Factors that influence the development of cultured neurons from the brain of the moth Manduca sexta

During metamorphic adult development, neurons and glial cells in the developing olfactory (antennal) lobes of the moth undergo characteristic and extensive changes in shape. These changes depend on an interplay among these two cell types and ingrowing sensory axons. All of the direct cellular interactions occur against a background of changing steroid hormone titers. Antennal-lobe (AL) neurons dissociated from stage-5 (of 18 stages) metamorphosing animals survive at least 3 wk in primary cell culture. We describe here the morphological influences on AL neurons of (1) exposure to the steroid hormone 20-hydroxyecdysone, (2) exposure to sensory axons, and (3) interactions among the AL neurons. Cultured AL neurons respond only weakly, if at all, to 20-hydroxyecdysone. They do, however, show greater total outgrowth and branching when they had been exposed in vivo to sensory axons. Because there is no direct contact between some of the neuronal types and the sensory axons at the time of dissociation, the increase in outgrowth must have been mediated via a diffusible factor(s). When AL cells (neurons and glia) are plated at high density in low volumes of medium, or when the cells are plated at low density but in the presence of medium conditioned by high-density cultures, neurite outgrowth and cell survival are increased. Nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor-basic (bFGF), transforming growth factor- β (TGF_β) and insulin-like growth factor (ILGF) had no obvious effect on neuronal morphology and thus are unlikely to underlie these effects. Our results suggest that the mature shape of AL neurons depends on developmental interactions among a number of diffusible factors.