Tracing Synaptic Connectivity onto Embryonic Stem Cell-derived Neurons§

Authors

  • Isabella Garcia,

    1. Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA
    2. Medical Scientist Training Program, Baylor College of Medicine, Houston, Texas, USA
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  • Longwen Huang,

    1. Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA
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  • Kevin Ung,

    1. Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA
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  • Benjamin R. Arenkiel

    Corresponding author
    1. Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA
    2. Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA
    3. Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA
    4. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA
    • Department of Molecular and Human Genetics, Jan and Dan Duncan Neurological Research Institute, Baylor College of Medicine, Houston, Texas 77030, USA

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    • Telephone: 713-798-1960; Fax: 832-825-1240


  • Author contributions: I.G.: experimental design, data collection, and manuscript writing; L.H.: data collection and data analysis; K.U.: data collection; B.A.: experimental conception and design, manuscript writing, and financial support.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS July 31, 2012.

Abstract

Transsynaptic circuit tracing using genetically modified rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it is now possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem cells (ESCs) in vitro, and these properties are under intense investigation toward devising cell replacement therapies. Here, we report the generation of a novel mouse ESC (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ESC-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to synaptically connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest. STEM Cells2012;30:2140–2151

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