Down Syndrome (DS) is caused by triplication of human chromosome 21 (HSA21). It is a leading cause of intellectual deficits and a single genetic factor leading to the development of Alzheimer's disease (AD). However, the mechanisms leading to cognitive dysfunction in DS are still largely unknown. While HSA21 gene triplications affect all brain cells, behavioral and cognitive deficits ultimately reflect neuronal network dysfunctions. Mechanistic understanding of altered neuronal activity is limited, as functional studies in DS individuals are not feasible and the currently available animal models for DS have considerable shortcomings. To overcome these limitations and comprehensively assess the impact of trisomy on neuronal activity and connectivity in human cells, we utilized an induced pluripotent stem cell (iPSC)-derived cellular system of cortical organoids (COs). We regionally patterned isogenic COs to generate dorsal COs (dCOs) and ventral COs (vCOs) that primarily give rise to glutamatergic neurons and inhibitory GABAergic interneurons (InN), respectively. We previously identified cellular and transcriptomic aberrations in excitatory neurons in trisomic dCOs. In our preliminary studies using calcium imaging, we detected an increased correlation of firing patterns across individual neurons in trisomic dCOs. We further expanded our experimental model and generated assembloids through the fusion of vCOs and dCOs. This powerful platform enables us to study the contribution of InN to neuronal connectivity and network activity, assess their migration as well as their role in the development of AD pathology in DS (DS-AD). To examine the effect of trisomy on neuronal activity in a maturation-promoting environment, our team developed a multimodal recording platform where we engraft fluorescently labeled COs into the retrosplenial cortex of immunodeficient mice and monitor neuronal activity through implanted, optically transparent graphene microelectrode arrays. Xenografted COs become vascularized and establish synaptic connections with host neuronal circuits allowing us to perform longitudinal electrical and optical measurements for 6 months or longer. Using our advanced in vivo and in vitro systems, this study will determine cellular, transcriptomic, and motility- related changes driven by trisomy and how they pertain to the alterations in neuronal activity and connectivity and the development of DS-AD. In Aim 1, we will utilize euploid and trisomic assembloids to examine the effect of trisomy on cellular composition, InN migration, and activity of individual neurons and networks using electrophysiology, calcium imaging, as well as histological and biochemical methods. Next, we will perform dCO (Aim 2) and vCO (Aim 3) xenotransplantations to address how trisomy affects (a) development of spontaneous and stimulus-triggered neuronal activity over time; (b) cellular migration and integration with the host brain; (c) transcriptomic changes within engra...