Visual system development and synaptogenesis

描述(申请人提供)：我们将分析调节果蝇光感受器突触形成和功能的基因的作用。我们之前在这些基因可操作的模型光感受器终末和它们的目标中间神经元上的四分体突触的工作，有助于建立作为苍蝇功能对应的视网膜外丛状层的板层。我们的长期目标是了解多接触突触(二联体、三联体等)的功能组织，这些突触是如何形成的，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。目前的目标是研究基因突变，以调节四分体和反馈突触如何在光感受器终末形成，无论是在识别突触后靶点期间，还是后来当突触细胞器被靶向这种接触的位置时。我们将通过先进的共聚焦和电子显微镜方法，以及我们熟练的技术人员，在纯合子苍蝇或全眼马赛克中检查突变的光感受器。我们正在开发创新的电磁断层扫描和冷冻替代方法。我们将研究：a.在光感受器终端选择靶标的过程中，如Dscam和N-钙粘素等基因的功能作用；以及将突触细胞器靶向终末所需的iMac和dHIP14。B.当光感受器的传输被温度敏感的果蝇动力素结构可逆地阻止时，板层反馈突触之间的反应性突触变化。C.高分辨率EM断层扫描，以识别在突变突触处受到干扰的蛋白质复合体，我们将用Fab抗体片段对其进行标记。我们还将继续：D.鉴定在突触细胞器表达的蛋白质，使用免疫金标法，或通过标记识别的蛋白质并用ReAsH方法标记这些蛋白质。E.使用序列EM和靶向HRP表达，分析来自光感受器和板层细胞终末的第二神经堆或延髓远侧层的突触微电路。我们将确定当光感受器对Lar或Curta基因发生突变时，这些电路发生变化的方式。我们拟议的研究旨在产生一个适用于多接触突触的突触发生的基本模型，例如视网膜的二联体和三联体，并帮助识别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.