VISUAL SYSTEM DEVELOPMENT AND SYNAPTOGENESIS

本项目的总体目标是研究控制 光感受器突触的数量和质量组成 果蝇视叶的第一个神经桩，或层 还有苍蝇 我们特别想了解 多接触突触的发育组装（二分体、三分体等）， 苍蝇感受器四分体突触就是一个模型。 的模块 被称为药筒的薄层包括小的、固定数量的被识别的 神经元 我们将抽样这些：1）使用定量单节EM， 估计突触频率，测量突触接触部位和细胞 表面积。2)利用串行EM进行计算机三维建模 未成熟细胞形态的重建， 突触的形成 在发生快速变化之后， 光或温度的功能性逆转，我们将绘制位置 新的突触部位相对于旧的，检查的影响， 现有的网站上形成一个新的突触附近的网站，和 突触前带的形成方式。 我们还将 检查突触细胞器之间的膜的再循环，使用 果蝇变种shibire。 解剖突触的功能结果 将从ERG记录中寻找变化，并且 突触的形成将在具有受损的前或后突触的突变体中评估。 突触后板层功能 控制昼夜节律的调节， 层由两组分布广泛的神经元组成，一组被认为是释放5-HT， 其他肽PDF，将作为可塑性的进一步例子进行分析 在lamina。 我们将照射苍蝇作为脓包， 神经胶质细胞，包裹板盒，并探索突触 预期的从中间神经元发芽的结果， 完整弹壳与相邻弹壳之间的距离 通过视网膜光消融术切断传入神经 三重突变体rol/sol/mnb 会导致突触后神经元的不同组合的丢失， 四分体的贡献者，通过自发的细胞变性之前， 突触发生，并使存活的细胞有机会表现出 他们替代突触伴侣的能力 我们将利用 我们开发了一个组织培养系统来检查选择性肌束震颤 感光细胞轴突之间的相互作用及其与它们的板层细胞 目标，并在体外从共培养物中获得突触发生， 光感受器和视叶细胞。 与其他实验室合作，我们 将检查参与形成或 突触的维持。 我们将检查GAL 4果蝇系，其中 基因表达由外源报告基因标记，以鉴定 在已知促进突触发生的条件下发生的表达，如 以及将特定的分子标记物固定在识别的视叶上 细胞 我们还将研究两种可能的突触表达， 有希望的候选基因，irreC和demo，使用免疫EM。 研究 因为他们的目标是建立一个突触发生的基本模型 适用于多接触突触，例如二联体和三联体 广泛存在于视觉系统中，将有助于了解 疾病状态下视觉突触的扰动 在其生长和发育过程中易受影响。

The general aims of this project are to study the factors controlling the numerical and qualitative composition of photoreceptor synapses formed in the first neuropile, or lamina, of the optic lobe of the flies Drosophila and Musca. We especially want to understand the control of the developmental assembly of multiple-contact synapses (dyads, triads, etc.), for which the fly receptor tetrad synapses are a model. The modules of the lamina, called cartridges, comprise small, fixed numbers of identified neurons. We will sample these: 1) using quantitative single-section EM to estimate synaptic frequencies, measure synaptic contact sites and cell surface areas. 2) using serial EM to undertake computer 3-D reconstructions of immature cell morphologies, to relate dendritic growth to synapse formation. Under states of rapid change occurring after functional reversals of light or of temperature, we will plot the location of new synaptic sites relative to old ones, to examine the influence of an existing site on the formation of a new synaptic site nearby, and the presynaptic ribbon's mode of formation at the new site. We will also examine the recycling of membrane between synaptic organelles, using the Drosophila mutant shibire. The functional outcome of anatomical synaptic changes will be sought from ERG recordings, and the activity-dependence of synapse formation will be assessed in mutants with impaired pre- or postsynaptic lamina function. The control of circadian modulations in the lamina by two sets of widespread neurons, one thought to release 5-HT, the other the peptide PDF, will be analyzed as a further example of plasticity in the lamina. We will irradiate flies as pupae to kill the epithelial glial cells that envelop the lamina cartridges, and explore the synaptic consequences, and rapidly, of anticipated sprouting from the interneurons of intact cartridges to adjacent cartridges which have been acutely deafferented by retinal photo-ablation. The triple mutant rol/sol/mnb will procure the loss of differing combinations of postsynaptic contributors to the tetrads, through spontaneous cell degeneration prior to synaptogenesis, and allow surviving cells the opportunity to exhibit their ability to substitute synaptic partners. We will exploit the tissue-culture system we have developed to examine selective fasciculation amongst photoreceptor axons and their interactions with their lamina cell targets, and to procure synaptogenesis in vitro from co-cultures of photoreceptors and optic lobe cells. In collaboration with other labs, we will examine the expression of genes involved in either the formation or maintenance of synapses. We will examine GAL4 Drosophila lines, in which gene expression is marked by an exogenous reporter gene, to identify expression occurring under conditions known to promote synaptogenesis, as well as to secure specific molecular markers to identified optic lobe cells. We will also examine the possible synaptic expression of two promising candidate genes, irreC and demo, using immuno-EM. The studies proposed here because they aim to produce a basic model of synaptogenesis applicable to multiple-contact synapses, such as the dyads and triads found widely in visual systems, will contribute to a general knowledge of the perturbations in disease states to which visual synapses are susceptible during their growth and development.