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Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.

dc.contributor.author Arriaga, Gustavo
dc.contributor.author Macopson, Joshua J
dc.contributor.author Jarvis, Erich D
dc.coverage.spatial United States
dc.date.accessioned 2015-12-11T01:40:27Z
dc.date.issued 2015-09-14
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/26436639
dc.identifier.uri https://hdl.handle.net/10161/11146
dc.description.abstract Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).
dc.language eng
dc.publisher MyJove Corporation
dc.relation.ispartof J Vis Exp
dc.relation.isversionof 10.3791/50672
dc.subject Animals
dc.subject Biotin
dc.subject Cholera Toxin
dc.subject Dextrans
dc.subject Genes, Reporter
dc.subject Green Fluorescent Proteins
dc.subject Herpesvirus 1, Suid
dc.subject Mice
dc.subject Neural Pathways
dc.subject Neurons
dc.subject Pseudorabies
dc.subject Staining and Labeling
dc.subject Swine
dc.subject Synapses
dc.title Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.
dc.type Journal article
duke.contributor.id Jarvis, Erich D|0205264
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/26436639
pubs.issue 103
pubs.organisational-group Basic Science Departments
pubs.organisational-group Duke
pubs.organisational-group Duke Institute for Brain Sciences
pubs.organisational-group Institutes and Provost's Academic Units
pubs.organisational-group Neurobiology
pubs.organisational-group School of Medicine
pubs.organisational-group University Institutes and Centers
pubs.publication-status Published online
dc.identifier.eissn 1940-087X


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