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

以单细胞分辨率测量皮质突触连接的高通量方法

• 批准号: 10473009
• 负责人: Adam Granger
• 金额: $ 90万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473009
• 关键词: Animals; Bar Codes; Bipolar Disorder; Brain; Cells; Cerebral cortex; Classification; Consumption; Disease; Electrophysiology (science); Environment; Fluorescent in Situ Hybridization; Genetic Transcription; Goals; Image; Individual; Institutes; Interneurons; Label; Measures; Mental disorders; Methods; Molecular; Neurons; Pattern; Positioning Attribute; Rabies; Research; Resolution; Schizophrenia; Site; Synapses; Techniques; Time; Training; Viral; base; cell type; cost; design; information processing; innovation; insight; microscopic imaging; neuropsychiatric disorder; optogenetics; patch clamp; presynaptic neurons; programs; risk variant; technology development

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.

项目摘要 大脑中的信息处理是通过通过特定整合神经元活动来完成的 各种神经元亚型和连通性变化之间的突触连通性模式是 假设引起一系列神经精神疾病。但是，当前的测量方法 具有单细胞精度的突触连通性是费力的，耗时且昂贵的。主 我的研究计划的目标是开发用于测量模式的高通量方法 大脑皮质中的突触连通性。为此，我将采用三种单独的方法 使用病毒，功能和分子策略。首先，我将优化基于狂犬病的反式突触跟踪 为了标记局部皮质电路并将连接的神经元分类为多重，误差 - 强大的荧光原位杂交（Merfish）。接下来，我将通过 将单个突触前神经元的全息光遗传学刺激与多路复用 全细胞自动补丁钳，以增加可以探测的突触连接的数量 来自单个动物。最后，我将设计针对前后突触部位的分子条形码 可以用Merfish成像，以识别与转录的共同连接 细胞类型分类的信息。我将首先将这些方法应用于高信风险 与精神分裂症和躁郁症相关的基因，因为无序的皮质回路被认为是 引起这些疾病。该提案需要多个尖端的创新整合 技术和新的，不存在的技术的发展。鉴于我的培训 电生理学，光遗传学，高级显微镜和成像分析以及我的电流 在布罗德学院的斯坦利中心的环境中，我理想地有能力开发和验证 这些技术。成功完成此建议将为分析提供新的方法 突触连通性适用于广泛的神经科学问题，并提供有关 精神疾病的病理生理机制。