Enzyme-luminescence method: Tool for real-time monitoring of natural neurotoxins in vitro and l-glutamate release from primary cortical neurons

• We describe the applicability of our previously developed novel enzyme-luminescence method for rapid and sensitive detection of natural neurotoxins (e.g., shellfish and mushroom toxins) using brain model cells (C6 glioma cells) in vitro.
• A racemic mixtures of the gonyautoxins (GTX), including GTX2,3 and GTX1,4 were used for evaluating the inhibition effects of these toxins on glutamate release from the C6 glioma cells. The potency was compared based on IC50 values.
• The activation effect of ibotenic acid (a mushroom toxin) on glutamate release from C6 cells was also evaluated. The potency was compared based on EC50 values.
• We also tested the applicability of our system for real-time detection of glutamate release from primary rat cortical neurons instead of model cells.
• This novel detection technique may be also applicable in determining neuronal differentiation ratio as well finding glutamatergic neurons without immunostaining in situ.
• This sensing tool may also has a great potential for the investigation of the effects of various growth factors and chemicals on neuronal differentiation, neurotransmitter dynamics, neurodegeneration, and synaptogenesis.

Novel enzyme-luminescence method is used for the rapid and sensitive in vitro detection of natural neurotoxins (e.g., shellfish and mushroom toxins) using model brain cells. Paralytic shellfish poisons gonyautoxins (e.g., GTX2,3 and GTX1,4) were detected at 1 nM level by their inhibition of glutamate release from C6 glioma cells upon drug stimulation (IC50: GTX2,3 = 30 nM and GTX1,4 = 8 nM). Activation of glutamate release from C6 cells by ibotenic acid (a mushroom toxin) was also evaluated (EC50 = 10 nM). The method was tested for real-time detection of glutamate release from primary rat cortical neurons. Dose-dependent effects of KCl (0–200 mM) and NMDA on glutamate release from primary cortical neurons were studied. The effects of different culture conditions on K+-depolarization-induced glutamate release were also investigated. The method may be applicable to screening of drugs and toxins, and finding glutamatergic neurons in brain slices without in situ staining.