Conditional Transneuronal Tracing with PRV Figure 4. Fluorphor Expression in Spinal Cord IML. Infected SPN neurons in the thoracic IML after injection of PRV recombinants into the kidneys are illustrated. The approximate level of thoracic spinal cord illustrated in figures B–D is designated by the red arrow on the dissection of brain and spinal cord shown in figure A. Figure B & C illustrate infected SPNs in IML segments ipsilateral to kidneys injected with PRV-263 (B) or PRV-267 (C) in case 4. The default dTomato reporter fills the soma and proximal dendrites of SPNs infected only with PRV-263 while capsids tagged with the VP26-mRFP fusion protein densely label the nucleus (white arrows) of neurons only infected with PRV-267. Figure D illustrates the fluorophor profiles of infected SPNs in an IML segment ipsilateral to the kidney injected with PRV-267 (case 1). Labeled capsids are concentrated in the nuclei of infected SPNs but are also apparent in the cell cytoplasm (white arrow). In addition, IML neurons in this segment express cytoplasmic reporters of the recombined Brainbow cassette, either alone or with the VP26-mRFP reporter. The yellow arrows in d9–d0 illustrate a dual infected neuron expressing mRFP and cytoplasmic reporters of the recombined Brainbow cassette. The neuron labeled with the asterisk in d9 & d- expresses cytoplasmic reporters of the recombined Brainbow cassette, but no mRFP labeled capsids, and was infected by retrograde transneuronal passage of virus from dual infected neurons. Marker bars for figures B & C = 50 mm; marker bars for D and d9 = 20 mm. The magnification is equivalent for d9–d-. doi:10.1371/journal.pone.0021141.g004 reporter) and PRV-BaBlu (b galactosidase reporter) in this model. Such variations may be the result of differences in the density of sympathetic innervation of kidney or to variations in the number of infect cells within the kidney that become infected and amplify virus available for uptake and retrograde transport by sympathetic afferents, possibly due to differences in immune response to infection. In any event, the quantitative analysis in this study provided strong evidence that the invasion of preautonomic circuits from each kidney was equivalent for each recombinant within an animal, thereby optimizing the probability of achieving dual infection of collateralized neurons. Fluorescence profiles of infected neurons Neurons infected by PRV-267 express a red punctate signal that produces a dense labeling of cell nuclei early in viral replication (Figures 2B) followed by the appearance of red puncta in the cytoplasm later in infection (Figures 2C). This labeling is predicted by the demonstrated sequence of capsid assembly (nuclear) and envelopment (cytoplasmic) characteristic of viral replication and spread [31]. The expression profiles of fluorescent reporters resulting from infection of neurons with PRV-263, either alone or in the presence of Cre, have been characterized [17,18]. In the absence of Cre, the cytoplasm of infected cells fills with the dTomato (red) reporter expressed from the Brainbow cassette (Figures 2D and E). However, in the presence of Cre the red reporter gene is excised resulting in expression of either the yellow (EYFP) or cyan (mCerulean) reporters (Figures 2F–I). Dual infected neurons in the present study were marked by the punctate VP26-mRFP capsid marker of PRV-267 and the reporters of the recombined Brainbow cassette (white arrows in Figures 2F–H). Once Cre acts, no more recombination is possible thus rendering the expression characteristics of the resulting progeny virus permanent. Transneuronal passage of recombined PRV-263 will therefore produce either cytoplasmic cyan or yellow reporters. Whether transneuronally infected neurons express multiple cytoplasmic reporters of PRV-263 infection and also contain VP26-mRFP appears to depend upon the mixture of progeny virus that passes transneuronally from dual infected neurons (see Discussion for supporting literature). However, when the cytoplasmic reporters of the recombined Brainbow cassette are present in cells lacking punctate VP26-mRFP labeling (Figure 2I) the neurons should have been infected by retrograde transneuro- nal passage of virus from one or more dual infected neurons. Reporter gene expression in neurons replicating only one recombinant Reporter gene expression in sympathetic preganglionic neurons (SPNs) of thoracic spinal cord illustrated the distinctive reporters of neuronal infection with PRV-263 or PRV-267. Infection of SPNs in the IML revealed the largely lateralized sympathetic outflow to the kidneys and other autonomic targets; e.g., SPNs innervating the kidney injected with PRV-263 displayed homo- geneous expression of the dTomato reporter throughout the somatodendritic compartment (Figure 4A) while SPNs infected by retrograde transport of virus from the kidney injected with PRV- 267 contained dense concentrations of mRFP puncta in cell nuclei (Figure 4B). The largely lateralized organization of preautonomic circuitry innervating SPN outflow to each kidney predicts that the majority of neurons infected with only one virus will be concentrated on the side of the brain ipsilateral to the injected kidney. Examination of each group of infected neurons contributing to the preautonomic circuitry on the left and right sides of the brain confirmed this prediction. The majority of infected neurons in each node within the preautonomic network infected with only one virus displayed fluorescence profiles consistent with the genotype of virus injected into the ipsilateral kidney. Reporter gene expression in dual infected neurons We analyzed regions of the spinal cord, medulla and dien- cephalon previously shown to contain neurons infected through collateralized axonal projections to neural circuits innervating each kidney [20]. If neurons infected with PRV-267 are producing biologically active Cre then we should observe dual infected neurons expressing cyan and/or yellow reporters. Similarly, the presence or absence of the unique reporter of PRV-267 infection (punctate VP26-mRFP) in neurons expressing cyan and yellow fluorescence should allow us to discriminate neurons replicating both viruses from synaptically connected neurons infected by transneuronal passage of virus from dual infected neurons. We selected the thoracic spinal cord, RVLM, VMM, LC and PVN for this analysis because of the well-known collateralized connections PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21141