Visual system development and synaptogenesis

我们将分析调节感光突触形成和功能的基因在细胞中的作用。 果蝇我们以前在这些基因可操纵的模型光感受器上的四分体突触的工作 终末和它们的靶中间神经元，帮助建立作为苍蝇的功能对应物的板层， 视网膜的外丛状层。我们的长期目标是了解 多接触突触（二分体、三分体等），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。目前的目标是研究基因突变体， 四分体和反馈突触形成于感光器末梢，在识别突触后 目标或稍后，当突触细胞器被定向到这样的接触的网站。我们会检查变种人 光感受器，在纯合子苍蝇或在全眼马赛克，通过先进的方法共聚焦 和电子显微镜，以及我们的技术人员来实现这些。我们正在开发创新的EM 断层扫描和冷冻置换方法。我们将研究：A。基因的功能作用，如 Dscam和N-钙粘蛋白，在感光细胞终末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到末端。B。神经元间的反应性突触变化 当来自光感受器的传递被一种 果蝇发动蛋白温度敏感结构。C.高分辨率电磁层析成像， 我们将用Fab抗体片段标记突变突触处受到干扰的蛋白质复合物。 我们还将继续：D.识别在突触细胞器表达的蛋白质，使用免疫金 方法，或通过标记鉴定的蛋白质并通过ReAsH方法标记这些蛋白质。e.分析突触 第二神经桩或髓质的远端层中的微电路，其起源于感光器， 板层细胞终末，使用连续EM和靶向HRP表达。我们将确定这些方法 当光感受器的基因Lar或curta发生突变时，电路就会发生变化。我们建议的研究旨在 产生一个适用于多接触突触的突触发生的基本模型，如视网膜的 二联体和三联体，并帮助确定潜在的遗传基础的变化，导致先天性或 营养不良性疾病或视网膜损伤，以及视网膜突触中对这些疾病的反应性重排。

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.