Screening using split fluorescent protein tags for neurotransmitter receptors that define a synaptic balance in neuralcircuits

使用分裂荧光蛋白标签筛选神经递质受体，定义神经回路中的突触平衡

• 批准号: 10805112
• 负责人: Daichi Kamiyama
• 金额: $ 40.43万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10805112
• 关键词: 3-Dimensional; Affect; Automobile Driving; Biochemical; Brain; Cells; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Color; Communities; Complex; Consumption; DNA Repair; DNA cassette; Databases; Dendrites; Desire for food; Development; Drosophila genus; Drosophila inturned protein; Electron Microscopy; Equilibrium; Etiology; Excitatory Synapse; Future; Generations; Genes; Genetic; Goals; Homeostasis; Image; Immunohistochemistry; Impairment; Individual; Inhibitory Synapse; Insulin; Knock-in; Knowledge; Label; Lead; Libraries; Link; Maintenance; Maps; Mediating; Methods; Molecular; Molecular Analysis; Morphologic artifacts; Motor Neurons; Mutation; Neurologic; Neurons; Neurophysiology - biologic function; Neurotransmitter Receptor; Optics; Pattern; Persons; Physiology; Play; Population; Proteins; Research; Research Personnel; Resources; Role; Sampling; Signaling Protein; Stains; Synapses; System; Testing; Thick; Time; Tissues; Visualization; Work; cell type; cost effective; fluorescence imaging; fly; genetic analysis; genomic locus; interest; model organism; neural circuit; novel strategies; overexpression; pharmacologic; receptor; reconstitution; reconstruction; repository; response; screening; synaptic inhibition; synaptogenesis

The function of neural circuits relies on the number and distribution of synapses composed of neurotransmitter receptors, which establish and maintain a synaptic balance between excitation and inhibition. Mutations in these receptors or pharmacologic perturbations of their function can disrupt neural circuit function, leading to abnormal development and function of the brain. However, our knowledge of spatial patterning of the synaptic balance by neurotransmitter receptors is incomplete. The missing pieces of this puzzle include molecular characterizations that define excitatory and inhibitory (E and I) synapses in the developing brain. In the proposed project, we will develop a method to detect the presence of known neurotransmitter receptor subunits in Drosophila. We will generate a fly library in which endogenously expressed neurotransmitter receptor subunits are tagged by split fluorescent proteins (split FPs). A short region of the split FP coding sequence will be inserted into an array of neurotransmitter receptor subunit genes. The remainder of the FP will be expressed in particular cell types using available Drosophila expression lines. This will allow us to examine neurotransmitter receptor localization relative to individual dendritic branches. In Aim 1, we will optimize the split FP tags for multicolor imaging of endogenous proteins in Drosophila. We will also develop a cloning-free approach to insert the split FP tags into Drosophila genomic loci via CRISPR-mediated homology- directed DNA repair, which is cost-effective and scalable for library generation. In Aim 2, we will apply the knock-in approach to generate a fly library of 15 neurotransmitter receptor subunits tagged with split FPs. We will employ this library to delineate a map of neurotransmitter receptors along dendrites in the cells of neural circuits and ultimately study the molecular basis of E and I synapse formation in establishing circuit function. The proposed work will provide crucial new information on the distribution of neurotransmitter receptors relative to the establishment and maintenance of E and I synaptic balance as well as valuable resources for the scientific community. Moreover, the project will develop a transferrable novel approach for generating large- scale libraries of split FP-tagged molecules, giving researchers a means to localize proteins of interest in a variety of cell types in Drosophila.

神经回路的功能取决于由神经递质组成的突触的数量和分布 受体建立并维持激发和抑制之间的突触平衡。突变 这些功能的这些受体或药理扰动会破坏神经回路功能，导致 大脑的异常发育和功能。但是，我们对突触的空间图案的了解 神经递质受体的平衡不完整。这个难题的缺失包括分子 定义发育中大脑中兴奋性和抑制性（E和I）突触的特征。在 拟议的项目，我们将开发一种检测已知神经递质受体的存在的方法 果蝇中的亚基。我们将生成一个蝇库，内源表达的神经递质 受体亚基由荧光蛋白（分裂FPS）标记。拆分FP编码的一小段区域 序列将插入神经递质受体亚基基因的阵列中。 FP的其余部分 将使用可用的果蝇表达线在特定的细胞类型中表达。这将使我们能够 检查相对于单个树突分支的神经递质受体定位。在AIM 1中，我们将 优化果蝇中内源性蛋白的多色成像的分裂FP标签。我们还将开发一个 通过CRISPR介导的同源性插入分裂的FP标签的无克隆方法将分裂的FP标签插入果蝇基因组基因群中 定向DNA修复，对于图书馆的生成，它具有成本效益且可扩展。在AIM 2中，我们将应用 敲入方法生成一个用分裂FPS标记的15个神经递质受体亚基的飞行库。我们 将利用该库来描述沿着神经细胞中树突的神经递质受体图 电路并最终研究了E和I突触形成的分子基础，以建立电路功能。 拟议的工作将提供有关神经递质受体分布相对分布的至关重要的新信息 为了建立和维护E和我的突触平衡以及宝贵的资源 科学界。此外，该项目将开发一种可转让的新方法，以产生大型 分裂标签分子的比例库，为研究人员提供了一种将感兴趣的蛋白质定位在A中的方法 果蝇中的各种细胞类型。