3D spheroids have emerged as powerful drug discovery tools given their high-throughput screening (HTS) compatibility. The present invention presents a method for generating functional neural spheroids with differentiated human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes at cell type compositions mimicking specific regions of the human brain. Recordings of intracellular calcium oscillations were used as functional assays, and the utility of this spheroids system was shown through disease modeling, drug testing, and formation of assembloids, modeling connection of two unique brain regions, to model neurocircuitry. The invention further provides disease models applications such as for Alzheimer’s and Parkinson’s Disease through incorporating genetically engineered cells into spheroids, as well as Opioid Use Disorder by chronically treating spheroids with mu-opioid agonists.