A growth cone is the motile tip of a growing axon or dendrite. The proper development of neural circuits depends on the pathfinding activities of growth cones. Five behaviors of growth cones are critical in the formation of neural circuits: growth cone migration, turning, branching, retraction and synaptogenesis. Proper axonal pathfinding activities are important for repair of damage to neural circuits. The hypothesis presented here is that a growth cone is a sensory-effector machine that detects environmental cues and responds by regulating these five behaviors. Growth cone migration will be studied in two specific aims: 1. Chick embryo DRG neurons will be cultured on substrata containing alternating stripes of fibronectin (FN) and chondroitin sulfate proteoglycan (CSPG), which induces growth cones to turn away from the CSPG surface. Stable and dynamic microtubules, actin filament bundles and proteins of focal contacts will be localized. The distributions of these components will be related to growth cone behaviors at the CSPG border, resulting in turning away from this substrate. The turning of DRG growth cones toward a positive guidance cue, NGF, presented on 10ym diameter polystyrene beads, will also be examined. Stable and dynamic microtubules and actin filament bundles will be localized, and the distributions of these components related to growth cone behaviors after contact with an NGF-bead. The NGF receptor that mediates this response will be identified, and the cytoplasmic messenger system that may be involved in growth cone turning toward a point source of NGF will be probed. To precisely examine the temporal relationship between microtubule dynamics and growth cone migration, rhodamine-conjugated tubulin will be injected into neurons to visualize individual microtubules in living growth cones as they turn at a CSPG border and as they contact NGF-beads. 2. When DRG growth cones are exposed to 20 mMCa++, there are spikes in [Ca++]i and an inhibition of growth cone migration. Experiments will determine whether the actin filament content of growth cones is reduced after [Ca++]i spikes. To probe the role of the actin-binding protein gelsolin in the disassembly of actin filaments, neurons from the gelsolin mutant mouse will be cultured, and neurons will be exposed to 20 mM Ca++, which induces [Ca++]i spikes. To further examine the roles of several Ca++-regulated proteins, gelsolin, calcineurin, and N-type Ca++ channels will be localized in DRG growth cones at the ultrastructural level.
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