Cell-cell communications in neural circuit assembly

项目总结 神经生物学中的一个关键问题是单个神经元如何准确地与 其他在发育过程中形成功能电路。了解以下机制： 哺乳动物大脑中的神经回路组装可能为揭示脑血管疾病的病因提供线索。 人类大脑紊乱。在哺乳动物的大脑中，每个神经元平均形成 与数以千计的其他神经元连接。这些复杂电路的组装 在神经发育的许多步骤中依赖于细胞间的通讯。在 在这笔赠款的前两个周期，我们专注于开发MADM(MOSAIC 双标记分析)。分离了两种不同颜色的MADM标签 具有共同谱系的单个神经元或神经元组。同时， MADM可以使标记了某种颜色的纯合子突变体的神经元 目的基因和标记第二颜色野生型的神经元作为内对照。 MADM使研究人员能够检查哺乳动物神经中的基因功能 以单单元分辨率进行开发(以及其他流程)，并实现了许多 新的发现。在这次更新中，我们将利用MADM和我们拥有的其他工具 在之前的授予周期中开发，用于研究神经电路中的细胞间通信 在小鼠的大脑中组装。具体地说，我们关注两类蛋白质： 神经营养素受体和肌钙蛋白。 使用神经营养素受体TrkC的MADM分析，我们之前已经表明 浦肯野细胞中TrkC的稀疏但不是全局敲除减少了树突状细胞的生长 和分枝，提示了树枝状形态发生的竞争机制。我们 将研究TrkC介导的竞争树突状细胞的细胞机制 使用活体成像，测试突触后活动是否需要 浦肯野细胞树突状发育及其与TrkC活性的关系 发信号。我们还将探讨神经营养素受体TrkB在神经元中的功能。 形态发生。从我们对果蝇嗅觉回路组装的研究中，我们发现 两种在进化上保守的II型跨膜蛋白， 通过同性吸引指导突触伙伴匹配。我们将测试一下 Teneurins还在小鼠脑内神经回路组装中介导细胞间的相互作用 使用MADM分析、条件基因敲除、病毒介导的组合 错误表达和体外检测。

PROJECT SUMMARY A key question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly in the mammalian brain may provide insights into the etiology of human brain disorders. In the mammalian brain, each neuron on average forms connection with thousands of other neurons. The assembly of these complex circuits depends on cell-cell communication during many steps of neural development. In the previous two cycles of this grant, we have focused on developing MADM (Mosaic Analysis with Double Markers) in mice. MADM labels with two distinct colors isolated individual neurons or groups of neurons that share a common lineage. At the same time, MADM can render neurons labeled with one of the colors homozygous mutant for a gene of interest and neurons labeled with the second color wild type as internal controls. MADM has allowed researchers to examine gene function in mammalian neural development (as well as other processes) with single-cell resolution, and enabled many new discoveries. In this renewal, we will utilize MADM and other tools we have developed in the previous grant cycles to study cell-cell communications in neural circuit assembly in the mouse brain. Specifically, we focus on two classes of proteins: neurotrophin receptors and teneurins. Using MADM analysis of the neurotrophin receptor TrkC, we have previously shown that sparse but not global knockout of TrkC in Purkinje cells reduces dendritic growth and branching, suggesting a competitive mechanism for dendrite morphogenesis. We will investigate the cellular mechanisms by which TrkC-mediated competitive dendrite morphogenesis using in vivo imaging, test whether postsynaptic activity required for Purkinje cell dendrite development, and the relationship between activity and TrkC signaling. We will also explore the function of neurotrophin receptor TrkB in neuronal morphogenesis. From our studies in Drosophila olfactory circuit assembly, we identified two teneurins, which are evolutionally conserved type II transmembrane proteins, that instruct synaptic partner matching via homophilic attraction. We will test whether teneurins also mediate cell-cell interaction in neural circuit assembly in the mouse brain using a combination of MADM analysis, conditional knockout, virus-mediated misexpresssion, and in vitro assays.