High-throughput methods for measuring cortical synaptic connectivity at single-cell resolution

项目摘要 大脑中的信息处理是通过整合特定的神经元活动来完成的 不同神经元亚型之间的突触连接模式和连接的变化如下 推测会导致一系列神经精神障碍。然而，目前的测量方法 以单细胞精度进行突触连接既费时又费钱。主 我的研究计划的目标是开发高通量的方法来测量 大脑皮层的突触连接。为了实现这一点，我将采用三种不同的方法 使用病毒、功能和分子策略。首先，我将优化基于狂犬病的跨突触追踪 为了能够对局部皮质电路进行标记，并将连接的神经元与多路传输、错误- 强健的荧光原位杂交(MerFish)。接下来，我将通过以下方式筛选功能连接性 全息光遗传刺激对单个突触前神经元的复合作用 全细胞自动膜片钳增加可探测的突触连接数量 从一只动物身上。最后，我将设计针对突触前和突触后位置的分子条形码 可以用MerFish成像来识别突触连接与转录 用于细胞类型分类的信息。我将首先将这些方法应用于高置信度风险 与精神分裂症和双相情感障碍相关的基因，因为紊乱的皮质回路被认为是 导致这些疾病。这一提议需要多个尖端技术的创新集成 技术和开发新的、不存在的技术。考虑到我在 电生理学、光遗传学、高级显微镜和成像分析以及我的电流 在布罗德研究所斯坦利中心的环境中，我处于开发和验证的理想位置 这些技巧。成功完成这项建议将带来新的分析方法 适用于一系列神经科学问题的突触连接，并提供对 精神障碍的病理生理机制。

Project Summary Information processing in the brain is accomplished by integrating neuronal activity via specific patterns of synaptic connectivity between diverse neuronal subtypes, and changes in connectivity are hypothesized to cause a range of neuropsychiatric disorders. However, current methods to measure synaptic connectivity with single-cell precision are laborious, time-consuming, and costly. The main goal of my research program is to develop high-throughput methods for measuring the patterns of synaptic connectivity in the cerebral cortex. To accomplish this, I will pursue three separate approaches using viral, functional, and molecular strategies. First, I will optimize rabies-based trans-synaptic tracing to enable labeling of local cortical circuitry and classify connected neurons with multiplexed, error- robust fluorescent in situ hybridization (merFISH). Next, I will screen for functional connectivity by combining holographic optogenetic stimulation of individual pre-synaptic neurons with multiplexed whole-cell automated patch-clamp to increase the number of synaptic connections that can be probed from a single animal. Finally, I will design molecular barcodes targeted to pre- and post-synaptic sites that can be imaged with merFISH to identify synaptic connections in concert with transcriptional information for cell-type classification. I will first apply these methods towards high-confidence risk genes associated with schizophrenia and bipolar disorder, as disordered cortical circuitry is thought to cause these diseases. This proposal requires the innovative integration of multiple cutting-edge technologies and the development of new, non-existing techniques. Given my training in electrophysiology, optogenetics, advanced microscopy, and imaging analysis as well as my current environment at the Stanley Center at the Broad Institute, I am ideally positioned to develop and validate these techniques. Successful completion of this proposal will result in new methods for analyzing synaptic connectivity applicable to a wide range of neuroscientific questions and provide insight into the pathophysiological mechanisms of psychiatric disorders.