Generation of glutamatergic/GABAergic neuronal co-cultures derived from human induced pluripotent stem cells for characterizing E/I balance in vitro

Obtaining mechanistic insights into the disruptions of neuronal excitation and inhibition (E/I) balance in brain disorders has remained challenging. Here, we present a protocol for in vitro characterization of E/I balance. Using human induced pluripotent stem cells, we describe the generation of glutamatergic excitatory/GABAergic inhibitory neuronal co-cultures at defined ratios, followed by analyzing E/I network properties using immunocytochemistry and multi-electrode array recording. This approach allows for studying cell-type-specific contribution of disease genes to E/I balance in human neurons. For complete details on the use and execution of this protocol, please refer to Mossink et al. (2022)(1) and Wang et al. (2022).(2)