[3H]AMPA Binding

Adult male SD rats (220–250 g) were euthanized by decapitation. The brainstems were dissected and homogenized for 2 min in 15 volumes of ice-cold 50 mM Tris–HCl buffer (pH 7.4) at 4°C. The homogenates were centrifuged at 1,000 g for 10 min, and the supernatants were collected and centrifuged at 40,000 g for 20 min. The pellets were dispersed in 50 mM Tris-HCl pH 7.4 and centrifuged at 40,000 g for 20 min. The last procedures were repeated a third time with a total of four washes to eliminate endogenous glutamate from the tissue. The resulting pellets were suspended in eight volumes of 0.3 M sucrose, then stored at –70°C until use.

Tissue membranes were washed twice in 50 mM Tris-HCl pH 7.4, and the resultant membranes resuspended in 50 mM Tris-HCl pH 7.4 in the presence of 100 mM KSCN. The dispersed tissue membranes were incubated with 200 nM [³H]AMPA (58 Ci/mmol, PerkinElmer, Shanghai, China) and LCX001 at different doses (10^-9–10^-4 M) in a final volume of 1 ml. The binding reaction was performed in disposable glass culture tubes at 25°C for 60 min. Bound [³H]AMPA was separated from the free ligand by filtration under reduced pressure with GF/C filters presoaked in 0.2% polyethyleneimine. The pellets were washed with ice-cold Tris-HCl pH 7.4 three times and dried with a piece of tissue paper. Nonspecific binding was determined by 1 mM nonradioactive glutamate. The binding activity on the filters was detected using a LS6500 scintillation counter (Beckman, Shanghai, China). For saturation binding with [³H]AMPA, LCX001 (10^-4 M) was added in the resuspended assay with changing concentrations of [³H]AMPA from 1 to 400 nM.