Mouse stem cell-derived neurons, NMDA receptor, AMPA receptor, Neurotoxicity, Apoptosis, CNS lesions, Nerve agent-induced seizure

Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca, culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78– 200 mM glutamate evoked reproducible timeand dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 mM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg or substitution of Ba for Ca. Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78–200 mM glutamate resulting in apoptosis and 3000 mM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dosedependent fashion. These data indicate that ESNs replicate many of the excitogenic mechanisms observed in primary neuron culture, offering a moderate-throughput model of excitotoxicity that combines the verisimilitude of primary neurons with the flexibility and scalability of cultured cells. ESNs therefore offer a physiologically relevant platform that exhibits characteristic NMDAR responses, and appears suitable to evaluate molecular mechanisms of glutamate-induced excitotoxicity and screen for candidate therapeutics. Citation: Gut IM, Beske PH, Hubbard KS, Lyman ME, Hamilton TA, et al. (2013) Novel Application of Stem Cell-Derived Neurons to Evaluate the Timeand DoseDependent Progression of Excitotoxic Injury. PLoS ONE 8(5): e64423. doi:10.1371/journal.pone.0064423 Editor: Christopher Mark Norris, Univ. Kentucky, United States of America Received August 3, 2012; Accepted April 15, 2013; Published May 14, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was funded by the National Institutes of Health National Institute of Allergy and Infectious Diseases (IAA number AOD12058-0001-0000) and the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division (grant number CBM.THRTOX.01.10.RC.021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]