Visual system development and synaptogenesis

描述（申请者提供）：我们将分析果蝇中调节光感受器突触形成和功能的基因的作用。我们之前对这些遗传可操纵的模型光感受器终端及其目标中间神经元的四分体突触的研究，有助于建立果蝇的层作为视网膜外丛状层的功能对应物。我们的长期目标是了解多接触突触（二联、三联等）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。目前的目标是研究调节四分体和反馈突触如何在光感受器末端形成的基因突变，无论是在突触后靶点识别期间，还是在突触细胞器靶向这种接触位点时。我们将通过共聚焦和电子显微镜的先进方法，在纯合子果蝇或全眼马赛克中检查突变的光感受器，并由我们的技术人员实施这些。我们正在开发创新的EM层析成像和冷冻替代方法。我们将研究：A. d - scam和N-cadherin等基因的功能作用，它们在光感受器末端的靶标选择过程中起作用；以及imac和dHIP14，它们是将突触细胞器靶向到末梢所必需的。B.当来自光感受器的信息传递被果蝇动力蛋白的温度敏感结构可逆地阻断时，在层反馈突触中发生的反应性突触变化。C.高分辨率EM断层扫描，以识别突变突触中受干扰的蛋白质复合物，我们将用Fab抗体片段标记。我们还将继续：D.鉴定在突触细胞器中表达的蛋白质，使用免疫金方法，或通过标记已鉴定的蛋白质并通过ReAsH方法标记这些蛋白质。E.利用序列电镜和靶向HRP表达分析第二神经堆或髓质远端层的突触微回路，这些微回路起源于光感受器和层状细胞终端。当光感受器突变为Lar或curta基因时，我们将确定这些回路的变化方式。我们提出的研究旨在建立一个适用于多接触突触（如视网膜的二联体和三联体）的突触发生的基本模型，并帮助确定先天性或营养不良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对这些变化的反应性重排。

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.