Steroid hormone enhancement of neurite outgrowth in identified insect motor neurons involves specific effects on growth cone form and function.

Dramatic reorganization of dendrites and axonal terminals is a hallmark of neuronal remodeling during metamorphosis in the hawkmoth, Manduca sexta. The dendritic and axonal arbors of leg motor neurons regress in late larval stages, then regrow during adult development. Ecdysteroids, the insect steroids that trigger metamorphosis, control both regression and outgrowth in vivo and stimulate neuritic growth in cultured pupal leg motor neurons. To identify subcellular targets of ecdysteroid action in these neurons, we examined the dynamic and structural features of branching and their modulation by ecdysteroids in vitro. Delayed treatment of pupal leg motor neurons with ecdysteroid led to a robust enhancement of neuritic branch accumulation accompanied by a subtle effect on total neuritic length. Repeated imaging revealed that branch formation occurred almost exclusively at the growth cone; interstitial branching was extremely rare. Ecdysteroid treatment significantly enhanced both the formation and retention of branches at the growth cone. Branches formed via two distinct processes: engorgement (of fine protrusions) and condensation (of lamellae) with the relative contributions of these mechanisms being unaltered by ecdysteroid. Confocal imaging of the cytoskeleton demonstrated that growth cones consisted of microtubule-based domains fringed by actin-based filopodia. Treated growth cones were larger and displayed increased numbers of microtubule-based branches, whereas filopodial density was unaffected. These findings indicate that ecdysteroid enhances neuritic branching by altering growth cone structure and function, and suggest that hormonal modulation of cytoskeletal interactions contributes significantly to neuritic remodeling during metamorphosis.