A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.

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 recom...

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Autores principales: J Patrick Card, Oren Kobiler, Ethan B Ludmir, Vedant Desai, Alan F Sved, Lynn W Enquist
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Publicado: Public Library of Science (PLoS) 2011
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spelling oai:doaj.org-article:eb0b53f0ca3146de9cd224ca901596922021-11-18T06:51:58ZA dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.1932-620310.1371/journal.pone.0021141https://doaj.org/article/eb0b53f0ca3146de9cd224ca901596922011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21698154/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Replication 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.J Patrick CardOren KobilerEthan B LudmirVedant DesaiAlan F SvedLynn W EnquistPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 6, p e21141 (2011)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
J Patrick Card
Oren Kobiler
Ethan B Ludmir
Vedant Desai
Alan F Sved
Lynn W Enquist
A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
description 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.
format article
author J Patrick Card
Oren Kobiler
Ethan B Ludmir
Vedant Desai
Alan F Sved
Lynn W Enquist
author_facet J Patrick Card
Oren Kobiler
Ethan B Ludmir
Vedant Desai
Alan F Sved
Lynn W Enquist
author_sort J Patrick Card
title A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
title_short A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
title_full A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
title_fullStr A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
title_full_unstemmed A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
title_sort dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
publisher Public Library of Science (PLoS)
publishDate 2011
url https://doaj.org/article/eb0b53f0ca3146de9cd224ca90159692
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