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

Abstract

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.

Keywords

• Neurotoxins
• Cell-based assay
• Enzyme-luminescence method
• C6 glioma cells
• Primary cortical neurons
• l-Glutamate release
• Real-time monitoring