Transmitter Release From Mammalian Horizontal Cells.

说明(申请人提供)：光感受器突触三联体由光感受器终末、双极细胞树突和水平细胞末端组成，是一种特殊的突触复合体，具有重要意义。从生理上讲，这是光感受器最初传递视觉信息的地方，信息传递的保真度对视觉处理至关重要。水平细胞在双极细胞树突和光感受器终末介导视网膜外区域的抑制性反馈；然而，哺乳动物水平细胞释放递质的机制尚不清楚。例如，突触三联体中的水平细胞末端有相对较少的小而清晰的核心小泡，它们缺乏传统上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，包括囊泡型GABA转运体(VGAT)和SV2a，以及与胞吐相关的突触蛋白，包括SNAP-25和Complin，以及L类型的钙通道，表明GABA是通过囊泡机制释放的。这一机制不同于先前为非哺乳动物水平细胞所描述的GABA质膜转运蛋白机制。 这个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。这项应用将通过检验哺乳动物水平细胞中存在介导囊泡递质释放的细胞结构和生化机制的假设来解决这一目标。这一假设将被检验如下：具体目标1：描述突触三联体中水平细胞末端的小泡的特征。用常规电子显微镜观察支配锥体蒂的水平细胞树突和支配杆状小球的轴突末梢中的囊泡类型、数量和分布。具体目的2：通过RT-PCR和免疫组织化学方法检测a)VGAT和b)VAMP亚型、SV2A、突触素1和2、Rab3亚型以及空泡质子泵在水平细胞末端参与调节胞吐作用的主要囊泡蛋白的分布。具体目的3：通过RT-PCR和免疫组织化学方法检测Synaxin、SNAP-25和Complin 1-4、MUNC 13-1和18以及RIM1的表达，检测参与囊泡对接、启动和融合的关键突触蛋白在水平细胞终末的分布。 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部的功能作用。这些发现将有助于开发治疗与黄斑变性等视网膜疾病相关的视网膜外部病变的治疗策略。

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.