Transneuronal circuit analysis with pseudorabies viruses

Over the past decade there has been a dramatic increase in the use of viruses as transneuronal tracers of neuronal circuitry. The method exploits the propensity of neurotropic viruses to invade neurons and then produce infectious progeny that cross synapses to infect other neurons within a circuit. The protocols and commentaries included in this unit focus upon the use of the swine alpha herpesvirus known as pseudorabies virus (PRV) for polysynaptic analysis. Here, the aspects of experimental design that have the greatest import for successful use of viruses in circuit definition are presented. Accordingly, the protocols included in this unit can be applied in concert with methods in which the use of classical tract tracers has been detailed. A procedure for retrograde infection of CNS circuits in the rat CNS by peripheral injection of virus is detailed, while transneuronal analysis by intracerebral injection is also described. A variant of these procedures, transneuronal analysis with multiple recombinant strains, is also described along with methods for growing and titering viral stocks, and procedures for single and dual immunohistochemical localization of viral antigens in fixed brain tissue.