Culturing neurons from the snail Helisoma

The large neurons of the freshwater snail Helisoma trivolvis provide a unique preparation to study cytoskeletal mechanisms involved in neuronal growth and axon guidance. When placed into culture, these neurons form large growth cones in which cytoskeletal components and their dynamics can be analyzed with high-spatial resolution. Moreover, these growth cones display all of the dynamic features characteristic of growing axons, including advance, pause, collapse, and turning, allowing the correlation of cell biological mechanisms with growth cone motility. This chapter describes complete procedures for culturing Helisoma neurons, including snail dissection, enzymatic treatments, removal of neurons, and necessary solutions, equipment, and supplies. Techniques are presented to culture Helisoma neurons by the extraction and transfer of individual neurons to culture dishes. A newer technique to dissociate neurons from whole ganglia is also described. In addition, methods to culture neurons on two substrates are presented. Culturing on polylysine in defined medium produces large, but nonmotile growth cones for cytoskeletal analysis, whereas culturing on polylysine in conditioned medium allows growth and motility for behavioral analysis. Recent tests suggest a new, simpler formulation for the medium used to culture Helisoma neurons that does not require the special-order medium that was previously used for cultures. These procedures make it feasible for someone inexperienced to successfully culture Helisoma neurons for use in a variety of experiments.