Conditional Transneuronal Tracing with PRV Figure 7. Fluorophor Expression in Diencephalon and PVN. The distribution of infected neurons in diencephalon at the level of the paraventricular hypothalamic nucleus (PVN) is illustrated. Figure A maps infected neurons detected by immunoperoxidase localization of viral antigens in case 1. Each red dot indicates the position of an infected neuron and the position of the section with respect to Bregma (b) is indicated below the schematic. Figure B illustrates the fluorescent profiles of neurons in parvicellular PVN subdivisions in a section adjacent to that shown in A. The fluorescence in figure B is a composite of that revealed by the filters specific for dTomato, mCerulean and EYFP and the boxed area is shown at higher magnification in the inset. Figures b–b0 show the fluorescence for individual channels in the same area as the inset in figure B. White arrows mark dual infected neurons and asterisks mark neurons that only express reporters of the recombined Brainbow cassette. The schematic diagram is adapted from the atlas Brain Maps: Structure of the Rat Brain [28]. Marker bar in figure B = 50 mm and the marker bar in the inset of figure B = 10 mm. Figures b–b0 are of the same magnification and the marker bar in b = 20 mm. doi:10.1371/journal.pone.0021141.g007 following injection of PRV-152 (EGFP reporter) and PRV-BaBlu (b-galactosidase reporter) into separate kidneys. Retrograde transneuronal infection of neurons in the PVN occurred in the parvicellular subdivisions of this diencephalic cell group. Only scattered infected neurons were found in the PVN in animals analyzed four days following injection of virus into the kidneys. Five days following kidney injection we observed numerous infected neurons in the dorsal, medial and posterior parvicellular subfields (Figure 7A). Figure 7B illustrates the distribution and phenotype of neurons typically observed in animals exhibiting the most robust infection of preautonomic circuitry (e.g., cases 1 and 4). In each case, infected neurons were present within in both the dorsal and medial parvicellular subfields of PVN. Dual infected neurons (e.g., white arrows in 7b) were a subset of a larger population infected only with the recombinant injected into the ipsilateral kidney. Transneuronal infection from dual infected neurons The expression of conditional reporters of the Brainbow cassette throughout the preautonomic network, while confirming the presence of collateralized neurons, also revealed new insights into the synaptic organization of preautonomic synaptology. For example, we observed infected neurons that replicated the recombined Brainbow cassette (expressing cyan and yellow cytoplasmic reporters), but did not express punctate VP26-mRFP labeling. Since the presence of punctate VP26-mRFP marks cells infected with PRV-267, neurons that exclusively express cytoplas- mic reporters of the recombined Brainbow cassette should have been infected by virtue of their synaptic linkage to dual infected neurons (i.e., are presynaptic to dual infected neurons). This synaptic relationship cannot be distinguished in dual infection approaches that do not involve conditional reporter expression (e.g., injection of PRV152 & PRV-BaBlu). Neurons of this phenotype (marked by asterisks in Figures 4, 5, 6, 7) were observed in each of the cell groups analyzed in this study and their prevalence appeared to vary among cell groups. For example, neurons displaying this phenotype were prevalent within RVLM, VMM and LC but were rarely observed within raphe pallidus. A more detailed analysis incorporating a larger sample size and quantitative analysis is necessary to determine the relative proportions of these neurons within individual cell groups of the renal preautonomic network. Nevertheless, the ability to discrim- inate these neurons from dual infected cell groups provides another level of insight into the synaptology of neural networks identified in dual infection paradigms. Discussion The findings reported in this manuscript document a new viral transneuronal tracing approach that can be used to identify connections to neurons within a complex network whose axons collateralize to influence separate targets. To test the utility of this approach, we used a well documented dual infection animal model in which PRV recombinants that express unique reporters are injected into separate kidneys [20]. The use of PRV-263 and PRV-267 in this model system provides unique insights into the synaptic organization of complex circuits that cannot be resolved in dual infection studies employing isogenic PRV recombinants that constitutively express unique reporters (e.g., PRV-152 & PRV- BaBlu). Particularly important in this regard is the ability to discriminate neurons presynaptic to dual infected neurons. Nevertheless, it is important to emphasize that the method does not permit a definitive identification of all neurons providing synaptic input to dual infected collateralized neurons and thereby provides a qualitative rather than quantitative approach for identifying these neurons. The method builds upon recent studies in which we reported the construction and characterization of PRV-263 [17] and demon- strated the ability of lentivirus mediated Cre expression to produce conditional reporter expression from a Brainbow cassette [19] carried by PRV-263 in targeted populations of neurons [18]. Here we describe the construction and use of PRV-267, which serves both as a transneuronal tracer and a vector for circuit related expression PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21141