The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis

Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20- hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, we hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB- restricted reporter gene expression, we identified Kenyon cells in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2-4 d. Morphometric analysis demonstrated that MB neurons exposed to 20E had significantly greater total neurite length and branch number compared with that of MB neurons grown without hormone. The relationship between branch number and total neurite length remained constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis.