Characterization of transsynaptic tracing with central application of pseudorabies virus

Although transsynaptic tracing with peripheral injection of pseudorabies virus (PRV) has been extensively characterized, several methodological issues related to central application of this tracer have not been addressed. In the present study, we addressed the following three issues by using microinjection of a cocktail containing PRV (Bartha strain) and cholera toxin subunit B (CTb) into different sites in the rat brain. First, we estimated PRV diffusion by examining injection sites at different times after application. Second, we tested whether PRV is taken up by fibers of passage following injections into the olivocerebellar pathway. Third, we developed criteria for leakage of PRV into cerebral ventricles. Our data indicate that (i) centrally injected PRV diffuses very little and produces focal injection sites; (ii) PRV is taken up and transported by fibers of passage, although less prominently than found for Ctb; (iii) PRV produces specific and easily identifiable ependymal cell as well as neuronal labeling following ventricular injection. This labeling can be used as a criterion for determining if labeling obtained was due to injected tracer leaking into brain ventricles. In summary, the present study provides new and important information about using PRV to trace central multisynaptic circuitry.