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.

神经回路的功能依赖于由神经递质组成的突触的数量和分布 受体，建立和维持兴奋和抑制之间的突触平衡。突变 这些受体或其功能的药理学干扰可破坏神经回路功能，导致 大脑发育和功能异常。然而，我们对突触空间模式的了解， 神经递质受体的平衡是不完全的。这个谜题中缺失的部分包括分子 在发育中的大脑中定义兴奋性和抑制性（E和I）突触的特征。在 建议的项目，我们将开发一种方法来检测已知的神经递质受体的存在 果蝇中的亚基。我们将建立一个果蝇文库， 受体亚单位被分裂荧光蛋白（分裂FP）标记。分裂FP编码的短区域 序列将被插入到神经递质受体亚基基因的阵列中。FP的其余部分 将使用可获得的果蝇表达系在特定细胞类型中表达。这将使我们能够 检查神经递质受体相对于单个树突分支的定位。在目标1中，我们 优化分裂FP标签，用于果蝇内源蛋白质的多色成像。我们还将开发一个 通过CRISPR介导的同源性将分裂的FP标签插入果蝇基因组基因座的无克隆方法- 定向DNA修复，这是成本有效的和可扩展的文库生成。在目标2中，我们将应用 敲入方法产生15个用分裂的FP标记的神经递质受体亚基的果蝇文库。我们 将利用这个库描绘神经递质受体沿着神经细胞树突的地图， 电路，并最终研究建立电路功能的E和I突触形成的分子基础。 这项工作将为神经递质受体的分布提供重要的新信息。 E和I突触平衡的建立和维持，以及对神经系统的宝贵资源。 科学界。此外，该项目还将开发一种可转移的新方法，用于产生大规模的 分裂FP标记分子的规模库，为研究人员提供了一种将感兴趣的蛋白质定位在 果蝇中的各种细胞类型。