In the presence of Cre, the red reporter gene is removed and either the cyan (mCerulean) or yellow (EYFP) reporter is expressed. It is important to emphasize that each virus can express only a single reporter (before or after Cre-recombination) but that infected neurons can replicate more than one viral genome, resulting in a mixed reporter phenotype of some infected neurons. We recently documented the utility of this approach for circuit analysis by combining PRV-263 infection with lentivirus-mediated expression of Cre in phenotypically-defined, anatomically localized, and projection-specific populations of neurons [18]. In this report we document a dual infection transneuronal tracing approach to identify neurons synaptically linked to collateralized neurons within complex networks. The method takes advantage of the Cre-conditional reporter expression of PRV-263 and a new strain of PRV (PRV-267) that expresses both Cre and an mRFP-capsid fusion protein (VP26-mRFP). Injection of PRV-267 and PRV-263 into separate kidneys using a well- characterized dual infection paradigm [20] produced unique markers of collateralized neurons synaptically linked to both kidneys (nuclear mRFP and conditional fluorescent reporter expression from the Brainbow cassette) as well as neurons infected by replication and transneuronal passage of progeny virus from those neurons (only Brainbow reporters). This approach expands the utility of dual infection viral transneuronal tracing paradigms by providing a means of distinguishing collateralized neurons within complex networks from the neurons that are antecedent to them presynaptically. Materials and Methods Ethics Statement All experimental procedures involving animals conformed to regulations stipulated in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh IACUC (protocol number: 0909666), Recombinant DNA Committee (reference number: 112-09), and Division of Environmental Health and Safety (protocol number: 0909666). The in vitro experiments used to construct and characterize PRV- 267 were conducted at Princeton University and approved by the Recombinant DNA Technology Committee (MUA # 912). Animals Adult male rats (Harlan Sprague-Dawley) weighing 250 to 320 grams at the time of viral injection were used for in vivo experiments conducted in a Biosafety Level 2 certified laboratory. Animals were single housed and lived within this facility after virus injection. Photoperiod (12 hours light; light on at 0700) and temperature (22–25uC) were standardized and animals had free access to food and water. PRV Recombinants The genomic organization of the recombinants and related strains of PRV are illustrated in Figure 1. The preparation of PRV-263, a PRV-Bartha recombinant carrying the Brainbow 1.0L cassette in the US4 (gG) locus (Figure 1C), has been previously described [17]. PRV-267, a PRV-Bartha recombinant expressing Cre-recombinase and a red fluorescent protein-tagged VP26 capsid protein, is a new virus constructed for this study. Preparation of a Cre-containing plasmid (pEL2) and construction of the virus are described below. To construct the pEL2 plasmid, the Cre-recombinase coding region, including an N-terminal nuclear localization signal and 133-base-pair synthetic intron was amplified by PCR from pBecker3 (a self-recombining bacterial artificial chromosome) as Conditional Transneuronal Tracing with PRV previously described [21]. Two PCR primers were designed with a KpnI restriction site in the sequence homologous to the 59 Cre open reading frame (ORF) (59-GGGGTACCATGCCCAAGAA- GAAGAGGAAG) and an XbaI site in the sequence complemen- tary to the 39 Cre ORF (59-GCTCTAGACATATCGCCATC- TT CCAGCAG). The eGFP ORF of pEGFP-N1 was removed through KpnI/XbaI digestion, followed by ligation with the am- plified Cre ORF. The resulting plasmid, pEL2, contained the Cre- recombinase coding region under immediate-early human cyto- megalovirus (hCMV) promoter control. Restriction fragment analysis and nucleotide sequencing verified the structure of the plasmid. PRV-267 was constructed as follows. We first constructed PRV- 266, a PRV-Bartha strain expressing mRFP-VP26 and diffusible eGFP, through co-infection of porcine kidney epithelial (PK-15) cells with PRV-152 (a PRV-Bartha strain encoding a diffusible eGFP under an immediate-early hCMV promoter from the US4 locus, [22]) and PRV-756 (a mRFP-VP26 fusion protein, [23]). VP26 is a surface capsid protein encoded by the UL35 gene and the fusion protein incorporating mRFP labels viral capsids intensely, thereby providing a unique marker of neurons replicating PRV-267. We selected viral recombinants expressing both mRFP-VP26 and diffusible eGFP; sequential rounds of plaque purification of this virus, PRV-266, were then performed. We then co-transfected PRV-266 nucleocapsid DNA (purified as previously described, [24]) with linearized pEL2 in PK15 cells. Homologous recombination resulted in PRV-267. We selected viral recombinants expressing mRFP-VP26, but not eGFP, to distinguish PRV-267 from PRV-266 and conducted sequential rounds of plaque purification to isolate PRV-267. Experimental design The design of the experiment is illustrated in Figure 2A. Eight animals were included in the study. Animals were injected in pairs on different days and fresh aliquots of virus from the same viral stock were thawed for each pair of injections. Animals were deeply anesthetized using isoflurane and each kidney was exposed by a retroperitoneal approach. A total of 2 ml of PRV-263 was injected into the parenchyma of the left kidney (4 injections of 0.5 ml per site) using a 10 ml Hamilton syringe. An equivalent volume of PRV-267 was injected into the right kidney using the same procedure. We made an effort to standardize injections between animals by injecting at four similar sites along the greater curvature of each kidney and by marking the needle of the Hamilton syringe so that it penetrated the kidney parenchyma approximately 4 mm at each injection site. After injection, surgical incisions were sutured closed and animals received a subcutaneous injection of analgesic (Ketofen; 2 mg/kg). Upon full recovery from anesthesia animals were returned to their home cages in the BSL 2 laboratory where they lived for the balance of the experiment. The purified stocks of the viruses had concentrations of 3.46108 pfu/ ml (PRV-263) and 56108 pfu/ml (PRV-267). Tissue Preparation Animals deeply anesthetized with sodium pentobarbital were perfused transcardially with paraformaldehyde-lysine-periodate fixative [25] four (n = 2) and five (n = 6) days post inoculation. Aldehyde fixed tissues were postfixed and cryoprotected. The brain was sectioned with a freezing microtome at 35 mm/section through its rostrocaudal extent. The spinal cord was divided into cervical, thoracic and lumbosacral divisions and sectioned horizontally at 40 mm/section. Tissue was stored in cryoprotectant [25] at 220uC prior to immunocytochemical analysis. Details of all of these procedures have been published [26]. PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21141