A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits

Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS ONE (Impact Factor: 3.23). 06/2011; 6(6):e21141. DOI: 10.1371/journal.pone.0021141
Source: PubMed


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.

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    • "The availability of these reporter viruses opened the door for dual infection experiments that define neurons whose axons collateralize within complex circuits to innervate multiple neurons (Billig et al. 2000; Cano et al. 2004; Toth et al. 2008). In addition, the recent exploitation of Cre-lox technology to construct alpha herpesviruses that replicate conditionally or carry conditional reporter genes now permits the identification of neural networks synaptically connected to phenotypically defined neurons (Card et al. 2011a, b; Lo and Anderson 2011; DeFalco et al. 2001; Campbell and Herbison 2007a, b; Yoon et al. 2005; Braz et al. 2002, 2009). While it is noteworthy that the host range of PRV includes all mammals and some birds, it does not infect higher primates, as does the human pathogen herpes simplex virus (HSV) (Pomeranz et al. 2005; Gustafson 1975). "
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