Modeling GABAergic hyperexcitability in sleep bruxism patient-derived brainstem neurons using a multielectrode array platform.

OBJECTIVES

Sleep bruxism (SB) involves involuntary jaw movements during sleep and has been linked to impaired inhibitory regulation of brainstem circuits (particularly those involving GABAergic neurons). While previous studies using patch-clamp electrophysiology have demonstrated intrinsic hyperexcitability in neurons differentiated from SB patient-derived human induced pluripotent stem cells (hiPSCs), the low-throughput nature of this technique limits large-scale phenotypic screening. We aimed to establish a robust, high-throughput, multielectrode array (MEA)-based platform capable of quantitatively assessing electrophysiological phenotypes of SB-derived neurons.

METHODS

hiPSCs from three patients with SB and three healthy controls were differentiated to become ventral brainstem-like neurons. Their neuronal composition was evaluated using immunocytochemistry for total neurons (TUBB3+) and GABAergic neurons (GAD1/2+). Spontaneous neuronal firing was assessed using MEA under steady-state conditions and immediately after medium change. Extracellular GABA levels were quantified via ELISA to indirectly assess GABAergic hyperexcitability.

RESULTS

Differentiation efficiency to TUBB3+ or GAD1/2+ neurons did not differ among cell lines. Under steady-state conditions, the weighted mean firing rate (wMFR) did not differ between groups. The SB group showed significantly elevated extracellular GABA concentrations and a significant increase in wMFR immediately after medium change (GABA removal), suggesting that GABA hypersecretion concealed the underlying hyperexcitability of SB-derived neurons.

CONCLUSIONS

We developed a robust, high-throughput MEA platform that reliably quantifies SB-related electrophysiological phenotypes. Our findings indicate that SB-derived GABAergic neurons exhibit constitutive hyperexcitability and excessive GABA release, providing mechanistic insights and a scalable framework for therapeutic discovery.