Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro

Charles-Francois V Latchoumane,Maysam Ghovanloo,Kayvan F Tehrani,Ladonya Jackson,Mohammad S Eslampanah Sendi,Luke J Mortensen,Steven L Stice,Lohitash Karumbaiah

doi:10.1038/s41598-018-29069-3

Charles-Francois V Latchoumane, Maysam Ghovanloo + Show 6 more

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https://doi.org/10.1038/s41598-018-29069-3

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Abstract

Functional electrical stimulation (FES) is rapidly gaining traction as a therapeutic tool for mediating the repair and recovery of the injured central nervous system (CNS). However, the underlying mechanisms and impact of these stimulation paradigms at a molecular, cellular and network level remain largely unknown. In this study, we used embryonic stem cell (ESC)-derived neuron and glial co-cultures to investigate network maturation following acute administration of L-glutamate, which is a known mediator of excitotoxicity following CNS injury. We then modulated network maturation using chronic low frequency stimulation (LFS) and direct current stimulation (DCS) protocols. We demonstrated that L-glutamate impaired the rate of maturation of ESC-derived neurons and glia immediately and over a week following acute treatment. The administration of chronic LFS and DCS protocols individually following L-glutamate infusion significantly promoted the excitability of neurons as well as network synchrony, while the combination of LFS/DCS did not. qRT-PCR analysis revealed that LFS and DCS alone significantly up-regulated the expression of excitability and plasticity-related transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit (NR2A), brain-derived neurotrophic factor (BDNF) and Ras-related protein (RAB3A). In contrast, the simultaneous administration of LFS/DCS down-regulated BDNF and RAB3A expression. Our results demonstrate that LFS and DCS stimulation can modulate network maturation excitability and synchrony following the acute administration of an inhibitory dose of L-glutamate, and upregulate NR2A, BDNF and RAB3A gene expression. Our study also provides a novel framework for investigating the effects of electrical stimulation on neuronal responses and network formation and repair after traumatic brain injury.

Highlights

Traumatic brain injury (TBI) is a complex neuropathology commonly affecting healthy youth and war veterans[30,31], with a wide range of severity and phenotypic outcomes[32]
Following L-glutamate treatment, we found that low frequency stimulation (LFS) stimulation significantly increased the rate of maturation in neuronal excitability measures (# of cell bursting; one-way ANOVA, p = 0.039; LFS-LFS/direct current stimulation (DCS): p = 0.046; post-hoc Dunn correction) and marginally significantly for weighted mean firing rate (wMFR) compared to the NoStim L-glutamate group (Fig. 6C)
In order to understand the underlying effects of electrical stimulation of untreated cells and following L-glutamate treatment, we investigated the change in expression of transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit A and B (NR2A and NR2B; transcripts encoding two receptors linked to glutamate excitation and the modulation of long-term potentiation and depression), brain-derived neurotrophic factor (BDNF; transcript encoding a protein that promotes synaptogenesis and neurogenesis) and RAS-related protein (RAB3A; transcript encoding a protein related to synaptic vesicles transport)

Summary

Introduction

Traumatic brain injury (TBI) is a complex neuropathology commonly affecting healthy youth and war veterans[30,31], with a wide range of severity and phenotypic outcomes[32]. The secondary phase that follows involves inflammation, oxidative stress, metabolic dysregulation and excitotoxic release of neurotransmitters such as glutamate[32,33,34]; resulting in significant brain tissue loss and functional impairments. To this date, no therapeutic approaches have proven to be effective in treating TBI32. We used embryonic stem cell (ESC)-derived neuron and glia co-cultures in combination with current stimulation protocols (i.e. DCS and LFS) and MEA recordings to investigate and modulate the response of neuronal networks to L-glutamate induced inhibition. We quantified the effects of stimulation on changes in network responses, and the change in excitability and synaptic function-related expression of NR2A, NR2B, BDNF, and RAB3A transcripts using qRT-PCR

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