Visual system development and synaptogenesis

描述（由申请人提供）：我们将分析调节果蝇感光突触形成和功能的基因的作用。我们以前的工作在这些遗传操纵模型感光末梢和它们的目标中间神经元的四分体突触，帮助建立作为苍蝇的功能对应的视网膜的外丛状层的层。我们的长期目标是了解多接触突触（二分体，三分体等）的功能组织，它们是如何形成的，它们表现出的反应性变化，以及神经活动在这些事件中的作用。目前的目标是研究遗传突变体，调节四分体和反馈突触如何形成在感光末梢，无论是在识别postysynaptic目标或以后，当突触细胞器有针对性的网站，这样的接触。我们将研究突变的光感受器，在纯合子苍蝇或全眼马赛克，通过先进的共聚焦和电子显微镜的方法，和我们的技术人员来实现这些。我们正在开发创新的EM断层扫描和冷冻替代方法。我们将研究：A。Dscam和N-cadherin等基因的功能作用，在感光末梢的靶选择过程中起作用;以及imac和dHIP 14，需要将突触细胞器靶向末梢。B。当光感受器的传递被果蝇发动蛋白的温度敏感结构可逆地阻断时，发生在板层反馈突触之间的反应性突触变化。C.高分辨率电磁断层扫描，以确定蛋白质复合物是在突变突触扰动，我们将标记与Fab抗体片段。我们还将继续：D.识别在突触细胞器中表达的蛋白质，使用免疫金方法，或通过标记已识别的蛋白质并通过ReAsH方法标记这些蛋白质。E.使用连续EM和靶向HRP表达分析第二神经桩或髓质远端层中源自感光细胞和板层细胞末端的突触微回路。我们将确定当光感受器的基因Lar或curta突变时，这些电路发生变化的方式。我们提出的研究旨在产生适用于多接触突触的突触发生的基本模型，例如视网膜的二分体和三分体，并帮助确定先天性或营养不良性疾病或视网膜损伤引起的变化的潜在遗传基础，以及视网膜突触之间的反应性重排。

DESCRIPTION (provided by applicant): We will analyze the actions of genes regulating the formation and function of photoreceptor synapses in Drosophila. Our previous work on tetrad synapses at these genetically manipulable model photoreceptor terminals and their target interneurons, helps establish the lamina as the fly's functional counterpart to the retina's outer plexiform layer. Our long-term objective is to understand the functional organization of multiple-contact synapses (dyads, triads, etc.), how these form, the reactive changes they manifest, and the role of neural activity in these events. Current objectives are to study genetic mutants that regulate how tetrad and feedback synapses form at photoreceptor terminals, either during recognition of postysynaptic targets or later, when synaptic organelles are targeted to sites of such contact. We will examine mutant photoreceptors, in homozygous flies or in whole-eye mosaics, by means of advanced methods of confocal and electron microscopy, and our skilled personnel to implement these. We are developing innovative EM tomographic and freeze-substitution methods. We will examine: A. The functional role of genes such as Dscam and N-cadherin, acting during target selection by photoreceptor terminals; as well as imac and dHIP14, required to target synaptic organelles to the terminals. B. The reactive synaptic changes among the lamina's feedback synapses that occur when transmission from photoreceptors is reversibly blocked by a temperature-sensitive construct of Drosophila dynamin. C. High-resolution EM tomography, to identify protein complexes that are perturbed at mutant synapses, which we will label with Fab antibody fragments. We will also continue to: D. Identify proteins expressed at synaptic organelles, using either immuno-gold methods, or by tagging identified proteins and labeling these by the ReAsH method. E. Analyze synaptic micro-circuits in the distal strata of the second neuropile, or medulla, that originate from photoreceptor and lamina cell terminals, using serial-EM and targeted HRP expression. We will identify ways in which these circuits change when photoreceptors are mutant for the genes Lar or curta. Our proposed studies aim to produce a basic model of synaptogenesis applicable to multiple-contact synapses, such as the retina's dyads and triads, and help identify the underlying genetic bases for changes that result from congenital or dystrophic diseases, or retinal damage, and reactive rearrangements to these among the retina's synapses.