Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing

Abstract

Event Abstract Back to Event Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing Alex S. Ferecskó1*, Zsolt Boldogköi2, Dóra Tombácz2, Balázs Ördög2, Hajime Hirase3, Paul Tiesinga4 and Attila Sík1 1 University of Birmingham, Neuronal Networks Group, School of Clinical and Experimental Medicine, United Kingdom 2 University of Szeged, Department of Medical Biology, Faculty of Medicine, Hungary 3 RIKEN Brain Science Institute, Hirase Research Unit, Japan 4 Radboud University Nijmegen, Donders Centre of Neuroscience, Department of Computational Neuroscience/Neuroinformatics, Netherlands Transsynaptically spreading neurotropic viruses are widely used for analyzing complex neural circuits. The attenuated Bartha (Ba) strain of pseudorabies virus is used as a retrograde transneuronal tracer which is transported to the cell body through axons. We have developed a genetically engineered strain of Ba virus allowing tracing of presynaptic monosynaptic neuronal connections. To restrict labelling to primary connections, the thymidine kinase (TK) gene of the virus has been deleted (TK is a key enzyme for the replication of virus DNA). Cultured cortical neurons were co-transfected with an amplicon-GFP (amp-GFP) construct along with a TK-expression cassette and subsequently infected with the TK-defected Ba (Ba-dTK) virus. The amp-GFP plasmid is packaged into the viral particles as concatamers, and crosses synapses together with the virus labelling presynaptic neurons with GFP. The virus replicates only in the neurons which were transfected with a plasmid containing a TK expressing cassette prior to virus infection. The TK gene containing plasmid does not cross the synapse which results both in the inability of the TK-defected virus to replicate and the inability of the amplicon to be encapsidated in the presynaptic neurons. Amp-GFP was used to visualize presynaptic neurons connected to the targeted neurons. Neurons were cultured into multifluidic cell platforms allowing pre- and postsynaptic neurons to be spatially segregated. Neurons are connected via microgrooves that are only accessible for axons, permitting complete fluidic isolation of neighbouring wells. Fluorescent microscopy revealed amp-GFP expressing transsynaptically labelled neurons contralateral to the infected well. Quantitative assessment of the retrogradely labelled neurons confirmed that the number of labelled neurons were constant and did not show further increases 48-72hours after Ba-dTK infection indicating monosynaptic transsynaptic spreading of the virus. This work was supported by HFSP. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Abstracts Citation: Ferecskó AS, Boldogköi Z, Tombácz D, Ördög B, Hirase H, Tiesinga P and Sík A (2010). Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00203 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 May 2010; Published Online: 03 May 2010. * Correspondence: Alex S Ferecskó, University of Birmingham, Neuronal Networks Group, School of Clinical and Experimental Medicine, Birmingham, United Kingdom, a.ferecsko@bham.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Google Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Google Scholar Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík PubMed Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.