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 an...