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

神经回路的功能依赖于神经递质组成的突触的数量和分布 受体，建立并维持兴奋和抑制之间的突触平衡。突变在 这些受体或其功能的药理学扰动会破坏神经回路功能，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 定义发育中大脑中的兴奋性和抑制性（E 和 I）突触的特征。在 拟议的项目，我们将开发一种方法来检测已知神经递质受体的存在 果蝇中的亚基。我们将生成一个果蝇库，其中内源表达神经递质 受体亚基由分裂荧光蛋白（分裂 FP）标记。分割 FP 编码的短区域 序列将被插入一系列神经递质受体亚基基因中。 FP 的剩余部分 将使用可用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各个树突分支的定位。在目标 1 中，我们将 优化果蝇内源蛋白多色成像的分割 FP 标签。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位点的无克隆方法 定向 DNA 修复，对于文库生成来说具有成本效益且可扩展。在目标 2 中，我们将应用 敲入方法生成带有分裂 FP 标记的 15 个神经递质受体亚基的果蝇文库。我们 将利用这个库来描绘神经递质受体沿神经细胞树突的图谱 电路并最终研究 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相对分布的重要新信息 建立和维持 E 和 I 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来产生大量 分裂 FP 标记分子的规模文库，为研究人员提供了一种方法来定位感兴趣的蛋白质 果蝇中的多种细胞类型。

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.