Functional evaluation of biological neurotoxins in networked cultures of stem cell-derived central nervous system neurons

Journal of Visualized Experiments : JoVE
Kyle S HubbardPatrick McNutt

Abstract

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or bot...Continue Reading

Citations

Nov 29, 2015·Toxicological Sciences : an Official Journal of the Society of Toxicology·Phillip H BeskePatrick M McNutt
Jan 1, 2019·Environmental Sciences Europe·J B LegradiH Hollert
Jul 25, 2021·International Journal of Molecular Sciences·Juliette Duchesne de LamotteCamille Nicoleau

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BETA
Assay
Flow cytometry

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MIST

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