Tracing synaptic connectivity onto embryonic stem cell-derived neurons.

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.

SUBMITTER: Garcia I 

PROVIDER: S-EPMC3458586 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature