iCell GABANeurons (Cellular Dynamics International, USA) were plated at a density of 60,000 cells per well in 96-well plates coated with poly-l-ornithine and laminin (Sigma-Aldrich, UK). iCell GABANeurons were maintained according to the manufacturer’s instructions. iCell GABANeurons were fed every 3 to 4 days by replacement of half media.

GABA release was assessed in iCell GABANeurons at DIV 16 to 18. Cells were treated with neurotoxins for 24 hours at 37°C. Following removal of neurotoxin, cells were washed three times in Neurobasal medium containing 1% B27 and 0.5 mM GlutaMAX (assay medium). Cells were loaded with [3H]-GABA (2 μCi/ml; PerkinElmer, UK) in assay medium for 120 min at 35°C. Following removal of [3H]-GABA, cells were washed three times with assay medium. Basal and stimulated [3H]-GABA release was established by incubation at 35°C for 5 min with assay medium (50 μl per well) containing low potassium (5.3 mM KCl) or high potassium (60 mM KCl), respectively. To determine retained [3H]-GABA, cells were lysed by adding radioimmunoprecipitation assay buffer (50 μl per well; Sigma-Aldrich, UK). Superfusates and cell lysates were transferred into 96-well IsoPlates (PerkinElmer, UK), and OptiPhase Supermix scintillation fluid (200 μl per well) was added. Radioactivity was quantified using a MicroBeta2 plate reader (PerkinElmer, UK).

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