Transmitter Release from Mammalian Horizontal Cells

描述（由申请人提供）：光感受器突触三联体是一个非常重要的突触复合体；从生理上讲，这是视觉信息初始传递的场所，信息传递的保真度对视觉加工至关重要。突触三联体的特征是光感受器末端、上双极细胞树突和横向分布的水平细胞突。水平细胞在产生光感受器和双极细胞周围的抑制性接受野以及其他下游视网膜神经元方面发挥核心作用。水平细胞参与视网膜神经元的光适应、中心周围感受野的形成和颜色对立的产生。囊泡介导的递质释放和质子分泌是外视网膜水平细胞输出的主要候选因素，尽管细胞机制及其作用部位尚未完全了解。该计划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作用。这一目标将通过测试以下假设来解决：1)水平细胞过程及其末端的GABA释放和质子分泌受调节的囊泡机制，以及2)水平细胞通过V- atp酶参与对光感受器的ph敏感作用，以及水平细胞和双极细胞的GABA抑制作用。特异性目标1将测试a)囊泡与质膜的融合是否依赖于Ca2+，并确定b)水平细胞中介导Ca2+信号传导的Ca2+通道亚型的细胞定位和功能特性。特异性目标2将测试a)囊泡与质膜的融合是否依赖于SNARE蛋白，并确定b)介导囊泡胞外分泌的关键囊泡和SNARE蛋白（包括v - atp酶）在水平细胞中的细胞定位和结合伙伴。特异性目的3将测试水平细胞囊泡性胞外分泌是否介导a)通过v - atp酶插入突触囊泡和质膜对光感受器的ph敏感作用，以及b)水平细胞和双极细胞的GABA作用。实验将a)评估水平细胞末端的v - atp酶活性，b)确定ph敏感的水平细胞作用是否由v - atp酶依赖的质子和/或其他ph调节膜蛋白介导，c)确定GABA是否介导水平细胞自分泌和双极细胞前馈作用。利用形态学、电生理学和细胞成像方法的研究将初步阐明水平细胞调节囊泡释放和质子分泌的分子和细胞基础，并将确定水平细胞对视网膜外神经元反应特性的作用。这些研究将进一步了解外视网膜视觉处理的细胞机制，这将有助于开发治疗眼病和损伤的治疗策略。这些目标与国家眼科研究所制定治疗和预防视网膜疾病的治疗方法的健康相关目标是一致的。

DESCRIPTION (provided by applicant): The photoreceptor synapse triad is a synaptic complex of great importance; physiologically, this is the site of the initial transfer of visual information, and the fidelity of information transfer is critically important for visual processing. The synaptic triad is characterized by the photoreceptor terminal, ON-bipolar cell dendrite and laterally distributed horizontal cell processes. Horizontal cells play a central role in generating the inhibitory receptive-field surrounds of photoreceptors and bipolar cells, as well as other, downstream retinal neurons. Horizontal cells participate in light adaptation, formation of center-surround receptive fields and generation of color opponency in retinal neurons. Vesicular-mediated transmitter release and proton secretion are leading candidates underlying horizontal cell output in the outer retina, although the cellular mechanisms and their sites of action are not yet fully understood. The long-term objective of this program is to understand the role of mammalian horizontal cells in visual information processing. This objective will be addressed by testing the hypotheses that 1) a regulated vesicular mechanism underlies GABA release and proton secretion from horizontal cell processes and their endings, and 2) horizontal cells participate in a pH-sensitive action on photoreceptors via V- ATPase, and an inhibitory GABA action on horizontal and bipolar cells. Specific aim 1 will test if a) vesicle fusion with the plasma membrane is Ca2+-dependent, and determine the b) cellular localization and functional properties of the Ca2+ channel subtypes mediating Ca2+ signaling in horizontal cells. Specific aim 2 will test if a) vesicle fusion with the plasma membrane is SNARE-protein dependent, and determine the b) cellular localization and binding partners of key vesicular and SNARE proteins mediating vesicle exocytosis, including V-ATPase, in horizontal cells. Specific aim 3 will test if horizontal cell vesicular exocytosis mediates a) a pH-sensitive action on photoreceptors via V-ATPase insertion into the synaptic vesicle and plasma membrane and b) GABA action at horizontal and bipolar cells. Experiments will a) evaluate V-ATPase activity in horizontal cell endings, b) determine if pH-sensitive horizontal cell action is mediated by V-ATPase dependent protons and/or other pH-regulating membrane proteins and c) determine if GABA mediates horizontal cell autocrine and bipolar cell feed forward action. Studies using morphological, electrophysiological and cellular imaging approaches will initially elucidate the molecular and cellular basis of regulated vesicular release and proton secretion from horizontal cells, and will determine horizontal cell action on the response properties of outer retinal neurons. Relevance these studies will further the understanding of the cellular mechanisms underlying visual processing in the outer retina, which will aid in the development of therapeutic strategies for the treatment of eye disease and injury. These objectives are consistent with the health-related goals of the National Eye Institute for the development of therapeutic approaches for the treatment and prevention of retinal disease.