Use of pseudorabies virus to delineate multisynaptic circuits in brain: opportunities and limitations

Transsynaptic tracing with live virus is a powerful tool that has been used extensively to analyze central efferents that regulate peripheral targets. More recently, investigators have begun to use this new methodology with central injections to identify circuit anatomy within the brain. Although transsynaptic tracing with peripheral injection of pseudorabies virus has been extensively characterized, several methodological issues related to central application of this tracer have not been addressed. Here, we review the following issues relevant to the use of pseudorabies virus (PRV; Bartha strain) in experiments involving injection of virus into rat brain: (i) factors that determine the zone of viral uptake; (ii) uptake of pseudorabies virus by fibers of passage; (iii) viral invasion of the brain after leakage of virus into the brain ventricles; (iv) considerations for double labeling for PRV with peptides and neurotransmitters; (v) use of PRV with conventional retrograde tracers to anatomically identify relays in a multisynaptic pathway; and (vi) transport of PRV throughout the dendritic tree as a means of identifying inputs to distal dendrites. Collectively, the data demonstrate that PRV provides a powerful means of dissecting the synaptology of CNS circuitry when appropriate controls are incorporated into the experimental design. A set of recipes for various procedures are included at the end of this article.