificial chromosome (BAC) [2
], which enables easy genetic modification of the genome in bacteria using standard DNA recombination technology [3