Mouse embryonic stem cell (mESC)‐derived neurons are a renewable cell source for investigation of neuronal circuits. Engineering circuit‐tracing components into stem cells facilitates studies on mechanisms of synaptic coupling and circuitogenesis. This unit details methods for the generation of mESC‐derived neurons harboring trans‐synaptic viral tracing elements, which are used for investigation of synaptic connections within circuits in vitro, ex vivo, and in vivo. The first protocol describes procedures for feeder‐free passaging of mESCs, modified to carry reporter and rabies virus tracing elements. The second protocol describes in vitro generation of neurons from these ESCs. The last protocols describe the use of ESC‐derived neurons as “source cells” for rabies virus circuit‐tracing to identify inputs onto synaptically connected neurons. Given the broad applicability, these protocols can be applied to investigate the ability of in vitro‐derived neurons to establish/maintain synaptic connections in disease models, and/or with human‐induced pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 25:2D.15.1‐2D.15.18. © 2013 by John Wiley & Sons, Inc.

The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons, now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs toward investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.