Conditional Transneuronal Tracing with PRV case 3. The schematic diagrams are adapted from the atlas Brain Maps: Structure of the Rat Brain [28]. NTS = nucleus of the solitary tract; RVLM = rostroventrolateral medulla; VMM = ventromedial medulla. Marker bars for b–e and c9 = 50 mm; marker bars for b9, d9, and e9 = 10 mm. doi:10.1371/journal.pone.0021141.g005 of neurons in these cell groups within the renal preautonomic network. As noted above, we observed large numbers of infected SPNs in the thoracic spinal cord that were marked by only PRV-267 or PRV-263 and were concentrated in IML segments ipsilateral to the injected kidney (Figure 4). However, as reported previously [20], we also observed interneurons within IML segments that were infected by retrograde transneuronal passage of virus from the contralateral IML. These neurons were most prevalent in cases with the most extensive transport through the preautonomic network and contained punctate VP26-mRFP labeling in the nucleus and cytoplasm as well as yellow and blue cytoplasmic reporters of the recombined Brainbow cassette (yellow arrows in Figures 4d9–4d0). This pattern of reporter gene expression is consistent with dual infection of neurons by PRV-263 and PRV- 267 through collateralized axons that synapse upon SPNs bilaterally in thoracic spinal cord. The profile of gene expression in RVLM neurons is predicted by the known connectivity of the RVLM, which is characterized by a large projection to the ipsilateral IML and lesser projections to the contralateral RVLM and IML. Data consistent with this prediction are shown in Figures 5B and D. We observed RVLM neurons expressing only the cytoplasmic dTomato reporter (PRV- 263 infection), neurons only expressing the punctate nuclear VP26-mRFP reporter (PRV-267 infection), and neurons express- ing the conditional cytoplasmic reporters (cyan and/or yellow) in combination with the punctate VP26-mRFP labeling of nuclei (dual infected neurons) (Figures 5b and d). The largest proportion of neurons expressing these phenotypes was concentrated in the rostral aspect of RVLM, which is the portion of this cell group that gives rise to the largest portion of the reticulospinal projection to thoracic cord. Reporter gene expression in VMM reflected documented descending reticulospinal projections to thoracic cord, reciprocity of connections to other nodes within the renal preautonomic network (e.g., RVLM), and local circuit connections within the VMM (Figure 5C). Direct descending projections to the thoracic cord were marked by cytoplasmic localization of the dTomato reporter or punctate VP26-mRFP labeling in neurons replicating only one virus, with the largest proportions of each of these groups present on the side of the brain ipsilateral to the injected PRV recombinant. We also observed neurons that contained both markers and were therefore infected by collateralization of axons to efferent pathways synaptically linked to both kidneys (white arrows in Figure 5c). Single and dual infected neurons were present in all subdivisions of the VMM, including raphe pallidus, but were most prevalent in the rostral third of this cell column, with the highest concentration occurring in the areas immediately lateral to the pyramids (Figure 5C and E; 5c and e). Retrograde transneuronal infection of the LC also produced a pattern of infected neurons that conformed to that previously documented after injection of virus into the kidney [20]. The cases that displayed more limited invasion of preautonomic circuits (e.g., animals surviving 4 days and cases 2 and 3 from the 5 day survival group) exhibited neurons largely confined to the ventral third of the LC. The majority of infected neurons in a single cell group were infected by the recombinant injected into the ipsilateral kidney, but a subset was infected by both recombinants. In cases with the most extensive transport of virus through preautonomic circuitry, infected neurons were observed throughout the dorso- ventral extent of the LC bilaterally (Figure 6A). However, neurons replicating both recombinants remained confined to the ventral third of the LC (white arrows in Figures 6B and C). These finding confirm and extend those reported by Cano and colleagues Figure 6. Fluorphor Expression in Rostral Brainstem and LC. The distribution of infected neurons in rostral brainstem at the level of the locus coeruleus (LC) is illustrated. The schematic sagittal section in the upper left of figure A illustrates the position of the plane sampled in the rostrocaudal axis of the brain. The coronal schematic in figure A maps the distribution of infected neurons detected by immunoperoxidase localization of viral antigens in case 1. Each red dot indicates the position of an infected neuron in a section 210.10 mm relative to Bregma. Figures B and C, also from case 1, illustrate the fluorescent profiles of LC neurons in sections adjacent to that shown in A. The box in A marks the relative position of images B and C, which illustrate reporter fluorescence revealed by the filters specific for dTomato, mCerulean and EYFP. Fluorescence signal in the boxed areas of B & C is shown at higher magnification in b9–b- and c9–c-. White arrows in B & C indicate neurons dual infected neurons. Cells marked by the asterisks are expressing reporters of the recombined Brainbow cassette but do not contain VP26-mRFP labeled capsids. The schematic diagram is adapted from the atlas Brain Maps: Structure of the Rat Brain [28]. Marker bars in B & C = 50 mm; marker bars in b- and c- = 20 mm. doi:10.1371/journal.pone.0021141.g006 PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e21141