Pooled Optical Screens of Synaptic Function

尽管我们对突触蛋白质组成的理解和对突触参与神经元活动的认识， 精神疾病中的突触机制都在继续增长，我们研究突触功能的能力， 低通量、高开销的测定仍然阻碍了基因的表达。我们建议采取以下措施， 在培养的神经元中开发遗传扰动对突触功能的影响的合并筛选。 与阵列筛选相比，合并筛选对条件间变异性更稳健，因此 更适合研究高度依赖培养的表型，如突触传递。尽管如此 然而，由于其优势，目前还不清楚如何将汇集筛选技术从研究细胞存活或生长转化为研究细胞存活或生长的技术。 将表型与动态功能表型耦合，如参与神经元功能的表型。我们提出 通过利用新的光学方法来解决这个问题的创新方法。我们的方法是 在培养的神经元池上进行条形码遗传扰动，以对其进行时间分辨成像测定。 突触后或突触前功能，并随后使用多重RNA或蛋白质读出方法， 读取条形码并将突触表型与扰动相匹配。突触后功能将使用 同时电压成像和光遗传学，而突触前功能将通过监测囊泡读出 再循环与pH敏感的荧光蛋白。突触后和突触前协议是独立的， 它们相互独立，构成各自的具体目标。我们将广泛描述两者的性能 分析并对在裂殖吸虫全外显子组中鉴定的50个基因的突触功能进行初步筛选 测序研究。实现这些目标是可能的创新表达策略，测量 协议和条形码方法。这项工作的主要意义是使突触功能的研究成为可能 在研究新基因家族的发现过程中更早出现。此外，试点屏幕我们 将产生有关精神分裂症相关的基本神经生物学功能的有价值的信息。 基因.我们将用传统的膜片钳方法验证这些结果，它们将作为 这是进一步研究这些基因作用机制的起点。

Though our understanding of the protein composition of the synapse and our recognition of the involvement of synaptic mechanisms in psychiatric disease both continue to grow, our ability to study the synaptic function of genes is still hampered by low throughput, high overhead assays. We propose to address this need by developing pooled screens of the effect of genetic perturbations on synaptic function in cultured neurons. Compared to arrayed screens, pooled screens are more robust to condition-to-condition variability and are thus better suited for studies of highly culture-dependent phenotypes such as synaptic transmission. Despite this advantage, it has been unclear how to translate pooled screening technology from studying cell survival or growth coupled phenotypes to dynamical functional phenotypes like those involved in neuronal function. We propose an innovative way around this problem by harnessing new optical methods. Our approach will be to perform barcoded genetic perturbations on a pool of cultured neurons, to perform time-resolved imaging assays of their postsynaptic or presynaptic function, and to subsequently use multiplexed RNA or protein readout methods to read out barcodes and match synaptic phenotypes to perturbations. Postsynaptic function will be read out using simultaneous voltage imaging and optogenetics, while presynaptic function will be read out by monitoring vesicle recycling with a pH sensitive fluorescent protein. The postsynaptic and presynaptic protocols are independent of each other and constitute separate specific aims. We will extensively characterize the performance of both assays and perform a pilot screen on the synaptic functions of 50 genes identified in Schizophrenia whole exome sequencing studies. Accomplishing these aims is possible by innovations in expression strategies, measurement protocols, and barcoding approaches. The chief significance of this work is to enable studies of synaptic function to occur earlier in the discovery process when studying new families of genes. In addition, the pilot screen we perform will generate valuable information about the basic neurobiological function of Schizophrenia associated genes. We will validate these results with traditional patch clamp approaches, and they will serve as the launching point for further investigations into the mechanisms of action of these genes.