A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits J. Patrick Card1*., Oren Kobiler2., Ethan B. Ludmir2, Vedant Desai1, Alan F. Sved1, Lynn W. Enquist2 1 Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Molecular Biology and the Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, United States of America Abstract Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners. Citation: Card JP, Kobiler O, Ludmir EB, Desai V, Sved AF, et al. (2011) A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits. PLoS ONE 6(6): e21141. doi:10.1371/journal.pone.0021141 Editor: Eric J. Kremer, French National Centre for Scientific Research, France Received March 16, 2011; Accepted May 20, 2011; Published June 16, 2011 Copyright: 2011 Card et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by NIH grants 1RC1NS068414, 1RO1HL093134, and P40 RR018604 and NSF grant 0918867. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: card@pitt.edu . These authors contributed equally to this work. Introduction Neurotropic viruses represent popular and powerful tools for defining the identity and organization of synaptically connected neurons [1,2,3,4,5,6,7,8,9]. The method exploits the tropism of these viruses for neurons and their tendency to replicate and spread from neuron-to-neuron via the intimate synaptic contacts through which neurons communicate. Pseudorabies virus (PRV), a DNA swine alpha herpesvirus, is one of the most widely applied viruses for polysynaptic circuit analysis in the rodent nervous system. The extensive use of PRV in such studies is related to the availability of strains of reduced virulence that are transported selectively in the retrograde direction through neural circuits and also express unique reporter proteins (e.g., fluorescent proteins). Restricting PRV replication to defined populations of neurons through cell-specific gene expression is a recent advance of viral transneuronal tracing technology [10]. In an early report, DeFalco and colleagues exploited Cre-Lox site-specific recombination [11] of the viral genome to restrict PRV replication to phenotypically defined neurons and synaptically connected circuits [12]. Repli- cation of the virus constructed for that study (PRV-2001) requires Cre-mediated recombination of the viral genome to remove a floxed stop cassette that prevents transcription of a thymidine kinase gene essential for viral replication in non-mitotic cells. Once the stop cassette is removed, the virus is permanently replication competent and passes transneuronally to infect synaptically linked neurons. This approach has subsequently been employed to define neural circuits synaptically linked to LHRH- [13,14,15] and serotonin- [16] containing neurons. The aforementioned conditional approach is limited by the requirement that Cre be present in first-order neurons infected by PRV-2001. The approach cannot be used if the goal is to define synaptic connections specific to a population of Cre-expressing neurons embedded within a larger circuit, while maintaining target cell specificity of the circuit (e.g., Cre-expressing neurons separated from the injection site by more than one synapse). The recent development of a replication competent PRV recombinant that changes the profile of reporter gene expression when exposed to Cre has circumvented this problem [17,18]. The conditional recombinant, PRV-263, carries the Brainbow 1.0L cassette developed by Lichtman and colleagues [19] and expresses a red dTomato cytoplasmic reporter unless it has been exposed to Cre. PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21141