Conditioning factor(s) produced by several molluscan species promote neurite outgrowth in cell culture.

The ability to correlate the behaviour of neurones in intact ganglia with events in isolation as has been shown in the leech (Ready & Nicholls, 1979; Fuchs, Nicholls & Ready, 1981), may provide insight into the intrinsic properties of individual neurones. The culture of molluscan neurones is relatively new and the exact procedures for the control of environmental conditions necessary for obtaining neurite outgrowth have not been uniformly established. In early experiments, Chen, von Baumgarten & Takeda (1971) isolated viable identified Aplysia neurones, but were not able to obtain neurite outgrowth. Geletyuk (1977), using dissociated neurones from Lymnaea, demonstrated for the first time the regeneration of neurites in cell culture. Outgrowth has been reported by Kaczmarek, Finbow, Revel & Strumwasser (1979) and Dagan & Levitan (1981) for neurones isolated from several ganglia in Aplysia. Recently we reported (Wong, Hadley, Kater & Hauser, 1981) that isolated neurones from adult central ganglia olHelisoma required the presence of co-cultured, intact Helisoma brains or brain-conditioned medium in order for significant neuritic outgrowth to occur. The need for a growth-promoting factor was also suggested by Proshansky, Schacher & Camardo (1981), who found that addition of Aplysia haemolymph was required for optimal outgrowth of Aplysia neurones. This communication assesses the species-specificity of conditioned medium (CM) for Helisoma, Lymnaea, Biomphalaria and Aplysia. To make CM, isolated brains (two central ganglionic rings per ml lor Helisoma, Biomphalaria or Lymnaea or one entire CNS per 10 ml for Aplysia) were placed in serum-free 50 % Liebowitz medium (L-15) for 72 h with the salts appropriate for each species. For experiments with Helisoma, Biomphalaria or Lymnaea, salt concentrations of the media were: NaCl, 40 mMjKCl, l-7rrtM; CaClz, 4-1 mM; MgCk , 1-5 nun; 5mM-Hepes [4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid, pH7-3]. For Aplysia, salt concentrations were: NaCl, 460mM; KC1, lOmM; CaCh, HITIM; MgCb, 55 HIM; 5 mM-Hepes (pH 7-3). Isolated neurones for culture were obtained