Plus microscope slides (Fisher Scientific, Pittsburgh, PA), dehy- drated in graded alcohols, cleared in xylenes, and coverslipped with Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). The details of these procedures as applied in our laboratories have been published [26]. Fluorescence microscopy Sections of brain and spinal cord adjacent to those used for the immunoperoxidase analysis were analyzed using fluorescence micros- copy. Sections were mounted on gelatin-coated slides, air dried, and coverslipped using Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA). The fluorophor profile of infected neurons was determined using an Olympus BX51 epifluorescence microscope equipped with filters specific for reporter proteins encoded by the dTomato, mCerulean, and EYFP genes as described previously [18]. Digital micrographs of each region were captured with a Hamamatsu camera (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed using the procedures detailed in the next section. Data analysis We first characterized the extent of viral invasion of renal presympathetic circuits using immunoperoxidase localization of infected neurons in brain and spinal cord. The goals of this analysis were to determine if the invasiveness of each virus was equivalent and conformed to the distribution documented in our prior dual infection analysis of renal preautonomic circuitry [20]. To accomplish this we mapped the location of infected neurons in coronal sections through selected coronal planes through the neuraxis using StereoInvestigator image analysis software (version 8; Microbrightfield, Williston, VT). We selected 24 coronal sections that thoroughly sampled the renal preautonomic network across a 6.23 mm portion of the brain stem and five sections through a 0.92 mm portion of diencephalon that contained the PVN (Figure 3). Care was taken to encode the laterality of sections (e.g., left and right) to ensure accurate recording of viral invasion of neural circuits innervating the left (PRV-263) and right (PRV-267) kidneys. Similarly, we matched the rostrocaudal levels of sections to ensure an accurate comparison of viral labeling between cases. These maps allowed a quantitative comparison of the neuroinva- siveness of each virus from the injected kidney (Figure 3) and also revealed the pattern of viral spread through the preautonomic network. To illustrate these maps we faithfully transferred labels of individual infected neurons to templates from the Brain Maps: Structure of the Rat Brain compiled by Swanson [28]. With these quantitative data in hand we then documented the fluorescence profile of infected neurons in the thoracic spinal cord, ventromedial medulla (VMM), rostroventrolateral medulla (RVLM), locus coeruleus (LC), and the paraventricular hypothalamic nucleus (PVN). These regions were selected for analysis because prior investigations demonstrated that they would contain neurons synap- tically dedicated to the ipsilateral injected kidney as well as neurons that collateralize to circuits linked to both kidneys. Comparable coronal sections through these regions in each animal were examined and photographed. The fluorophor profile of all photographed neurons was determined using Adobe Photoshop software; fluorescence emitted by the protein products of the dTomato, mCerulean and EYFP genes was determined by examining color channels selective for each fluorophor. In this manner it was possible to determine with certainty the fluorophors expressed by each infected neuron. Results Our experimental design takes advantage of a well character- ized dual infection paradigm that results in predictable retrograde Conditional Transneuronal Tracing with PRV transneuronal passage of PRV recombinants from the kidneys [20]. The pattern of infection and the distribution of collateralized neurons observed in the present analysis recapitulated the findings documented in that foundational study, which used PRV recombinants expressing unique reporters (PRV-152; EGFP and PRV-BaBlu; b galactosidase) injected into separate kidneys. The predictable pattern of infection produced in this model system provided a strong foundation for the proof-of-principle observa- tions reported in the following sections. Invasive Profiles of PRV Recombinants There is a finite time period after initial infection of a neuron by PRV (about 6 hours) when the cell is permissive to infection by a second strain of PRV [29,30]. Accordingly, we first determined the invasive profiles of PRV-263 and PRV-267 by conducting a quantitative analysis of the spread of each recombinant through the preautonomic network in dual injected animals. We localized infected neurons using immunoperoxidase procedures and ob- tained counts of neurons on each side of the brain using an image analysis system. Immunoperoxidase localization of viral antigens does not distinguish between the recombinants infecting individual neurons but does provide an informed evaluation of the extent of spread of each recombinant through the predominantly lateralized circuitry synaptically linked to each kidney. The temporal kinetics and pattern of invasion of renal preautonomic circuitry for PRV-263 and PRV-267 recapitulates that documented in our prior studies. Replication and transneu- ronal passage of each recombinant produced infection of sympathetic preganglionic neurons in the intermediolateral cell column (IML) of thoracic spinal cord and subsequent retrograde transneuronal passage through synaptically connected neurons in the renal preautonomic network. At four days after kidney infection, PRV immunopositive neurons were largely confined to areas in the brain stem that give rise to direct descending projections to the thoracic spinal cord (e.g., RVLM and A5; data not shown). One day later, the number of infected neurons in regions infected at 4 days increased and the infection spread transneuronally to neurons in other regions of the medulla, midbrain and forebrain (Figure 2A). In every case, the distribution of infected neurons conformed to that previously documented in prior viral transneuronal tracing studies. Importantly, the absence of infected neurons in the dorsal motor nucleus of the vagus (a parasympathetic cell group innervating the visceral) demonstrated that organ-specific transport of virus was not compromised by leakage of viral inoculum into the peritoneal cavity. Figure 3 illustrates the number of infected neurons on the left and right side of the brain in multiple coronal planes sampling the renal preautonomic network in the six cases processed 5 days after kidney injection. The number of infected neurons on the left and right sides of the brain were remarkable consistent in each animal, indicating that both PRV-267 and PRV-263 invaded the preautonomic network at similar rates and extents from each kidney. Additionally, each recombinant infected the same cell groups (e.g., Figures 4, 5, 6, 7). Interestingly, the extent to which the preautonomic network was infected varied to a considerable extent between animals surviving five days. The post inoculation survival interval for these animals ranged from 116 to 119 hours and the animals were all injected and perfused midway through the light phase of the circadian cycle (between 11 am and 3 pm). All injections were made from the same stock of virus of constant titer and a freshly thawed aliquot of virus was used to inject each pair of animals. Similar variations in the magnitude of infection between animals were noted in the study by Cano et al. [20] employing PRV-152 (EGFP PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21141