title: Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.
abstract: Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Phillip H
authors.initials: PH
authors.lastname: Beske
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Bradford
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Justin O
authors.initials: JO
authors.lastname: Grynovicki
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Elliot J
authors.initials: EJ
authors.lastname: Glotfelty
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Hoffman
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Kyle S
authors.initials: KS
authors.lastname: Hubbard
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
authors.forename: Kaylie M
authors.initials: KM
authors.lastname: Tuznik
authors.affiliation: Cellular and Molecular Biology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland patrick.m.mcnutt2.civ@mail.mil.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McNutt
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000633:toxicity*; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D006801:Humans; D008666:Metalloendopeptidases / Q000633:toxicity*; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects*; D050825:Synaptosomal-Associated Protein 25 / Q000032:analysis; D013744:Tetanus Toxin / Q000633:toxicity*
chemical_list: D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; C035979:tetanospasmin; D016291:Dizocilpine Maleate; D008666:Metalloendopeptidases; D001905:Botulinum Toxins
keywords: botulinum toxins; electrophysiology; neurons; spontaneous postsynaptic currents; synaptic transmission; tetanus toxin