Axonal branching and growth cone structure depend on target cells

Growth cones provide crucial guidance to neurons in response to appropriate targets and other environmental cues. We have cocultured ciliary neurons with myotubes and utilized antibodies to GAP-43 (a neuron-specific, growth-associated phosphoprotein) and MAP-2 (a cytoskeletal marker for dendrites) together with immunofluorescence microscopy to characterize the changes in patterns and polarity that ciliary nerve growth cones undergo when they contact a "proper" target. Our results show that ciliary neurons plated alone or cocultured with fibroblasts have one or two axons, but, when cocultured with myotubes, most cells have 4 or 5 axons showing GAP-43 immunoreactivity. The mean number of axons per cell soma was 1.9 +/- 0.9 micron2 (SEM) when ciliary neurons were plated alone and 3.4 +/- 0.1 when ciliary neurons were cocultured with myotubes. Differences in growth cone size were readily apparent in these two types of cultures. In coculture with myotubes the neuronal growth cone lamelopodia occupied 20-30 microns2 with an average area of 25.0 +/- 2.3 microns2 (SEM) whereas those neurons plated alone or in the presence of fibroblasts occupied an average area of 56.3 microns2 +/- 4.1 microns2 (SEM). The number of growth cone filopodia depended on culture condition. In nerve-muscle cocultures, the average number of filopodia per growth cone was 3.6 +/- 0.2 (SEM), whereas in ciliary cultures plated in the absence of myotubes the number of filopodia was 6.8 +/- 0.4 (SEM). These results indicate that muscle cells or the factors they release can regulate the growth and topography of axons and their growth cones.