UNIT 3.20 Dissection and Culture of Embryonic Spinal Commissural Neurons Simon W. Moore 1 and Timothy E. Kennedy 1 1 McGill University, Montreal, Quebec, Canada ABSTRACT Studies of spinal commissural neurons have provided substantial insight into the mech- anisms that regulate axon guidance. Explants of embryonic spinal cords and isolated spinal commissural neurons have been important experimental tools for the identifica- tion and characterization of several guidance cues, including netrins, semaphorins, slits, sonic hedgehog, BMPs, and wnts. In this unit, protocols are provided for establishing these explant assays to assess the outgrowth and turning capacity of commissural axons. In addition, methods are included for preparing cultures highly enriched with embryonic commissural neurons, which have been used to probe the biochemical signaling mecha- nisms regulating axon guidance. Curr. Protoc. Neurosci. 45:3.20.1-3.20.17. C  2008 by John Wiley & Sons, Inc. Keywords: spinal commissural neurons  tungsten needle  hanging drop  collagen INTRODUCTION In the late nineteenth century, Santiago Ram´ on y Cajal observed commissural neurons extending axons ventrally towards the floor plate in fixed sections of embryonic chick spinal cord (Ram ´ on y Cajal, 1999). As part of his formulation of the chemotropic model of axon guidance—the theory that chemical cues guide axons—he proposed that the floor plate attracted these axons by secreting factors that guide their growth ventrally. Experimental validation of this hypothesis was obtained ∼100 years later, in part based on the results of the assays presented in this unit (see Basic Protocols 2 and 3; Tessier-Lavigne et al., 1988; Placzek et al., 1990; Kennedy et al., 1994; Serafini et al., 1994). Methods have since been developed to generate cultures highly enriched with embryonic spinal commissural neurons (see Basic Protocol 1), and these have been used in biochemical studies to address the intracellular signaling mechanisms underlying axon guidance to the midline of the embryonic CNS (Bouchard et al., 2004). NOTE: Published reports often employ an embryonic dating scheme where the day after fertilization is recorded as E0. Here, a more common method is used where the morning after fertilization is set as E1 (Bayer and Altman, 1995). Basic Protocols 1 and 2 require E14.5 rat, equivalent to Carnegie stage 17, embryos, while Basic Protocol 3 requires E12.5 rat, equivalent to stage 13, embryos (O’Rahilly et al., 1987). Although explant microdissection is described in this unit using E12.5 and E14.5 rat embryos, it is equally possible to use E11 and E13 mice embryos, or Hamburger & Hamilton stage 17 and 25 chick embryos. NOTE: All procedures using live animals must be reviewed and approved by an Institu- tional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the use and care of laboratory animals. Current Protocols in Neuroscience 3.20.1-3.20.17, October 2008 Published online October 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142301.ns0320s45 Copyright C  2008 John Wiley & Sons, Inc. Cellular and Developmental Neuroscience 3.20.1 Supplement 45