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 将神经元植入电路确保了正常的功能。在视网膜中，双极神经元之间多样但精确的连接 细胞（BCs）和视网膜神经节细胞（RGC）是视觉信号回路处理的结构基础 从外层视网膜到内层视网膜。这些线路的缺陷会导致严重的视网膜疾病。最常见的 这些疾病的一个例子是青光眼，它给美国带来了巨大的社会和经济负担 人口开发有效疗法以使BC再生到RGC连线的主要障碍是我们的贫穷。 了解这些回路的结构以及确保BC和RGC连接的机制 没错在这个建议中，我们说明了功能特异性BC到RGC布线的细胞策略以及CD3是如何与RGC连接的。 选择性地指示RGC将各种BC类型作为布线的目标。为了说明特定功能的组装， 电路，我们开发了一种新的跨细胞标记技术来标记突触连接到功能特异性 RGC。我们对一种特殊类型的DS-RGCs，BD-RGCs的研究结果与EM连接组学的结果相当。 问题研究我们的研究结果还表明，两种不同的RGC类型与不同的BC类型突触，表明RGC BC类型的特定类型布线。这些研究使我们能够研究更多的RGC类型，并揭示 指导每个研资局类型与不同的BC类型组合的基本机制。为此，我们 发现CD 3 ζ（I类主要组织相容性复合体（MHCI）的受体）的突变消除了突触 在某些实施方案中，突触连接增加了视锥BCs与BD-RGC的突触连接，但增加了视锥BCs与J-RGC的突触连接，从而允许 RGC类型特异性突触调节。CD3 γ突变体中视锥BCs与BD-RGCs的突触连接缺陷 显著损害BD-RGC的光诱发反应，表明精确的BC-RGC突触连接 是确保RGC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 指定RGC功能。我们还将研究CD3 β如何指导突触的形成和这些突触的功能。 RGC。为了揭示CD3 β介导的突触组装的潜在机制，我们发现敲低CD3 β介导的突触组装可能是一种新的机制。 或在野生型小鼠的RGC中过表达CD3 β或CD3 β突变体仅在转导的RGC中诱导表型， 这表明是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CD3+是否是树突状细胞发育所特异性需要的。 RGC，它是否足以维持RGC的树突状发育，以及它是否需要维持RGC的生长。 成年人的树突稳定性。因此，我们将使用我们的新方法研究CD3 β在BCs到RGC连接中的作用。 产生了分子和基因工具。总的来说，我们的研究试图揭示的战略和机制， 控制RGC类型特定的电路形成。这些研究将是向前迈出的重要一步， 理解特定功能电路发展的机制。

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.