Revealing Neuronal Circuitry Using Stem Cell‐Derived Neurons

Abstract

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.

G gene-deficient single-round rabies viruses for neuronal circuit analysis

“…By gene targeting cDNA encoding the full complement of starter cell proteins, RABV G, TVA, and tdTomato were introduced at the ROSA 26 locus (Garcia et al, 2012(Garcia et al, , 2013. The derived embryonal stem cells (mESC) revealed high-efficiency differentiation into functional neurons in vitro, and the derived neurons had a high capacity to synaptically connect with primary neuronal cultures.…”

Loss of MeCP2 in immature neurons leads to impaired network integration

“…5B). Although RABV has been used for in vivo neuronal connectivity assays, only very few studies have used it for in vitro connectivity analysis and all of these studies are relatively immature human stem cell differentiated neurons (37)(38)(39)(40). The main reason is that cultured mouse neurons can establish a wide connection with large numbers of other neurons, therefore the number of 'starter neurons' must be kept in low number for effective analysis.…”

How neurons move during action potentials

“…Minute (nanometer-scale) cellular deformations accompanying the action potential have been observed in crustacean nerves (0.3~10 nm) [1][2][3][4] , squid giant axon (~1 nm) 5,6 and, recently, in mammalian neurons (0.2~0.4 nm) 7,8 . These findings illustrate that rapid changes in transmembrane voltage due to the opening and closing of the voltage-gated ion channels have mechanical consequences accompanying action potentials and other changes in cell potential, which may allow non-invasive label-free imaging of neural signals 9 .…”

“…These findings illustrate that rapid changes in transmembrane voltage due to the opening and closing of the voltage-gated ion channels have mechanical consequences accompanying action potentials and other changes in cell potential, which may allow non-invasive label-free imaging of neural signals 9 . However, detecting the nanometer-scale millisecond-long deformations in biological cells is very challenging, and previous observations were limited to a single spot on the axon [1][2][3][4][5]7 or to the boundary of the cell soma 8 . Until now, the entire picture of how neurons move during the action potential was unclear, and the underlying mechanism remains poorly understood.…”