[3H]MK-801 Binding Assay

Crude synaptic membranes were prepared from the cerebral cortex of Sprague–Dawley rats³⁸. Animals used procedures complied with NIH publication n° 80–23 revised in 1996 and approved by the Animals Care Commettee of the University of Pisa in compliance with the Directive 2010/63/EU on animal welfare. The pellets were stored at −80 °C for at least 24 h, and washed three more times with Tris–HEPES buffer (Tris 4.5 mM, HEPES 5 mM, pH 7.4) to remove the endogenous amino acids before the binding assay³⁸.

In the binding assay, 50 μL of membrane preparation (40–50 μg protein), and 10 μL of compound were mixed at 25 °C in the presence of 50 µM L-glutamate (10 μM) and 50 μL of glycine (10 μM). Then, 50 µl of [³H]MK-801 (final concentration 3 nM) were added to the preparation. Tris–HEPES buffer was added to a final volume of 0.5 mL³⁸. Following incubation time of 2 h at 25 °C, binding was terminated by rapid filtration. Radioactivity was measured using a PerkinElmer liquid scintillation counter. Nonspecific binding was determined in the presence of unlabeled 100 μM MK-801. The dissociation constant (Kd) of [³H]MK-801 in rat cortex membranes was 4.0 nM. For compound activity determination, aliquots of membrane pellets were incubated with different ligand concentrations of Memit or Memantine (10 nM-10 µM) in the absence or presence of 4 mM Cysteine for 30 min, and then incubated with 3 nM [³H]MK-801 for 2 h at 25 °C. Samples were then filtered, and the radioactivity was counted.