Assembly and characterization of human cortico-striatal neural networks

皮质-纹状体网络的破坏已经在许多人类大脑疾病中发现，包括帕金森氏症 疾病、亨廷顿氏病、自闭症、精神分裂症和重度抑郁症。不幸的是， 在这些疾病中，导致连接中断的分子缺陷非常困难 研究，因为有限的访问初级人脑组织和无法概括疾病相关的 动物模型中的网络缺陷。这是一个主要的问题，阻止了新的治疗方法的发现， 患者因此，本研究的目的是开发第一个强大的方法来产生人类皮质- 概括皮质-纹状体连接的纹状体类器官，并使用该系统研究细胞 以及负责人类皮质-纹状体网络的建立和成熟的分子机制。 为此，我们将使用我们的新方法来产生皮质，并首次从人类纹状体类器官 干细胞衍生的单神经元受体（SNRs）。在我们的初步实验中，我们发现SNR衍生的 皮质类器官由不同亚型的皮层神经祖细胞、深层和浅层皮质神经祖细胞组成， 兴奋性神经元，一小部分具有侧神经节隆起（LGE）样身份的抑制性神经元， 星形胶质细胞和少突胶质细胞，而SNR衍生的纹状体类器官由不同的亚型组成 结果显示，D1/D2中棘神经元、大部分抑制性神经元和LGE- 比如身份、星形胶质细胞和少突胶质细胞。此外，我们证明了5个月大的SNR- 衍生的类器官显示出成熟激发重复动作电位的功能和形态学证据， 接受兴奋性和抑制性突触输入，并显示出精致的树突分支和棘。我们 本研究的具体目的是（1）开发一种稳健的和可重复的用于组装皮质-纹状体 具有明确细胞组成和组织的类器官;（2）表征 人类皮质-纹状体类器官中的解剖和功能网络;以及（3）确定分子和 皮层和纹状体神经元的功能特性。重要的是，我们将使用 “尖端”技术，如单细胞mRNA测序，长期植入多电极探针， 狂犬病病毒追踪和光遗传学，以研究潜在的细胞和分子机制。在 总之，该项目将开发第一个用于组装功能性人类皮质-纹状体网络的协议， 为网络连接的细胞和分子机制提供了新的见解。因为大多数大脑 疾病会影响几个大脑区域，破坏区域间的大脑交流， 代表多个大脑区域的类器官复制了复杂的神经系统结构， 生理学将构成脑部疾病建模和药物发现的重大突破。

Disruption of the cortico-striatal network has been found in many human brain disorders, including Parkinson’s disease, Huntington’s disease, autism, schizophrenia, and major depression. Unfortunately, the cellular and molecular deficits responsible for the development of disrupted connectivity in these disorders are very difficult to study because of the limited access to primary human brain tissue and inability to recapitulate disease-related network deficits in animal models. This is a major problem that prevents the discovery of novel therapies for patients. Thus, the objectives of this study are to develop the first robust method for generating human cortico- striatal organoids that recapitulate cortico-striatal connectivity and to use this system to investigate the cellular and molecular mechanisms responsible for the establishment and maturation of human cortico-striatal networks. To this end, we will use our new method to generate cortical and, for the first time, striatal organoids from human stem cell-derived single neural rosettes (SNRs). In our preliminary experiments, we found that SNR-derived cortical organoids consist of different subtypes of pallial neural progenitors, deep and superficial layer cortical excitatory neurons, a small fraction of inhibitory neurons with lateral ganglionic eminence (LGE)-like identities, astrocytes, and oligodendrocytes, whereas SNR-derived striatal organoids are composed of different subtypes of subpallial neural progenitors, D1/D2 medium spiny neurons, a large fraction of inhibitory neurons with LGE- like identities, astrocytes, and oligodendrocytes. In addition, we demonstrated that neurons in 5-month-old SNR- derived organoids show functional and morphological evidence of maturity—firing repetitive action potentials, receiving excitatory and inhibitory synaptic inputs, and exhibiting elaborate dendritic branches and spines. Our specific aims in this study are (1) to develop a robust and reproducible protocol for assembling cortico-striatal organoids with well-defined cell composition and organization; (2) to characterize the establishment of anatomical and functional networks in human cortico-striatal organoids; and (3) to determine the molecular and functional properties of the cortical and striatal neurons that make the connections. Importantly, we will use “cutting-edge” techniques such as single-cell mRNA sequencing, chronically implanted multi-electrode probes, rabies virus tracing, and optogenetics to investigate the underlying cellular and molecular mechanisms. In summary, this project will develop the first protocol for assembling functional human cortico-striatal networks and provide novel insights into the cellular and molecular mechanisms of network connectivity. As most brain disorders impact several brain regions and disrupt interregional brain communication, the ability to generate organoids representing multiple brain regions that replicate the complex nervous system architecture and physiology will constitute a major breakthrough in brain disease modeling and drug discovery.