Transmitter Release From Mammalian Horizontal Cells.

描述（由申请人提供）：光感受器突触三联体由光感受器末端、双极细胞树突和水平细胞末端组成，是一种非常重要的特化突触复合体。在生理上，这是从光感受器的视觉信息的初始传输的网站和信息传输的保真度是至关重要的视觉处理。水平细胞介导的抑制性反馈外视网膜双极细胞树突和感光末梢，然而，哺乳动物水平细胞的递质释放的机制知之甚少。例如，在突触三联体中的水平细胞末端具有相对较少的小的、透明的核心囊泡，并且它们缺乏常规定义的突触前膜特化。相反，这些细胞表达已建立的突触囊泡蛋白，包括囊泡GABA转运蛋白（VGAT）和SV 2A，突触蛋白的补充，包括SNAP-25和复合蛋白，与胞吐和L型Ca 2+通道，表明GABA是通过囊泡机制释放的。这种机制不同于以前建立的GABA质膜转运机制描述的非哺乳动物水平细胞。 本项目的长期目标是了解哺乳动物水平细胞在视觉信息处理中的功能作用。本申请将解决这一目标，通过检查的假设，即介导囊泡递质释放的细胞结构和生化机制存在于哺乳动物水平细胞。具体目标1：表征突触三联体中水平细胞末端的囊泡。将使用常规电子显微镜检查支配视锥梗的水平细胞树突和支配视杆小球的轴突末端中的囊泡类型、数量和分布。具体目标2：通过RT-PCR和免疫组织化学测定a）VGAT和B）VAMP同种型、SV 2A、突触结合蛋白1和2、Rab 3同种型和空泡质子泵的表达，检查参与水平细胞末梢中调节胞吐作用的主要囊泡蛋白的分布。具体目标3：通过测定突触融合蛋白、SNAP-25和复合蛋白1-4、Munc 13-1和18以及RIM 1的表达，检查水平细胞末端中参与调节递质释放的囊泡对接、启动和融合的关键突触蛋白的分布。RT-PCR和免疫组织化学。 这些研究将进一步阐明水平细胞释放递质的结构和生化基础，从而更好地了解水平细胞在视网膜外层的功能作用。这些发现将有助于开发治疗策略，用于治疗与视网膜疾病（如黄斑变性）相关的外层视网膜病理变化。

DESCRIPTION (provided by applicant): The photoreceptor synaptic triad, consisting of a photoreceptor terminal, bipolar cell dendrites and horizontal cell endings, is a specialized synaptic complex of great importance. Physiologically, this is the site of initial transfer of visual information from photoreceptors and the fidelity of information transfer is critically important for visual processing. Horizontal cells mediate inhibitory feedback in the outer retina at bipolar cell dendrites and photoreceptor terminals; however, the mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. For instance, horizontal cell endings in the synaptic triad have relatively few small, clear-core vesicles and they lack conventionally defined presynaptic membrane specializations. In contrast, these cells express established synaptic vesicle proteins, including the vesicular GABA transporter (VGAT) and SV2A, a complement of synaptic proteins, including SNAP-25 and complexin, that are associated with exocytosis, and L-type Ca2+ channels, suggesting GABA is released by a vesicular mechanism. This mechanism differs from a previously established GABA plasmalemmal transporter mechanism described for non-mammalian horizontal cells. The long-term objective of this project is to understand the functional role of mammalian horizontal cells in visual information processing. This application will address this objective by examining the hypothesis that the cellular structure and biochemical machinery that mediate vesicular transmitter release are present in mammalian horizontal cells. This hypothesis will be examined as follows: Specific aim 1: Characterize vesicles in horizontal cell endings in the synaptic triad. Vesicle type, number and distribution in horizontal cell dendrites that innervate cone pedicles and axonal terminals that innervate rod spherules will be examined with conventional electron microscopy. Specific aim 2: Examine the distribution of the principal vesicular proteins which are involved in regulated exocytosis in horizontal cell endings by determining the expression of a) VGAT and b) VAMP isoforms, SV2A, synaptotagmin 1 and 2, Rab3 isoforms, and the vacuolar proton pump with RT-PCR and immunohistochemistry. Specific aim 3: Examine the distribution of key synaptic proteins, which participate in vesicle docking, priming and fusion in regulated transmitter release, in horizontal cell endings by determining the expression of syntaxin, SNAP-25 and complexin 1-4, Munc 13-1 and 18, and RIM1 with RT-PCR and immunohistochemistry. These studies will further elucidate the structural and biochemical basis of transmitter release from horizontal cells, thus providing a better understanding of the functional role of horizontal cells in the outer retina. These findings will aid in the development of therapeutic strategies for the treatment of pathological changes in the outer retina related to retinal disease, such as macular degeneration.