NEURONAL SURVIVAL AND DIFFERENTIATION IN RETINAL EXPLANT

发育中的神经元的生存和分化取决于一个 充足的可扩散的营养分子的供应。这些分子可能是 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。一个这样的营养分子家族 它存在于视网膜神经节细胞的中央靶点，以及 视网膜本身，就是神经营养因子。神经营养因子[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]已被证明影响存活和 体内多种视网膜神经元的表型分化。这个 拟议的研究将进一步表征特定的 神经营养因子对视网膜内神经元存活和分化的影响 在视网膜发育的体外模型中。这种独特的外植体模型 将提供一种系统来检查神经化学物质 中华绒毛虫的分化和树突形态发育 小白蛋白免疫反应(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 (暗箱)途径--对个别神经营养因子的反应。进一步 研究，使用细胞内注射荧光黄染料到 已鉴定的视网膜神经节细胞，将提供对 体外培养视网膜神经节细胞的树突状形态成熟 外植体培养在特定神经营养因子存在的情况下保持。 然后将进行研究，以探索神经营养因子的作用。 视网膜神经元的存活。在发育过程中，许多神经元衰竭 为了获得足够的营养支持经历细胞程序性死亡 (细胞凋亡)，一种需要表达特定基因的活跃事件 细胞死亡基因。视网膜外植体培养将被诱导进行 停用血清或钙通道治疗引起的细胞凋亡 对抗者。细胞死亡的程度将由DNA决定 片段化分析(TUNEL技术)。这种方法，也将被用于 检测特定神经营养因子保护神经元的能力 诱导细胞死亡。最后，研究将探索这一现象的机制。 通过检测抑制细胞的基因的作用来实现保护作用 死亡计划，BCL-2。Bcl2的表达将被治疗所阻断 将反义寡核苷酸定向到bcl-2mRNA，并 对神经营养素介导的存活的影响将被确定。全 了解神经营养因子的作用及其作用机制， 将为神经营养因子的潜在治疗用途提供深入的见解 治疗视网膜退行性疾病。

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.