Experimental models of the human developing brain are needed to investigate human-specific aspects of brain development, evolution, and neurological disease. Progress in the field has been hampered by the lack of models, considering that the endogenous developing human brain cannot be directly investigated; animal models often fail to recapitulate human disorders and cannot feasibly be used to study complex polygenic states spanning many genes. While reductionist in nature, stem-cell derived 3D human brain organoids offer a first-of- its-kind opportunity to study processes of human brain formation and wiring that are otherwise not accessible. However, there is an unmet need for organoid models that are cellularly complete and reproducible and for methodology to decode the establishment, connectivity and dynamics of neural circuits in organoids, at scale and with high fidelity. If we could map the activity and connectivity of organoids at scale, both to understand circuit function/dysfunction and to guide further development of organoids, we could close the loop on organoid design and application. Towards this goal, we have developed many molecular and imaging tools for high-throughput analysis of neural activity and connectivity, which we propose to apply to new, next-generation organoid models. Here, we propose a collaborative approach among four groups (Arlotta - brain organoids and human brain development; Boyden - circuit physiology and neural imaging technology; Lewis - material science and bioengineering and Insoo Hyun- bioethics) to pioneer a robust organoid system that combines the development of vascularized brain organoids incorporating more complete cell diversity and maturation with advanced high-throughput functional molecular and imaging tools to enable interrogation of circuit activity, connectivity, and molecular changes in cells participating in physiologically relevant circuits. We will build on a highly reproducible brain organoid model that we recently developed to promote the generation of cell types that are currently absent in organoids but needed for circuit maturation, refinement, and functionality. This work is intended to generate more advanced organoid models designed to promote maturation and robust network activity. In parallel, we will develop a pipeline to record neural activity from intact organoids using all- optical-electrophysiology techniques at scale, and optimize epitope-based barcoding and expansion microscopy to enable molecularly-annotated connectomics of brain organoids. The work proposed here will enable the use of human organoid models to study human circuit formation, plasticity, and function, analyses that are currently hampered by the lack of technologies and assays for high-throughput measurements of circuit physiology and connectivity in organoids. Beyond the work proposed here, these methods will directly enable investigation into how disease states alter information processing in the brain; for example, linking mutations in disease-associated genes with specific abnormalities in human neurons and circuits to inform the identification of molecular targets for therapeutic intervention.

Project Narrative Neurodevelopmental and neuropsychiatric disorders affect millions worldwide and are of enormous public health significance; however, we have a very limited understanding of the molecular, cellular and circuit-level abnormalities underlying these diseases. Progress in the field has been hampered by the lack of research models, given that animal models often fail to recapitulate the human disease, and that, while 3D human brain organoids offer experimental access to processes of human brain formation and wiring, methodology is needed to decode circuit function and connectivity in organoids, at scale and with high fidelity. We propose here to pioneer the development of next-generation, vascularized brain organoids and to apply novel, scalable molecular and imaging tools to enable investigation of human neural circuits in health and disease.