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。基因突变 这些受体或其功能的药物干扰会扰乱神经回路功能，导致 大脑的不正常发育和功能。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整的。这个拼图中缺失的部分包括分子 定义发育中大脑中兴奋性和抑制性(E和I)突触的特征。在 计划中，我们将开发一种方法来检测已知神经递质受体的存在 果蝇的亚基。我们将建立一个苍蝇文库，在其中内源性表达神经递质 受体亚基由裂解荧光蛋白(裂解FPS)标记。拆分FP编码的一小段 序列将被插入到神经递质受体亚单位基因阵列中。FP的其余部分 将使用现有的果蝇表达系在特定的细胞类型中表达。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，我们将 优化果蝇内源性蛋白质多色成像的裂解FP标签。我们还将开发一种 通过CRISPR介导的同源性将分裂的FP标签插入果蝇基因组座位的无克隆方法 定向DNA修复，对文库生成具有成本效益和可扩展性。在目标2中，我们将应用 用敲入的方法产生一个含有15个神经递质受体亚基的Fly文库，该文库带有分裂的FP标记。我们 将利用这个文库来描绘神经细胞中树突上的神经递质受体图 并最终研究E和I突触形成在建立回路功能中的分子基础。 这项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。 为建立和维持E和I突触平衡以及为 科学界。此外，该项目还将开发一种可转让的新方法，用于生产大型- 分裂的FP标记分子的规模化文库，为研究人员提供了一种在 果蝇体内细胞类型的多样性。

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.