Primary culture of crustacean stomatogastric ganglion neurones in a defined medium.

The crustacean stomatogastric ganglion, consisting of about 30 identifiable neurones, produces several distinct patterned outputs governing rhythmic movements of the stomach. This system has become a model for addressing cellular properties and synaptic interactions underlying pattern generation in neural networks (see Selverston and Moulins, 1987). Studies of the system have been limited by the need to use acutely isolated intact or semi-intact ganglion preparations. Also, interesting membrane responses and synaptic interactions occur in the neuropile, but recording from sites other than the somata has been difficult using an intact preparation. Primary culture of dissociated neurones from the ganglion may yield new insights on regional membrane properties and longterm (days) responses to neuromodulators of individual neurones that have regenerated neuritic processes. This paper presents our culture methods and some observations of outgrowth from neurones that have been dissociated from the stomatogastric ganglia of two evolutionarily diverse species, the Maine lobster Homarus americanus (Fig. 1) and a tropical semi-terrestrial crab Cardisoma carnifex (Fig. 2). The only other study on stomatogastric neurones in culture of which we are aware used a freshwater crayfish (Pacifastacus leniusculus), in which neurones were plated in media supplemented with serum (Krenz and Fischer, 1988). The culturing procedure is based on that developed for crustacean X-organ neurones (Graf et al. 1988; Cooke et al. 1989) with a number of modifications that optimize conditions for culturing the larger and more delicate stomatogastric neurones. Ganglia were removed from adult animals with 0.5 cm lengths of connective, and submerged for 5min in an antibiotic crab or lobster saline containing lOOunitsml" penicillin-G, 75 units ml" streptomycin (both from frozen powder) and 0.25/imolml" fungizone (all from Gibco), pH7.6. The dissection and plating were performed in a laminar horizontal flow hood (Baker) using a Wild M5 dissecting microscope equipped with fibre optic illumination. Dissected ganglia were transferred to 35 mm culture dishes containing an