Neurites in explant cultures of adult goldfish retina derived from ganglion cells.

We recently described a technique for obtaining neurite outgrowth from explants of larval Xenopus I and adult goldfish retinal As is true of explants from other neural tissues, such as dorsal root ganglion a and spinal cord 12,18, these cultures are comprised of a heterogeneous population of neurons, any of which might give rise to neuritic processes. There was inferential evidence that the neurites in the retinal explants were derived from ganglion cells: they are the only retinal neurons with long, extrinsically projecting axons in vivo, whereas all of the other neuronal types are characterized by short processes confined to the retina. In addition, optic nerve crush prior to explan-tation stimulates neurite production 1,7. This finding further implicates the retinal ganglion cells, since they are then in the process of regenerating their severed axons, and are thus already 'primed' for growth at the time of retinal explantation. Since we wished to pursue further studies using the retinal explant system as a model for optic nerve regeneration, it was essential to establish the identity of the neurites definitively. In the present study we have employed the folowing approaches to determine the source of the neurites: (1) an examination of the histological integrity of the explants, relying on the laminar structure of the retina as an aid in identifying cell types; (2) reduced silver staining to visualize the neurons and their processes; (3) scanning electron microscopic (SEM) observations; and (4) tracing the neurites back to their cells of origin, using the horseradish peroxidase (HRP) technique. Retinal explants from adult goldfish (Carassius auratus), 6-7 cm body length, were prepared as described previously 7. In some cases, 5-fluorodeoxyuridine (Sigma, 10-4 M) was added to the medium to inhibit non-neuronal cell proliferation 4. The substrata used were glass coverslips (Corning, 22 mm) or plastic tissue culture dishes (Nunclon, 35 × 10 mm) coated with either a collagen film 2 or with poly-L-lysine 4,9. Ten to 14 days following optic nerve crush, retinas were removed and cut through their full thickness (approximately 150/~m) into 100/zm wide strips, or, alternatively , into 500 #m squares by means of a Mcllwain chopper as described previously 7. The square pieces were placed on the substratum with the vitreal or photoreceptor surface up, whereas the strips were oriented with the cut surface apposed to the sub