DEVELOPING AND ADULT VISUAL SYSTEM

视网膜脱离是导致视力损害的一个严重原因。获得了一个 对其细胞效应的了解有助于改善对 疾病，并有助于我们对视网膜及其相关的基础知识 与色素上皮的关系。我们最近确认了 长期脱离中的特定细胞和生化变化 动物模型。进一步了解这些更改，请使用 结构、生化和分子技术的结合是一个主要的 这个项目的目标。具体来说，我们将确定：1)何时 中间丝蛋白的上调和蛋白表达的下调 米勒细胞中存在酶和维生素A结合蛋白(通过使用 免疫细胞化学和免疫印迹分析)；2)如果mRNA水平，例如 这些类别中的每一类中的蛋白质随着 蛋白质表达(通过Northern和狭缝印迹分析；适当 MRNAs的细胞表达将通过原位杂交进行研究)； 3)光感受器中视素重新分布的开始和程度(通过 免疫细胞化学)；4)视素合成和外节段更新 继续(通过免疫沉淀和荧光素黄带置换 分析)；5)如果视蛋白mRNA水平改变以及如果视蛋白mRNA分布 视蛋白重新分布或其形态发生变化的细胞 严重干扰(通过Northern、狭缝杂交和原位杂交 研究)；6)增殖反应的程度和鉴定 所涉及的细胞类型(通过连续递送~3H-胸腺嘧啶核苷、LM和EM 放射自显影)；7)如果涉及生长因素(通过 免疫细胞化学)，或者它们是否能模拟脱离的效果(通过 将生长因子注入眼内或将其添加到培养物中 穆勒细胞)；8)如果蛋白质表达或重新分布的变化或 增殖性反应被视网膜阻止或改变 重新连接。 对人类视网膜的突触组织有更深入的了解 将通过用连续切片电子研究突触电路而获得 成人和发育期人视网膜的显微镜观察及高尔基体染色分析 浸渍整装坐骑。在后者中，我们将：1)研究被污染的 用标准高尔基体描述法描述75个现存整体的神经元 技术；2)分析高尔基体染色细胞的突触输入 通过电子显微镜(通过从供体眼睛获得新的死后组织 并使用各种固定方案来开发更好的结构 视网膜的保存)。

Retinal detachment is a serious cause of visual impairment. Gaining an understanding of its cellular effects can help improve management of the disease and contribute to our basic knowledge of the retina and its relationship to the pigment epithelium. We have recently identified specific cellular and biochemical changes in long-term detachments using an animal model. Gaining a further understanding of these changes using a combination of structural, biochemical, and molecular techniques is a major goal of this project. Specifically we will determine: 1) when the upregulation of intermediate filament proteins and the downregulation of enzymatic and vitamin A binding proteins occurs in Muller cells (by using immunocytochemistry and immunoblot analyses); 2) if the mRNA levels,for one protein in each of these classes, changes in concert with the changes in protein expression (by Northern and slot blot analyses; appropriate cellular expression of the mRNAs will be studied by in situ hybridization); 3) the onset and extent of opsin redistribution in photoreceptors (by immunocytochemistry); 4) if opsin synthesis and outer segment renewal continues (by immunoprecipitation and Lucifer yellow band displacement analyses); 5) if opsin mRNA levels change and if opsin mRNA distribution changes in cells that have redistributed opsin or whose morphology is severely disrupted (by Northern, slot blot and in situ hybridization studies); 6) the extent of the proliferative response and identification of the cell types involved (by continuous delivery of 3H-thymidine, LM and EM autoradiography); 7) if growth factors are involved (by immunocytochemistry) or if they can mimic the effects of detachment (by injection of growth factors into the eye or their addition to cultures of Muller cells); 8) if the changes in protein expression or redistribution or the proliferative response are arrested or modified by retinal reattachment. A greater understanding of the synaptic organization of the human retina will be gained by studying synaptic circuitry by serial-section electron microscopy of adult and developing human retinas and the analysis of Golgi- impregnated whole-mounts. In the latter, we will: 1) study the stained neurons in the 75 existing whole-mounts by standard Golgi descriptive techniques, and 2) analyze the synaptic input into the Golgi stained cells by electron microscopy (by obtaining new postmortem tissue from donor eyes and using a variety of fixation protocols to develop better structural preservation of the retina).