NEURONAL SURVIVAL AND DIFFERENTIATION IN RETINAL EXPLANT

发育中的神经元的存活和分化取决于 充足的可扩散营养分子供应。这些分子可以 由神经元的轴突靶组织提供，或者由 周围的神经元或神经胶质细胞。其中一个营养分子家族 它存在于视网膜神经节细胞的中心靶点， 视网膜本身就是神经营养素。神经营养素[即神经 生长因子（NGF），脑源性神经营养因子（BDNF）， 神经营养因子-3（NT-3）和NT-4]已显示影响存活， 表型分化的各种视网膜神经元，在体内。的 拟议的研究将进一步描述具体的影响， 神经营养因子对视网膜内层神经元存活和分化的影响 在视网膜发育的体外模型中。 这种独特的外植模型 将提供一个系统来检查神经化学物质 分化和树突状形态发育 小清蛋白免疫反应（IR）、AII无长突细胞和蛋白激酶 C-IR，视杆双极细胞--视杆的中间神经元 （暗视）通路-响应于个体神经营养因子。 进一步 研究，使用细胞内注射荧光黄染料， 确定视网膜神经节细胞，将提供一个分析， 视网膜神经节细胞体外树突状形态成熟 在特定神经营养因子存在下维持外植体培养物。 然后将进行研究，以探讨神经营养素的作用， 对视网膜神经元存活的影响在发育过程中，许多神经元 接受足够的营养支持，经历程序性细胞死亡 细胞凋亡（apoptosis）是一种需要表达特异性的细胞凋亡的活跃事件。 细胞死亡基因将诱导视网膜外植体培养物经历 通过血清撤回或用钙通道处理的细胞凋亡 拮抗剂细胞死亡的程度将由DNA决定 片段化测定（TUNEL技术）。这种方法也将用于 检查特定神经营养因子保护神经元免受 诱导细胞死亡。最后，研究将探讨这一机制 通过检测抑制细胞的基因的作用来研究保护作用 死亡程序bcl-2 bcl-2的表达将被治疗阻断 用针对bcl-2 mRNA的反义寡核苷酸， 将确定对神经营养因子介导的存活的影响。所有 了解神经营养因子的作用及其作用机制， 将为神经营养素的潜在治疗用途提供见解 视网膜变性疾病的治疗。

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.