Male Wistar rats (6–8 weeks old, 140–160 g, obtained from Harlan Ibérica, Barcelona, Spain) were used and were handled according to EU guidelines for use of experimental animals, the rats being anesthetized under halothane atmosphere before being killed by decapitation. The synaptosomes were prepared by centrifugation of homogenized striatal tissue (dissected striatum corresponded mostly to the nucleus caudate–putamen) in sucrose medium (see Rodrigues et al. 2005). For immunochemical analysis (see Rodrigues et al. 2005), the striatal synaptosomes were placed onto coverslips previously coated with poly-l-lysine, fixed with 4% paraformaldehyde for 15 min and washed twice with phosphate-buffered saline (PBS). The synaptosomes were permeabilized in PBS with 0.2% Triton X-100 (Sigma) for 10 min and then blocked for 1 h in PBS with 3% bovine serum albumin and 5% normal rat serum. The synaptosomes were then washed twice with PBS and incubated with either rabbit anti-adenosine A1 receptor (1 : 500; from Upstate Biotechnology, Golden, CO, USA) or rabbit anti-synaptophysin antibodies (1 : 200; from Zymed Laboratories, Lisbon, Portugal) together with either rat anti-dopamine transporter (1 : 500; from Chemicon, Southampton, UK) or mouse anti-tyrosine hydroxylase antibodies (1 : 500; from Chemicon) for 1 h at 20°C. The synaptosomes were then washed three times with PBS with 3% bovine serum albumin and incubated for 1 h at 20°C with AlexaFluor-488 (green)-labeled goat anti-rabbit and either AlexaFluor-594 (red)-labeled goat anti-rat or AlexaFluor-594 (red)-labeled goat anti-mouse antibodies (1 : 200 for all, from Molecular Probes, Leiden, The Netherlands). The selectivity of the A1 receptor antibody was confirmed by the lack of signal obtained in synaptosomes derived from A1 receptor knockout mice tissue (generously supplied by Bertil B. Fredholm, Karolinska Institutet, Sweden). We also confirmed that none of the secondary antibodies produced any signal in preparations to which the addition of the corresponding primary antibody was omitted. After washing and mounting on slides with Prolong Antifade, the preparations were visualized in a Zeiss Axiovert 200 inverted fluorescence microscope (Zeiss, Göttingen, Germany) equipped with a cooled CCD camera and analyzed with MetaFluor 5.0 software (Universal Imaging Co., Downingtown, PA, USA). Data represent means ± SEM of three experiments and in each experiment, using different synaptosomal preparation from different animals, four different fields acquired from two different coverslips were analyzed. Each coverslip was analyzed by counting two different fields and in each field, a total amount of 500 individualized elements. For the release experiments (see Köfalvi et al. 2005), the synaptosomes were labeled with 5 μCi of [7,8-3H]-dopamine (41.0 Ci/mmoL from Amersham, Buckinghamshire, UK) for 5 min at 37 C, layered over Whatman GF/C filters and superfused (flow rate: 0.7 mL/min) with Krebs solution for 20 min before starting collection of the superfusate every 2 min. The synaptosomes were stimulated for 1 min with 20 mmol/L K+ (isomolar substitution of NaCl by KCl in the Krebs solution) at 4 and 16 min (first and second stimulation periods, S1 and S2, respectively) after starting sample collection, triggering a release of tritium in a Ca2+-dependent manner that was mostly 3H-dopamine, gauged by HPLC (data not shown). The A1 receptor agonist N6-cyclopentyladenosine (CPA; Sigma) was added 6 min before the start of S2 and its effect was quantified by the modification of S2/S1 ratio versus control (i.e., absence of drugs), whereas the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; Sigma) was added 15 min before starting sample collection and did not modify the S2/S1 ratio versus control. The concentrations of A1 receptor agonists and antagonists used in synaptosomal experiments were previously shown to be selective for the A1 receptor (Ciruela et al. 2006). Radioactivity was expressed in terms of fractional release, i.e., percentage of tritium released as a function of the total amount of tritium retained in each chamber. A paired Student’s t-test was used to test the significance of the effect of a drug versus control with ‘n’ representing the number of experiments carried out using different animals, always in duplicate. When making comparisons from different sets of experiments with control, one-way anova was used followed by Dunnett’s test.