Assembly and characterization of human cortico-striatal neural networks

在包括帕金森氏症在内的许多人类大脑疾病中都发现了皮质-纹状体网络的破坏 疾病、亨廷顿病、自闭症、精神分裂症和严重抑郁症。不幸的是，细胞和 在这些疾病中，导致连接中断的分子缺陷是非常困难的 由于获取原代人脑组织的途径有限，无法总结与疾病相关的信息，因此需要进行研究 动物模型中的网络缺陷。这是阻碍发现新的治疗方法的一个主要问题。 病人。因此，这项研究的目标是开发第一种健壮的方法来产生人类皮质- 纹状体有机体，概括皮质-纹状体连接，并使用这个系统来研究细胞 以及负责人类皮质-纹状体网络建立和成熟的分子机制。 为此，我们将使用我们的新方法从人类中首次产生皮质和纹状体有机化合物 干细胞来源的单个神经花环(SNR)。在我们的初步实验中，我们发现SNR派生出 皮质类器官由不同亚型的皮质神经前体细胞组成，皮质深层和表层 兴奋性神经元，一小部分具有外侧神经节隆起(LGE)特征的抑制性神经元， 星形胶质细胞和少突胶质细胞，而SNR来源的纹状体有机体由不同的亚型组成 在大脑皮质下神经前体细胞中，D_1/D_2中的棘神经元中，有很大一部分抑制神经元具有LGE- 比如身份、星形胶质细胞和少突胶质细胞。此外，我们还证明了5个月龄SNR中的神经元- 衍生的有机化合物显示成熟激发重复动作电位的功能和形态证据， 接受兴奋性和抑制性突触输入，并显示精细的树突分支和棘突。我们的 本研究的具体目标是(1)开发一种健壮的、可重复性的组装皮质纹状体的方案 具有明确的细胞组成和组织的有机化合物；(2)表征 人类皮质纹状体器官的解剖和功能网络；以及(3)确定分子和 建立联系的皮质和纹状体神经元的功能特性。重要的是，我们将使用 单细胞信使核糖核酸测序、长期植入的多电极探针、 狂犬病病毒追踪和光遗传学研究潜在的细胞和分子机制。在……里面 总结，该项目将开发第一个组装功能性人类皮质纹状体网络的协议，并 为网络连接的细胞和分子机制提供新的见解。因为大多数人的大脑 障碍会影响几个大脑区域，并扰乱区域间的大脑交流，产生 代表多个大脑区域的有机化合物，这些区域复制复杂的神经系统结构和 生理学将成为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.