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

Abstract

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.

The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth ofDrosophilaMushroom Body Neurons Isolated during Metamorphosis

“…Very short neurites (less than ϳ8 m) could not be measured reliably because of limitations in the precision of cursor placement at the ends of a measured segment. Furthermore, on Manduca motor neurons in vitro such short neurites are transient, actin-based filopodia that rarely contain microtubules (Matheson and Levine, 1999) that stabilize neuritic branches (Ahmad et al, 1993;Smith, 1994). Therefore, those neurites with a measured length Ͻ7.8 m were not included in the length and branch number totals for a cell but were tallied separately.…”

“…When the dishes were flooded with medium 2 hr after plating, one dish from each pair received medium containing 1 g/ml 20E (2.1 ϫ 10 Ϫ6 M). This concentration of 20E has been shown to elicit a significant response by Manduca motor neurons in culture (Prugh et al, 1992;Matheson and Levine, 1999) and is within the physiological range estimated to occur in vivo during metamorphosis in Drosophila (Fristrom and Fristrom, 1993). Whether hormone was in a dish was unknown to the individual who scanned them for ␤gal-expressing MB neurons and who later performed the measurements on the photographic images of those cells.…”

“…The relationship between branch number and total neurite length remains constant for Kenyon cells in culture regardless of treatment Manduca pupal motor neurons respond to 20E in vitro by increased branching at growth cones (Matheson and Levine, 1999), resulting in increased branch complexity (Prugh et al, 1992). To determine whether 20E also influences branch complexity of cultured Drosophila pupal Kenyon cells, we examined the relationship between branch number and total neurite length of individual cells grown in the presence or absence of 20E.…”

“…Hence, 20E does not affect branch complexity of cultured Drosophila pupal MB neurons. This contrasts with Manduca motor neurons, which respond to 20E by increased branching at growth cones (Matheson and Levine, 1999). The difference may reflect cell type-specific, hormone-dependent growth responses that fulfill the distinct requirements of these two neuronal populations during metamorphosis.…”

Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons

“…The properties of many classes of Manduca neuronsmotoneurons, interneurons, neurosecretory neurons and sensory neurons -have been characterized in vivo during the different life stages. This work is increasingly complemented by investigations of the same neurons in vitro (e.g., reviewed by Levine and Weeks 1996;see Hayashi and Hildebrand 1990;Zufall et al 1991;Prugh et al 1992;Stengl et al 1992;Streichert et al 1997;Grüne-wald and Levine 1998;Matheson and Levine 1999;Zee and Weeks 2001). For example, it has been demonstrated that the segment-specific pattern of programmed cell death of accessory planta retractor (APR) motoneurons during metamorphosis is a direct, cell-autonomous response to 20E by placing APRs in primary cell culture and exposing them to 20E Hoffman and Weeks 1998;Zee and Weeks 2001).…”