Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.
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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).
Published Version (Please cite this version)
Arriaga, Gustavo, Joshua J Macopson and Erich D Jarvis (2015). Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling. J Vis Exp(103). 10.3791/50672 Retrieved from https://hdl.handle.net/10161/11146.
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Dr. Jarvis' laboratory studies the neurobiology of vocal communication. Emphasis is placed on the molecular pathways involved in the perception and production of learned vocalizations. They use an integrative approach that combines behavioral, anatomical, electrophysiological and molecular biological techniques. The main animal model used is songbirds, one of the few vertebrate groups that evolved the ability to learn vocalizations. The generality of the discoveries is tested in other vocal learning orders, such as parrots and hummingbirds, as well as non-vocal learners, such as pigeons and non-human primates. Some of the questions require performing behavior/molecular biology experiments in freely ranging animals, such as hummingbirds in tropical forest of Brazil. Recent results show that in songbirds, parrots and hummingbirds, perception and production of song are accompanied by anatomically distinct patterns of gene expression. All three groups were found to exhibit vocally-activated gene expression in exactly 7 forebrain nuclei that are very similar to each other. These structures for vocal learning and production are thought to have evolved independently within the past 70 million years, since they are absent from interrelated non-vocal learning orders. One structure, Area X of the basal ganglia's striatum in songbirds, shows large differential gene activation depending on the social context in which the bird sings. These differences may reflect a semantic content of song, perhaps similar to human language.
The overall goal of the research is to advance knowledge of the neural mechanisms for vocal learning and basic mechanisms of brain function. These goals are further achieved by combined collaborative efforts with the laboratories of Drs. Mooney and Nowicki at Duke University, who study respectively behavior and electrophysiological aspects of songbird vocal communication.
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