Membrane Preparation and Binding Assays

Total membranes and synaptic membranes (from a synaptosomal preparation) were obtained by isopicnic and gradient centrifugations of homogenized brain tissue, as previously described (Rebola et al., 2005; Pliássova et al., 2016). To determine the enrichment and basic binding characteristics of A₁R in cortical synapses, we compared saturation binding isotherms of the selective A₁R antagonist ³H-DPCPX (0.1–10 nM; specific activity of 102.1 Ci/mmol; from DuPont NEN) in total and synaptosomal membranes (72–164 μg) incubated for 2 h incubation at room temperature in a buffer containing 50 mM Tris, 1 mM EDTA, 2 mM EGTA, pH 7.4, with adenosine deaminase (4 U/ml, Roche) before filtration through Whatman GF/C filters (Millipore), as previously described (Rebola et al., 2003; Coelho et al., 2006). To estimate the binding affinity of caffeine, we carried out displacement curves of ³H-DPCPX binding with caffeine (0.1–300 μM; from Sigma), as previously described (Coelho et al., 2006). Results are expressed as specific binding, determined by subtraction of the non-specific binding, which was measured in the presence of 2 μM 8-{4-[(2-aminoethyl)amino]carbonylmethyloxyphenyl}xanthine (XAC, a mixed A₁R/A_2AR antagonist; from Tocris) and normalized per amount of protein (bicinchoninic acid assay). To derive the binding parameters from saturation curves (K_D and B_max values) the data were fitted by a rectangular hyperbola using the GraphPad Prism software. For displacement binding curves, IC₅₀ values were converted to K_i values by non-linear fitting of the semi-logarithmic curves derived from the competitions curves.