Dl threo β benzyloxyaspartate tboa

Unless otherwise indicated, chemicals and drugs were purchased from Sigma-Aldrich (Milan, Italy). Stock solutions of tetrodotoxin (TTX; Tocris Bioscience, Bristol, UK), 2-(3-carboxypropyl)-6-(4-methoxyphenyl)-2,3-dihydropyrazidin-3-iminium bromide (gabazine), dl-threo-β-benzyloxyaspartate (TBOA; Tocris Bioscience), and cytosine β-d-arabinofuranoside (AraC) were prepared in distilled water and stored at −20°C. On the day of the experiment, stock solutions were diluted at their final concentration with our culture medium.

DL-threo-β-benzyloxyaspartate (TBOA) and (S)-4-Carboxy-phenylglycine (CPG) were obtained from Tocris Bioscience, Inc., (Bristol, UK). The drugs were frozen in 2.5 mM and 50 mM stocks, respectively, in 1 × phosphate buffered saline (PBS) and diluted as needed. All other chemicals were purchased from Sigma-Aldrich, Inc (St Louis, MO, USA).

Glutamate levels were measured using an Amplex Red Glutamic Acid/Glutamate Oxidase Assay kit (Thermo Fisher, A12221) as described previously (Wang et al., 2010 (link)). Briefly, neurons were cultured in a 12-well plate in triplicate until DIV14. Samples were collected before stimulation, after 15-min depolarization in a buffer containing 90 mM KCl, and poststimulation. All wells were treated with 500 μM DL-threo-β-benzyloxyaspartate (TBOA; Tocris) to prevent glutamate reuptake. Control wells were incubated with the calcium channel blocker cadmium chloride to prevent glutamate release. Samples were incubated with Amplex Red reagent per manufacturer’s instructions at 37°C for 30 min, and measured with a BioTek Synergy H1 Hybrid Multi-Mode microplate reader (BioTek) at excitation/emission 530 nm/590 nm. Fluorescence measurements were normalized to a blank well, and glutamate concentrations were calculated using a standard curve ranging from 0.5 to 30 μM. Total glutamate released was calculated by summing the basal, stimulated, and poststimulation glutamate concentrations for each experimental condition.