Journal of oral biosciences · 2026
Modeling GABAergic hyperexcitability in sleep bruxism patient-derived brainstem neurons using a multielectrode array platform.
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Onishi Mayu, Yamaguchi Akihiro, Abe Yuka, Sato Taro, Fujishima Miho, Akamatsu Wado, Baba Kazuyoshi
Аннотация
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.
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.