Growth cones of developing retinal cells in vivo, on culture surfaces, and in collagen matrices

The outgrowth of axons from the early retina in vivo is compared with that from retinal explants in two types of culture systems. The normal time course of axonal growth along the primordial optic pathway to the optic tectum is characterized, using tritiated proline and horseradish peroxidase (HRP) as anterograde tracers. The rate of axonal elongation in vivo is estimated to be about 32 micron/hr at 22 degrees C. The HRP technique allows visualization of retinal growth cones in vivo. Observations can thus be made on their microanatomy and on the environment through which they navigate. The growth cones of retinal ganglion cells in the embryo have lamellipodia and fairly short filopodia (approximately 10 micron) which are directed forward. The growth cones are found near the pial surface of the brain but do not seem to maintain contact with it. Two culture systems were developed to investigate axonal pathways in vitro. In the first, different substrates and culture media were explored. Results indicate that growth cones prefer a polyornithine substrate over a collagen one. The media that promotes the best neurite outgrowth consists of L15 (60%), fetal calf serum (10%) and Xenopus embryo extract (1 mg/ml). Time-lapse video monitoring of substrate cultures reveals an average rate of outgrowth of about 18 micron/hr with great variability. The growth cones in these cultures are large, flattened, and complex compared to those in vivo, and their filopodia extend in many different directions. The second culture system is a collagen gel infiltrated with growth medium. In these conditions neurite outgrowth more closely mimics that in vivo. The rate is faster than on substrates, and the growth cones appear morphologically similar to those in the embryo. Preliminary experiments using the gel culture system to test for chemotaxis of retinal axons toward their targets failed to demonstrate such an effect.