CELL COMMUNICATION IN RETINA

视网膜中的细胞通讯

• 批准号: 2710826
• 负责人: RAMON F DACHEUX
• 金额: $ 24.39万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2710826
• 关键词: amacrine cells; cone cell; evoked potentials; fluorescent dye /probe; intercellular connection; laboratory rabbit; light intensity; neuroanatomy; neurophysiology; neurotransmitters; retina; retinal bipolar neuron; retinal ganglion; rod cell; synapses; tissue /cell culture; visual phototransduction; voltage /patch clamp

This study is directed at elucidating the morphological and physiological organization of the inner plexiform layer of the mammalian retina. It focuses on examining the ultrastructure of physiologically identified ganglion cells in the rabbit retina, and will help interpret our existing morphological data pertaining to the synaptic profiles of cone bipolar axonal terminals with amacrine and ganglion cells. Intracellular recordings are obtained under visual observations using infrared illumination and DIC optics on an isolated, superfused retina preparation. These recordings will determine: whether the cells respond to the onset or termination of light in a sustained or transient manner; the nature of their photoreceptor input; and whether the receptive field properties undergo any reorganization with changes in the adaptive state of the cells. Once physiologically identified, the cells are injected with Lucifer Yellow to verify their morphology with the light microscope; the Lucifer is photoconverted to an osmophilic precipitate, which can be visualized with the electron microscope; and serial micrographs of the labeled cell are used in computer graphics to reconstruct the synaptic connections of several major dendrites. Special attention will be focused on ascertaining a relationship between the amount of amacrine and cone bipolar cell input the ganglion cell receives with respect to its physiological responses to light. A new technique has been developed for the simultaneous, ultrastructural examination of two different morphologically and physiologically identified neurons to demonstrate that they form the pre- and postsynaptic processes of a synapse. This method employs the use of separate intracellular injections of HRP and a fluorescent dye (Lucifer Yellow) into different cells which have overlapping dendritic trees and are suspected of synapsing with each other. The tissue is sequentially reacted under two different conditions using two different chromogens: henzidine dihydrochloride is used to react the HRP, and diaminobenzidine is used to form the reaction product during the photoconversion of Lucifer. The fluorescent dye cannot be photoconverted in the presence of any chromogen unless it is maximally excited with its specific wavelength light. These two chromogens produce distinctly different appearing reaction products that can be differentiated at the light and electron microscope. This method will be used to determine whether the DAPI-accumulating, flat cone bipolar cells synapse with off-ganglion cells in sublamina alpha of the IPL, and if the starburst amacrine cells synapse with the on-off directionally selective (DS) ganglion cells. Variations in the synaptic profiles among amacrine, bipolar and DS ganglion cells, for which the preferred-null axis has been identified, will be examined to evaluate the morphological substrate for the physiological models of DS ganglion cells. Finally, by obtaining intracellular recordings from DAPI-accumulating cone bipolar cells, and injecting them with Lucifer to correlate their morphology with the previously examined cells, this study will attempt to provide conclusive evidence that the are off-bipolar cells.

这项研究旨在阐明形态和生理学 哺乳动物视网膜内丛状层的组织。它 专注于检查生理鉴定的超微结构 兔视网膜中的神经节细胞，将有助于解释我们现有的 与锥双极的突触轮廓有关的形态数据 轴突末端带有无链氨酸和神经节细胞。细胞内 在使用红外线的视觉观察下获得录音 在孤立的，超级融化的视网膜制备上进行照明和DIC光学。 这些记录将确定：细胞是对发作还是 以持续或短暂的方式终止光；的本质 他们的感光体输入；以及是否接受场特性 进行任何重组，随着自适应状态的变化 细胞。一旦在生理上鉴定出来，就会注射细胞 Lucifer黄色以光学显微镜验证其形态；这 路西法会光电转换为渗透性沉淀物，这可以是 用电子显微镜可视化；和串行显微照片 标记的单元格在计算机图形中用于重建突触 几个主要树突的连接。 特别关注 确定悬脂和锥量之间的关系 双极细胞输入神经节细胞相对于其接收 生理对光的反应。已经开发了一种新技术 同时进行两种不同的超微结构检查 在形态学和生理上鉴定出神经元，以证明 它们形成了突触的突触前和突触后过程。此方法 采用单独的HRP细胞内注射和A 荧光染料（Lucifer Yellow）成不同的细胞 重叠的树突树，涉嫌与每棵 其他。组织在两个不同条件下依次反应 使用两种不同的染色体：使用Henzidine二氢氯化物来反应 HRP和二氨基苯胺用于形成反应产物 路西法的光转换。荧光染料不能 在任何发色原的存在下都会复制，除非它最大 激发其特定波长光。这两个染色体会产生 可以是明显不同的出现反应产品 在光和电子显微镜下分化。这个方法将是 用于确定dapi蓄积的平锥双极细胞是否用于 IPL的Sublamina alpha中与伴有链球细胞的突触，如果是 Starburst amacrine细胞与on On-Off方向选择性突触 （DS）神经节细胞。无链氨酸之间突触分布的变化， 双极和DS神经节细胞，首选的无轴已成为 确定，将检查以评估形态基材的 DS神经节细胞的生理模型。最后，通过获得 来自DAPI蓄积的锥双极细胞的细胞内记录，并 向他们注入路西法，以将其形态与 先前检查的细胞，本研究将尝试提供结论性 证据表明是双极细胞的。