Flexible 3D multimodal organoid interface reveals functional subnetworks and coordinated circuit activity in cortical organoids

The balance between excitatory and inhibitory (E/I) neurons is fundamental to cortical function, and its disruption underlies a wide range of neurological disorders, including epilepsy, Alzheimers disease and autism spectrum disorder. Cortical organoids offer a promising model to study this balance, as they recapitulate key features of brain development, including the emergence of E/I circuitry. However, no existing technology can simultaneously resolve neuronal identity, spatial organization, and dynamic network activity within intact organoids, limiting precise interrogation of E/I circuit function. Here, we report INFORM, an INtegrated Flexible Organoid Recording Multimodal neural interface that conformally wraps organoids of any size and shape, enabling long-term recordings over 371 days with above 90% of channels detecting spikes. INFORM interface combines four distinct modalities; electrophysiology, two-photon calcium imaging, targeted electrical microstimulation, and chemical intervention to monitor and modulate neural activity across multiple spatial and temporal scales spanning from action potentials from single neurons to synchronized bursts from large-scale populations. Such high-resolution interrogation enables probing of emergence of functional connectivity in fusing assembloids and resolves spatially discrete functional subnetworks that can be selectively recruited and modulated by INFORM. Neurotransmission blockade confirms that stimulus-evoked suppression of neuronal spiking is mediated by GABAergic inputs providing direct functional evidence of the presence of inhibitory signaling in organoids. This work allows single-cell dissection of functional networks in brain organoids, providing a powerful platform for modelling and studying cortical development, neurological diseases, and the E/I imbalances underlying circuit dysfunction.