Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson’s disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson’s disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain–striatum–cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

腹侧中脑多巴胺能神经元投射到纹状体和皮质，参与运动控制和奖励相关认知。在帕金森病中，黑质纹状体中脑多巴胺能神经元退化并导致典型的帕金森病运动相关障碍，而中皮质边缘中脑多巴胺能神经元的功能障碍与成瘾和神经精神疾病有关。然而，由于缺乏合适的模型和人体材料的获取，对人类多巴胺能系统的发育和选择性神经变性的研究受到限制。在这里，我们开发了一种人体体外模型，该模型概括了纹状体和皮质多巴胺能神经支配的关键方面。这些空间排列的腹侧中脑-​​纹状体-皮质类器官（MISCO）可用于研究多巴胺能神经元的成熟、神经支配和功能，对细胞治疗和成瘾研究具有重要意义。我们详细介绍了腹侧中脑、纹状体和皮质类器官的生长方案，并描述了它们在放置在定制嵌入模具中时如何以线性方式融合。我们报告了 MISCO 中与纹状体和皮质组织功能性远程多巴胺能连接的形成，并表明注射的腹侧中脑模式祖细胞可以成熟并支配该组织。使用这些组合体，我们检查了多巴胺能回路扰动，并表明长期可卡因治疗会导致长期持续的形态、功能和转录变化，这些变化在停药后仍持续存在。因此，我们的方法为研究人类多巴胺能细胞移植和电路重建以及药物对人类多巴胺能系统的影响开辟了新途径。