Uncovering NMDA receptor-mediated high firing activity in excitatory human neurons

Human iPSC-derived glutamatergic (iGlut) neurons provide a promising platform for studying neuronal function and modeling CNS diseases, but assessing large populations of neurons from multiple donors remains challenging. We developed a protocol that targets N-methyl-D-aspartate receptors (NMDA-Rs) and enhances neuronal activity, revealing functional phenotypes. Using the calcium indicator GCaMP8f, we demonstrate that Mg²⁺-free ACSF significantly increases neuronal activity, and is enhanced by glycine but inhibited by the NMDA-R antagonist AP-V. Multi-electrode array recordings also show robust firing in Mg²⁺-free ACSF. Lastly, patch-clamp electrophysiology confirms the higher firing rates in Mg²⁺-free ACSF across multiple donor lines, uncovering donor-specific firing phenotypes. This protocol facilitates functional analyses of iGlut neurons while preserving single-cell resolution, enabling detailed characterization of iGlut neurons in diverse applications such as CNS disease modeling and drug screening. This protocol establishes a versatile framework for large-scale studies of neuronal network dynamics and individual excitability in iPSC-derived iGlut neurons.