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，以及连接的变化， 据推测会导致一系列神经精神疾病然而，目前的测量方法 具有单细胞精度突触连接是费力、耗时且昂贵的。主要 我的研究计划的目标是开发高通量的方法来测量的模式， 大脑皮层的突触连接为了实现这一目标，我将采用三种不同的方法 使用病毒、功能和分子策略。首先我会优化狂犬病跨突触追踪 为了能够标记局部皮层电路并对连接的神经元进行分类， 鲁棒荧光原位杂交（merFISH）。接下来，我将通过以下方式筛选功能连接性： 将单个突触前神经元的全息光遗传学刺激与多路复用 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点的分子条形码 可以用merFISH成像，以识别与转录相关的突触连接， 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风险 与精神分裂症和双相情感障碍相关的基因，因为混乱的皮层回路被认为是 导致这些疾病。这一建议需要创新性地整合多个前沿 技术和开发新的、不存在的技术。鉴于我的训练， 电生理学，光遗传学，先进的显微镜和成像分析，以及我目前的 在布罗德研究所斯坦利中心的环境中，我非常适合开发和验证 这些技术。成功完成这项提案将导致新的分析方法 突触连接适用于广泛的神经科学问题，并提供了深入了解 精神疾病的病理生理机制

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.