A Novel Approach for Cell-Type Classification and Connectivity in the Human Brain

 描述(申请人提供)：人脑可以说是最复杂的生物结构。了解人脑中存在多少种不同类型的细胞并绘制神经连接图，是更好地了解大脑的发育和功能的关键任务。由于处理死后组织的固有局限性，这在人脑中尤其具有挑战性。这笔赠款专门针对人脑以及与之密切相关的非人类灵长类动物恒河猴和普遍研究的哺乳动物生物老鼠的这些任务。这项建议的目的是使用新的方法和途径，对发育中的人类和成人、猕猴和小鼠的前额叶皮质(PFC)的细胞类型和连接进行系统的清查/普查。我们之所以选择PFC进行这个项目，是因为它在高级认知功能中的重要性，以及在某些精神和神经障碍中观察到的PFC的变化。为了对PFC中的组成细胞进行普查，我们将使用单细胞RNA-SEQ来描述通过病毒介导的核糖体标记和轴突跟踪方法以特定细胞类型分离的单个细胞的转录本。为了将这些先进的技术应用于死后人脑，我们将实施一种新的组织处理方案[冬眠-冷冻保存结合脉冲灌流冬眠系统]来保持死后大脑的长时间冬眠。这可以稳定核酸，同时收集活的单细胞，并应用经典和先进的方法来识别神经元通路。我们还将开发不可知性和综合计算方法，以创建基于分子身份和连接性的细胞类型分类。这些特征将在不同物种、年龄和性别之间进行比较。这一应用主要有四个不同的方面：(1)实现新的方法，以扩展死后人脑的组织完整性和活性，以便我们能够(2)执行单细胞RNA-SEQ，(3)跟踪连接和检查死后人脑中的细胞形态，这是经典实验所不能接受的~和(4)开发新的分析工具和方法。这一试点项目和方法直接解决了大脑计划RFA的目标，旨在展示其实用性和可扩展性，最终完成对健康和疾病状态下整个人类大脑的全面细胞普查。

 DESCRIPTION (provided by applicant): The human brain is arguably the most complex biological structure. Understanding how many different cell types exist in the human brain and mapping neural connections are critical tasks to better understand the development and function of the brain. This is particularly challenging in the human brain due to inherent limitations of working with postmortem tissue. This grant is specifically addressing these tasks in the human brain as well as a closely related non-human primate, Rhesus macaque, and a commonly studied mammalian organism, the mouse. The objective of this proposal is to employ novel methods and approaches to generate a systematic inventory/census of cell types and connections in the developing and adult human, macaque monkey and mouse prefrontal cortex (PFC). We have chosen PFC for this project due both to its importance in higher cognitive functions as well as for the alterations observed in PFC in certain psychiatric and neurological disorders. To generate a census of component cells in the PFC, we will use single cell RNA-seq to profile the transcriptomes of single cells isolated in a cell-type specific way through the use of viral-mediated tagging of ribosomes and axonal tracing methods. To apply these advanced techniques to post-mortem human brain, we will implement a novel tissue processing protocol [Hibernation-Cryopreservation combined with a Pulsatile Perfusion Hibernation System] to keep post-mortem brains in prolonged hibernation. This allows for the stabilization of nucleic acids, the concurrent collection of live single cells, and the applicationof classical and advanced methods for the identification of neuronal pathways. We will also develop agnostic and integrative computational methods to create a taxonomy of cell types based on molecular identity and connectivity. These features will be compared across species, ages, and sexes. There are four major distinguishing aspects of this application: (1) implementation of novel approaches developed to extend tissue integrity and viability of the postmortem human brain such that we can (2) perform single cell RNA-seq, (3) trace connections and examine cell morphology in the postmortem human brain, which is not amenable to classical experimentations~ and (4) develop novel analytical tools and approaches. This pilot project and methodologies directly address the goals of this BRAIN Initiative RFA and are designed to demonstrate their utility and scalability to ultimately complete a comprehensive cell census of the entire human brain in healthy and disease states.