title: Advanced tracing tools: functional neuronal expression of virally encoded fluorescent calcium indicator proteins.
abstract: Pseudorabies virus (PrV) strains such as PrV-Bartha and its marker protein-expressing variants have been used in numerous studies as retrograde transneuronal tracing tools, defining the synaptic organization of mammalian neuronal circuits. However, the possibilities for functional examination of virus-infected neurons are limited to electrophysiological approaches or bulk loading strategies using calcium-sensitive dyes. Herein we report the generation and functional characterization of three PrV-Bartha-derived recombinant virus mutants that express different fluorescent calcium indicator proteins (FCIPs). All three generated virus recombinants are able to infect murine trigeminal neurons and express the corresponding FCIP (GCaMP2, camgaroo-2, or inverse pericam). Functionality of these virally expressed constructs was verified by using confocal Ca-imaging technologies. These FCIP-expressing virus recombinants provide a new tool for the functional analysis of whole circuits of synaptically connected neurons in vitro and in vivo.
authors.affiliation: Lehrstuhl für Zellphysiologie, Fakultät für Biologie und Biotechnologie, Ruhr-Universität, Bochum, Germany. Markus.Rothermel@rub.de
authors.forename: Markus
authors.initials: M
authors.lastname: Rothermel
authors.forename: Daniela
authors.initials: D
authors.lastname: Brunert
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.forename: Matthias
authors.initials: M
authors.lastname: Luebbert
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
authors.forename: Hanns
authors.initials: H
authors.lastname: Hatt
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D051379:Mice; D009434:Neural Pathways; D009445:Neuroanatomy / Q000379:methods*; D009474:Neurons / Q000502:physiology / Q000821:virology*; D009482:Neurophysiology / Q000379:methods; D018408:Patch-Clamp Techniques; D011557:Pseudorabies / Q000473:pathology / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013569:Synapses / Q000502:physiology / Q000821:virology; D012668:Trigeminal Ganglion / Q000166:cytology
chemical_list: D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D002118:Calcium