Regulation of neurite growth in purified retina neuronal cultures: effects of PNPF, a substratum-bound, neurite-promoting factor.

The responses of chick embryo retina neurons to the substratum-bound, neurite-promoting factor "PNPF" were studied using glia-free, purified neuronal monolayers. Polyornithine-coated dishes were exposed before cell seeding to either serum-containing culture medium (PNPF(-) substratum) or to the same medium supplemented with 25% rat schwannoma conditioned medium, a source of PNPF (PNPF(+) substratum). The dishes were thoroughly rinsed before receiving a suspension of 8 day chick embryo neural retina cells in serum-free medium. The presence of PNPF on the substratum determined a dramatic increase in the relative frequency of neurite-bearing cells in the cultures. After 6 hours in vitro PNPF(+) cultures contained 45% neurite-bearing cells as compared with 5-7% on PNPN(-) substrata. At 72 hours those values increased to 60% on PNPF(+) and to 40% on PNPF(-) substrata. PNPF(+) cultures also showed longer and/or more highly branched neurites, resulting in the formation of complex neurite networks. Moreover, a cell type characterized by the presence of a very long neurite could be seen on PNPF(+) but not on PNPF(-) substrata. Six hour cultures were used to analyze in more detail the response of retinal neurons to PNPF. Addition of fetal calf serum to the medium determined a concentration-dependent inhibition of neurite formation on PNPF(+) substrata. On the other hand, pretreatment of PNPF(+) substrata with concanavalin A also blocked the neurite-promoting effect of the factor. This concentration-dependent inhibitory effect of concanavalin A could be eliminated by the specific sugar alpha-methyl-D-mannoside. Wheat germ agglutinin, another lectin known to react with PNPF, did not cause any reduction in the neurite-promoting activity of this factor. Wheat germ agglutinin showed neurite-promoting properties of its own in control experiments using PNPF(-) substrata. The results indicate that the target spectrum of PNPF is broader than it was originally thought. Together with other reports from the literature, they also support the perception of neurite development as a cellular activity subject to complex regulatory mechanisms.