Transmitter Release from Mammalian Horizontal Cells

描述(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；从生理学上讲，这是视觉信息最初传递的地方，信息传递的保真度对视觉处理至关重要。突触三联体的特征是光感受器终末、双极细胞树突和横向分布的水平细胞突起。水平细胞在光感受器和双极细胞以及其他视网膜下游神经元周围的抑制性感受场环境中起着核心作用。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抗的产生。囊泡介导的递质释放和质子分泌是视网膜外水平细胞输出的主要候选因素，尽管细胞机制和它们的作用部位尚未完全清楚。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作用。为了达到这一目标，我们将检验以下假设：1)水平细胞突起及其末端的GABA释放和质子分泌是受调控的囊泡机制，2)水平细胞通过V-ATPase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。具体目标1将测试a)囊泡与质膜的融合是否依赖于钙离子，并确定b)水平细胞中介导钙信号的钙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SNARE蛋白，并确定b)在水平细胞中介导囊泡胞吐的关键囊泡和SNARE蛋白的细胞定位和结合伙伴，包括V-ATPase。特殊目的3将测试水平细胞囊泡吐出是否通过将V-ATPase插入到突触小泡和质膜来调节对光感受器的pH敏感作用，以及b)对水平和双极细胞的GABA作用。实验将a)评估水平细胞末端的V-ATPase活性，b)确定pH敏感的水平细胞的作用是否由V-ATPase依赖的质子和/或其他pH调节的膜蛋白介导，c)确定GABA是否介导水平细胞的自分泌和双极细胞的前馈作用。利用形态、电生理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。相关性这些研究将进一步了解外层视网膜视觉处理的细胞机制，这将有助于制定治疗眼部疾病和损伤的治疗策略。这些目标与国家眼科研究所开发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.