The survival and differentiation of developing neurons depends upon an adequate supply of diffusible, trophic molecules. These molecules may be provided by the axonal target tissue of neurons or alternatively, by surrounding neurons or glial cells. One such family of trophic molecules that is present in central targets of retinal ganglion cells, and in the retina itself, is the neurotrophins. The neurotrophins [namely, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4] have been shown to affect the survival and phenotypic differentiation of a variety of retinal neurons, in vivo. The proposed studies will further characterize the effects of specific neurotrophins on the survival and differentiation of inner retinal neurons in an in vitro model of retinal development. This unique explant model will provide a system in which to examine the neurochemical differentiation and dendritic morphological development of the parvalbumin-immunoreactive (IR), AII amacrine cell and the protein kinase C-IR, rod bipolar cell--well characterized interneurons of the rod (scotopic) pathway--in response to individual neurotrophins. Further studies, using intracellular injection of Lucifer yellow dye into identified retinal ganglion cells, will provide an analysis of the in vitro dendritic morphological maturation of retinal ganglion cells in explant cultures maintained in the presence of specific neurotrophins. Studies will then be undertaken to explore the role of the neurotrophins on retinal neuronal survival. During development, many neurons that fail to receive adequate trophic support undergo programmed cell death (apoptosis), an active event that requires the expression of specific cell-death genes. Retinal explant cultures will be induced to undergo apoptosis by serum withdrawal or treatment with a calcium channel antagonist. The degree of cell death will be determined by a DNA fragmentation assay (TUNEL technique). This method, will also be used to examine the ability of specific neurotrophins to protect neurons from induced cell death. Finally, studies will explore the mechanism of this protective effect by examining the role of a gene that suppresses the cell death program, bcl-2. Expression of bcl-2 will be blocked by treatment with antisense oligonucleotides directed to the bcl-2 mRNA, and the effects on neurotrophin-mediated survival will be determined. All understanding of the role of neurotrophins, and their mechanism of action, will provide insights into the potential therapeutic uses of neurotrophins in the treatment of retinal degenerative diseases.
