Functional characterization of brevetoxin-induced neurotoxicity in cortical neurons using membrane potential-sensitive fluorescent probes and cell signaling assays
Peterson, Jennifer Hoffman
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Brevetoxins represent a series of structurally related polyether neurotoxins that have been implicated in numerous epizootics and human exposures worldwide. Brevetoxins exert their effects by binding to voltage-gated sodium channels in the brain and altering their gating properties resulting in the propagation of continuous action potentials and the subsequent overactivation of N-methyl-D-aspartate receptors (NMDAR) in neurons. After optimizing assay conditions for the two membrane potential-sensitive fluorescent dyes DiBAC4(3) and the Membrane Potential Assay Kit (FMPblue, Molecular Devices Inc., blue dye) in intact neurons, we developed standard conditions that allowed us to quantify the magnitude of PbTx-2-induced depolarization through comparison to that produced by KCl in neocortical cells. We found that 300 nM PbTx-2 and 15 mM KCl produce equivalent levels of membrane potential in neocortical +cells. Using this data, we determined that both [Na]i and Src tyrosine kinase are involved in PbTx-2-induced upregulation of NMDARs.