DEVELOPMENT OF A NEURAL NETWORK FORMATION ASSAY FOR IPSC-DERIVED GLUTAMATERGIC NEURONS

Rapid advances in stem cell technology have led to widespread adoption for the development of in vitro models of neuron electrophysiology. And yet, the field lacks clear standards for evaluating the critical elements of neuronal models. In this study, we developed a neural network formation assay using either rat cortical neurons (RCNs) or varied mixtures of human iPSC-derived glutamatergic to GABAergic neurons cultured on microelectrode arrays (MEAs). Spontaneous activity was monitored over several weeks and cultures were sequentially dosed with a panel of pharmacological compounds to quantify functional synaptic activity and the balance of excitation and inhibition. The assay involved a three-step dosing protocol: (1) bicuculline to block GABA-mediated inhibition, (2) CNQX/ APV to block glutamatergic transmission via AMPR and NMDA receptors, and (3) 4-aminopyridine (AP) to block potassium channels. Post addition of bicuculline, RCNs demonstrated increased burst frequency and synchronized activity, indicating disinhibition of excitatory neurons. The addition of CNQX/APV silenced most of the functional neural activity with a 95% decrease in mean firing rate and 100% decrease in network burst frequency. On day 64 for iPSC-neurons, bicuculline increased burst frequency in mixed and inhibitory-poor cultures, while CNQX/APV suppressed all network activity across conditions. Unlike RCN cultures, CNQX/APV had less effect on overall mean firing rate of human iPSC-neurons, suggesting increased reliance on intrinsic excitability over synaptic connectivity. The differences observed between the primary and iPSC-derived neural populations underscore the need for standardized assays that consider cell-type specific timelines and analysis strategies to evaluate neural behavior.