Crude synaptosome preparation and glutamate uptake assay

Glutamate uptake assay was performed with crude synaptosome preparation from mouse cortices using the 0.32 M sucrose centrifugation method. After total protein determination by Bradford assay (Bio-Rad), 1 μCi L-³H glutamate and 100 μM non-labeled glutamate were mixed with Na⁺ uptake buffer (in mM; Tris 5, HEPES 10, NaCl 140, KCl 2.5, CaCl₂ 1.2, MgCl₂ 1.2, K₂HPO₄ 1.2, Glucose 10, total volume 275 μL) then added into 25 μL of each synaptosome sample in 96-well multiscreen High-throughput screening (HTS) filter plates (Millipore). After 6 min incubation, uptake was terminated by putting samples into an ice bath. Samples were then filtered using the Steriflip vacuum filtration system (Millipore) and washed 6x with ice-cold Phosphate-buffered saline (PBS) while continually filtering the samples. Each filtered 96-well membrane was excised out and transferred into vials for scintillation counting. Dihydrokainate (DHK, 500 μM) or DL-threo-β-Benzyloxyaspartic acid (DL-TBOA, 500 μM) was added into appropriate wells in glutamate uptake assay. Disintegration per min (DPM) value was normalized by total protein concentration and converted to fmol/μg/min unit. GLT1-dependent glutamate uptake was calculated by subtracting remaining glutamate uptake activity with DHK treatment from the total glutamate uptake activity.