Protocol

Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons

Department of Physiology and Cellular Biophysics and Department of Neuroscience, Columbia University, New York, NY 10032, USA

¹Corresponding author (abm1{at}columbia.edu)

INTRODUCTION

Dorsal root ganglion (DRG) neurons grow readily alone or in coculture with embryonic dorsal horn (DH) neurons and even appear to enhance survival of the DH cells. Furthermore, DRG neurons grow long neurites in the presence of nerve growth factor, forming many glutamatergic synapses onto DH neurons, if present. Otherwise (i.e., in monocultures), no synapses can be detected electrophysiologically. When preparing cocultures, use DRGs already removed from the same embryos used to prepare DH neurons, but after dissecting the dorsal horns; the DRGs are less sensitive to delay in dissection and dissociation, as long as they are left in culture medium. Microisland and mass cultures require different proportions of DRG and dorsal horn neurons. Synaptic interactions in the microisland culture of DRG and DH neurons are more intense than mass culture because the growing, branching DRG axons are confined to a limited area, and thus make many synapses onto each target DH neuron.