Electronic Expression of Background and Delayed Rectifier Currents via Dynamic Clamp Produces Physiological Action Potentials in Stem Cell Derived Glutamatergic Neurons

Abstract

Human stem-cell derived glutamatergic neurons (iPSC-GNs) are a model system for studying neurological disorders, such as epilepsy. However, progress for disease modelling in neuronal iPSC-GNs is hindered by the low expression of K+ currents. This results in depolarized resting membrane potentials (RMPs) which leads to the inability to evoke physiologically shaped action potentials (APs). Our goal was to overcome this limitation by developing a neuronal dynamic clamp system to electronically “express” K+ currents in real-time. iPSC-GNs (iCell GlutaNeurons from Fujifilm Cellular Dynamics, WI) were cultured according to the manufacturer's instructions and used 7-30 days after plating. Recordings were performed using the whole-cell ruptured patch clamp configuration. Dynamic clamp was implemented via the Cybercyte dynamic clamp system (Cytocybernetics, NY). iPSC-GNs had a peak sodium current at −20 mV (−198 ±21 pA/pF, n=32). The sodium current was blocked with tetrodotoxin. APs were triggered with 0.5-1.0 nA pulses for 0.3-1.5 ms at 0.5 Hz. Without electronic expression of a virtual K+ current, the RMP was depolarized (‑40 ± 2.1 mV, n=35). The electronic expression of an outwardly rectifying K+ leak current, modeled after Goldman-Hodgkin-Katz equation, allowed for the stabilization of the RMP to hyperpolarized potentials (∼55-60mV) and enabled the recording of evoked, stable and physiologically shaped APs. We also electronically expressed an A-type current which also caused hyperpolarization of the membrane potential and a physiologically shaped AP. The data shows that electronic addition of background and delayed rectifier K+ currents in hiPSC neurons allows for the unmasking of physiological AP properties that can be used for modelling neurological diseases.