Conditional Transneuronal Tracing with PRV Figure 2. Experimental Paradigm and Neuronal Phenotypes. The experimental paradigm used in this analysis (A) and the reporter phenotypes of neurons infected with PRV-263 and PRV-267, either alone or in combination (B–I), are illustrated. Each animal received separate injections of PRV-263 and PRV-267 into the left or right kidney (A). Prior studies have demonstrated that PRV-Bartha recombinants are transported from the kidney to infect postganglionic neurons in the inferior mesenteric ganglion. Subsequent replication and transneuronal passage of virus infects sympathetic preganglionic neurons in the IML and neurons of the renal preautonomic network. The preautonomic network linked to each kidney is largely lateralized but also contains neurons that collateralize to innervate circuits linked to each kidney. Neurons infected with both recombinants express unique reporters (cyan and/or yellow) in response to Cre-mediated recombination of the Brainbow cassette. The color-coding of neurons defined in the upper right box of A illustrates the various phenotypes possible in this injection paradigm. Neurons only infected with PRV- 267 are marked by the VP26-mRFP reporter, a capsid surface fusion protein that is differentially concentrated in the cell nucleus (B) but produces punctate labeling in the cytoplasm (C) as capsids migrate out of the nucleus to be incorporated into mature virions. Selective infection with PRV-263 in the absence of Cre results in default expression of the dTomato reporter and homogeneous cytoplasmic labeling (D & E). Cre-mediated recombination of the Brainbow cassette in neurons replicating both PRV-263 and PRV-267 results in VP26-mRFP capsid labeling and cytoplasmic labeling by the cyan and/or yellow reporters (white arrows in F–H). Neurons infected by transneuronal passage of virus containing recombined genomes (PRV-263re) from dual infected neurons only express the cyan and/or yellow cytoplasmic reporters (I). Figure B is from IML of thoracic spinal cord, figures C & H are from raphe pallidus, figures D–H are from VMM. Marker bars in B, C, F, and G = 20 mm and those in D, E, H, and I = 25 mm. doi:10.1371/journal.pone.0021141.g002 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21141