Mechanisms underlying CD3 guided assembly of retinal circuits

哺乳动物的神经系统由数十亿个神经元组成，这些神经元的精确组装 神经元转化为电路确保了正常的功能。在视网膜中，两极之间不同但精确的连接 细胞(BCs)和视网膜神经节细胞(RGC)是视觉信号电路处理的结构基础 从外视网膜到内视网膜。这些线路的缺陷会导致严重的视网膜疾病。最常见的 这些疾病的例子是青光眼，它给美国带来了重大的社会和经济负担 人口。开发有效的治疗方法来再生BC到RGC的一个主要障碍是我们的贫穷 了解这些电路的结构以及确保BCS和RGC接线的机制 正是如此。在这个方案中，我们说明了特定功能的BC到RGC连接的细胞策略以及CD3ζ是如何 有选择地指示RGC针对各种BC类型进行布线。要说明特定于函数的 电路，我们开发了一种新的跨细胞标记技术来标记与功能特定的突触连接的BCS RGC。我们在一种特定类型的DS-RGC，BD-RGC上的结果与EM Connectomic的结果相当 学习。我们的结果还表明，两种不同的RGC类型与不同的BC类型突触，提示RGC 使用BC类型的特定于类型的布线。这些研究使我们能够研究更多的研资局类型，并揭示 指导每种RGC类型与各种BC类型组装的基本机制。为此，我们 发现I类主要组织相容性复合体受体CD3ζ的突变可消除突触 锥体BCS与BD-RGC的联系，但增加了锥体BCS与J-RGC的突触联系，从而允许 RGC类型特异性突触调节。CD3型ζ突变体视锥细胞与视网膜节细胞突触连接的缺陷 显著损害BD-RGC的光诱发反应，提示精确的BC-RGC突触连接 是确保视网膜节细胞功能特异性所必需的。为了进一步测试这个想法，我们将识别连接的BC类型 另外4种RGC类型和这些RGC的突触功能，以揭示负责的细胞策略 指定RGC功能。我们还将研究cd3ζ如何指导突触的形成和功能。 RGC。为了揭示CD3ζ介导的突触组装的潜在机制，我们展示了击倒 或在野生型小鼠的视网膜节细胞中过表达CD3ζ或CD3ζ突变体仅在转导的视网膜节细胞中诱导表型， 暗示了一种细胞自主机制。为了进一步加深我们对这些机制的了解，我们将 进行一系列分析以确定CD3ζ是否是树突状细胞发育所特需的 视网膜节细胞，视网膜节细胞的树突状发育是否足够，是否需要维持 成人树突状细胞的稳定性。因此，我们将使用我们的新技术来研究CD3ζ在BCS到RGC连接中的作用 产生了分子和遗传工具。总而言之，我们的研究试图揭示战略和机制 控制RGC类型特定电路的形成。这些研究将成为#年向前迈出的重要一步。 了解特定功能电路发展的潜在机制。

The mammalian nervous system is composed of billions of neurons, and the precise assembly of these neurons into circuits ensures proper functionality. In the retina, the diverse but precise wiring between bipolar cells (BCs) and retinal ganglion cells (RGCs) serve as the structural basis for circuit processing of visual signals from outer retina to the inner retina. Defects in these wirings lead to severe retinal diseases. The most common example of these diseases is glaucoma, which imputes a significant social and economic burden on the US population. A major obstacle to developing effective therapies to regenerate BC to RGC wirings is our poor understanding of the structure of these circuits and the mechanisms ensuring BCs and RGCs to wire up precisely. In this proposal, we illustrate the cellular strategy of function specific BC to RGC wiring and how CD3ζ selectively instruct RGCs to target various BC types for wiring. To illustrate the assembly of function-specific circuits, we developed a novel transcellular labeling technique to label BCs synaptically wired to function-specific RGCs. Our results on a specific type of DS-RGCs, BD-RGCs, are comparable to those from EM connectomic studies. Our results also show that two different RGC types synapse with distinct BC types, suggesting RGC type-specific wiring with BC types. These studies position us to examine more RGC types and to reveal the underlying mechanisms guiding the assemble of each RGC type with various BC types. Toward this end, we found that mutation of CD3ζ, a receptor for class I major histocompatibility complex (MHCI), eliminates synaptic connections of cone BCs to BD-RGCs but increase synaptic connections of cone BCs to J-RGCs, thus allowing RGC type-specific synaptic regulation. The defects in synaptic wiring of cone BCs to BD-RGCs in CD3ζ mutants significantly impair the light-evoked responses of BD-RGCs, suggesting that precise BC-RGC synaptic wirings are necessary to ensure function specificity of RGCs. To further test this idea, we will identify the BC types wired to additional 4 RGC types and the synaptic function of these RGCs to reveal the cellular strategy responsible for specifying RGC function. We will also examine how CD3ζ instructs synapse formation and function of these RGCs. To uncover the mechanisms underlying CD3ζ-mediated synaptic assembly, we showed that knockdown or overexpress CD3ζ in RGCs of wildtype mice or CD3ζ mutants only induced phenotypes in transduced RGCs, suggesting a cell-autonomous mechanism. To further expand our understanding of the mechanisms, we will perform a series analyses to determine whether CD3ζ is specifically required for the dendritic development of RGCs, whether it is sufficient for the dendritic development of RGCs, and whether it is required to maintain dendritic stability in adults. Thus, we will examine the roles of CD3ζ in the BCs to RGCs wiring using our newly generated molecular and genetic tools. Collectively, our studies seek to reveal the strategy and mechanisms that control RGCs type-specific circuit formation. These studies will constitute a significant step forward in understanding the mechanisms underlying the development of function-specific circuits.