Cryo-electron tomography of functionally validated synapses in human induced neurons

Cryo-electron tomography (cryo-ET) has provided unparalleled insights into synaptic architecture, yet its application to human neurons has been limited by sample preparation challenges. Here, we establish an integrated platform that combines human induced neurons (iNs) cultured directly on electron microscopy (EM) grids with functional electrophysiological validation and cryo-ET imaging. Human iNs formed extensive neuronal networks on EM grids, exhibited reliable synaptic transmission and intrinsic excitability, and maintained short-term synaptic plasticity. While iNs exhibited intact synaptic transmission on EM grids, we found that EM grids acted as a current sink for extracellular electrical stimulation. Following vitrification, cryo-ET enabled direct visualization of ultrastructural features of excitatory synapses, including synaptic vesicle pools, active zone proximal vesicles, postsynaptic densities, and associated organelles. Together, our results establish a foundation for structurally and functionally dissecting synaptic organization between human iNs and provide initial insight into cryo-ET imaging of synapses from stem cell-derived human neurons.