Effects of ecdysteroid on the structure and function of growth cones of identified insect motor neurons

Abstract

Dramatic reorganization of dendrites and axonal terminals is a characteristic feature of neuonal remodelling during metamorphosis in the hawkmoth, Manduca sexta. In thoracic leg motor neurons, as in other neuronal populations, dendritic and axonal arbors regress in late larval stages then regrow during pupal development. Ecdysteroids, the insect steroids that trigger metamorphosis, control both regression and outgrowth in vivo, and stimulate neuritic growth and branching in vitro in leg motor neurons cultured at the beginning of pupal development. To identify potential subcellular targets of ecdysteroid action in these neurons, the present studies examined the dynamic and structural features of branching and their modulation by ecdysteroid in vitro. Delayed treatment of pupal leg motor neurons with ecdysteroid (after four days without treatment) led to a robust enhancement of neuritic branch accumulation accompanied by a subtle effect on total neuritic length. The effect on branching was significant after 4 days of treatment and was stage-dependent: larval leg motor neurons were unaffected. This culture regime accomplished the temporal dissociation of ecdysteroid-induced effects on branching and putative regenerative growth that occurs upon plating. Repeated imaging studies revealed that branch formation occurred almost exclusively at the growth cone, regardless of hormone treatment; interstitial branching was extremely rare. Ecdysteroid treatment significantly enhanced branch formation by growth cones observed for 12 hours, and increased branch retention in growth cone regions over the same interval. Branch formation occurred via two distinct processes, engorgement (of fine protrusions) and condensation (of lamellae); the relative contributions of these mechanisms were unaltered by ecdysteroid. Confocal imaging of the cytoskeleton demonstrated that growth cones consisted of separate microtubule- and actin-based domains, with actin-rich filopodia fringing the microtubule-based growth cone proper. Morphometric analysis revealed that ecdysteroid specifically increased growth cone complexity: treated growth cones were larger and more compact and displayed increased numbers of microtubule-based branches and filopodia. These findings indicate that ecdysteroid enhances neuritic branching by altering growth cone structure and function, and suggest that hormonal modulation of cytoskeletal interactions, directly or via well-known signal transduction cascades, contributes significantly to neuritic remodelling during metamorphosis.