The study of process regeneration by differentiated central neurons of the mammalian visual system would be aided by examining an identified cell in a controlled in vitro environment. This approach requires that cells be isolated, unequivocally identified, and cultured. For this purpose cultures of solitary rat and mouse retinal ganglion cells, identified with fluorescent probes, are being developed in this laboratory. Since ganglion cells are the only retinal neurons that project to other areas of central nervous system, they can be labelled by retrograde transport of fluorescent dyes injected into their projection sites, such as the superior colliculus and lateral geniculate nucleus. To confirm the identity of retrogradely labelled cells, a Thy-1 monoclonal antibody, that among retinal cells in vitro is specific for the ganglion cells, can also be used as a marker with immunofluorescence techniques. The labelled ganglion cells are plated on various culture substrates following dissociation of the retina with papain and trituration.
The specific aims of this proposal are-- (i) To characterize the effect of anti-Thy-1 monoclonal antibody that in preliminary studies has been shown to enhance process regeneration. A variety of monoclonal antibodies against different sites or epitopes on the well-characterized family of Thy-1 glycoproteins will be used to test if the growth-promoting effect is site-specific. Using electron microscopy, an attempt will be made to correlate structure and function by localizing the binding of anti-Thy-1 to nervous tissue at an ultrastructural level. In addition, an anti-idiotype monoclonal antibody against anti-Thy-1 will be used on the retina in an attempt to identify a """"""""naturally occurring"""""""" ligand that recognizes the Thy-1 determinant on the ganglion cells. If such a naturally occurring molecle exists it may enhance process regeneration since in some respects it would be the analog of anti-Thy-1. (ii) To study the effect on Thy- antibody-induced process regeneration of pharmacologically blocking or enhancing specific ionic currents in the cell membrane. Effects on specific ionic currents will also be monitored electrophysiologically using intracellular and patch-clamp recording. (iii) In collaboration with a separately-funded project, the Thy-1 antibodies that are found to promote regeneration in culture will be added to mechanical nerve guides that are used to increase regeneration of the optic nerve in the living animal.
| Krainc, D; Bai, G; Okamoto, S et al. (1998) Synergistic activation of the N-methyl-D-aspartate receptor subunit 1 promoter by myocyte enhancer factor 2C and Sp1. J Biol Chem 273:26218-24 |
| Leifer, D; Dreyer, E B; Lipton, S A (1991) Immunofluorescent characterization of retinal ganglion cell neurites cultured on substrates coated with antibodies against Thy-1. Exp Neurol 113:386-90 |
