(a,c,d) Predominant labeling in some somata (So), dendrites (Dn) and spines (Sp). Strongly labeled elements are indicated by So_L, Dn_L and Sp_L, while unlabeled or weekly labeled elements are indicated by So_U, Dn_U and Sp_U. (b) Negative control showing virtual lack of immunogold labeling for A_2A receptors in the striatum of A_2A receptor knockout mice. (e,f) Less predominant labeling in nerve terminals (NT) from asymmetric synapses (arrows) with unlabeled spines (f) and more rarely with labeled spines (e). (g) Quantitative analysis of immunolabeling density for A_2A receptors in the striatum of wild type (+/+) and A_2A receptor knockout mice (−/−); labeling of terminals is only counted for those forming asymmetric synapses with spines; results are indicated as mean+SEM; * and **: p<0.05 and p<0.01, respectively. Scale bars: 500 nm.

(a,b) Evidence for the existence of nerve terminals (NT) dually labeled for vGluT1 and adenosine A_2A receptor (VGLUT1+A2A-NT) that make asymmetric contact (arrows) on unlabeled dendritic spines (u-Dn). Glutamatergic (vGluT1-containing) nerve terminals are labeled with silver-intensified immunogold and A_2A receptor with immunoperoxidase. *: A_2A receptor-immunoreactive astrocytic process; A_2A-Dn: A_2A-immunoreactive dendrite. Scale = 0.4 µm.

(a) Normalized mean EPSC amplitudes recorded from direct-pathway MSNs were significantly enhanced by CGS21680 (1 µM) (n=7, p<0.05). (b) Average traces recorded at baseline (thin line) and after CGS21680 bath application (thick line). (c) The A_2A receptor antagonist SCH442416 (1 µM) significantly decreased EPSC amplitude (n=6, p<0.05) at direct-pathway MSNs. (d) CGS21680 did not alter IPSC amplitudes (n=5, p>0.05). (e) SCH442416 had no effect on normalized mean indirect-pathway EPSC amplitude (n=5, p>0.05). (f) CGS21680 had no effect on normalized mean indirect-pathway EPSC amplitude (n=5, p>0.05). (g) Summary of EPSC modulation by CGS21680 (CGS) and SCH442416 (SCH) at 20–25 min (direct pathway) or 15–20 minutes (indirect pathway) after drug application. (h) Mean-variance analysis of direct-pathway EPSCs following drug application (20–25 min). Asterisks denote p<0.05. Results are expressed as mean±SEM.

(a) Representative recordings of the input signal (current pulses) delivered from the stimulator in the orofacial area of the motor cortex (upper traces) and the EMG output signal obtained from the jaw muscles (lower traces), after the administration of either saline (left traces) or the A_2A receptor antagonist MSX-3 (3 mg/kg, i.p.; right traces). (b) Representative input stimulator signal power (time constant 0.01 sec; upper traces) and output electromyographic (EMG) signal power (time constant 0.01 sec; lower traces) after the administration of either saline (left traces) or MSX-3 (right traces). (c,d) Dose-dependent decrease in the power correlation coefficient (PCC) of the power of input and output signals induced by the administration of MSX-3 (c) and the D₁ receptor antagonist SCH23390 (d). Results represent means±SEM (n=6–8 per group); *,**,***: significantly different compared to saline (p<0.05, p<0.01 and p<0.001, respectively).

(a) Immunoblot analysis with striatal protein sample showing that anti-A_2A receptor antibody recognizes major (double arrowhead) and minor (single arrowhead) protein bands at about 44 and 62 kDa, respectively. (b, c) Single immunofluorescence of parasagittal brain sections showing an exclusive immunoreactivity in the striatum (St), nucleus accumbens (Ac) and olfactory tubercule (Tu) of the wild-type mice (WT) but not A_2A receptor knockout mice (KO); Cx, cortex; Hi, hippocampus; Th, thalamus; Cb, cerebellum; BS, brainstem. (d) Double immunofluorescence for A_2A receptor (A_2AR, red) and MAP2 (green) in the striatum showing a dense immunoreactivity for A_2A receptor on the surface of some MAP2-labeled somata (#) and dendrites (arrows); the remaining MAP2-labeled somata (*) and dendrites (arrowheads) did not express A_2A receptors. (e, f) Triple immunofluorescence for A_2A receptor (A_2AR, red), MAP2 (blue) and enkephalin (Enk, green) or substance P (SP, green) in the striatum showing a selective expression of A_2A receptors in Enk-positive medium spiny neurons (#). A_2A receptor is not detected in SP-positive medium spiny neurons (*). (g) Triple immunofluorescence for A_2AR (A_2AR, red), D₁ receptor (D₁R, blue), and D₂ receptor (D₂R, green) in the striatum showing a preferential distribution of A_2A in D₂ receptor-positive medium spiny neurons (#), but not in D₁ receptor-positive medium spiny neurons (*). Scale bars: (b,c) 1 mm; (d-g), 10 µm.

(a,b) Evidence for co-localization of A_2A receptors (arrows) and D₂ receptors (diffuse dark staining) in dendrites (Dn) and spines (Sp). In (b) there is an example of the uncommon A_2A receptor-labeled nerve terminal (NT; arrowheads) making asymmetric synaptic contact with a double-labeled spine. (c,d) Evidence for the lack of co-localization of A_2A receptors (arrows) and D₁ receptors (diffuse dark staining) in dendrites and spines (Sp). In (d) there is an example of an A_2A receptor-labeled nerve terminal (arrowheads) making an asymmetric synaptic contact with a D₁ receptor-labeled spine. Scale bars: 200 nm).

Representative triple labeling immunocytochemical co-localization of adenosine A_2A receptor (blue), D₁ receptor (red) and vGluT₁ (green) in synaptosomes and purified nerve terminals; in white, triple co-localization (arrows). There was a significantly higher triple expression of vGluT1 and A_2A and D₁ receptors in synaptosomes compared to purified nerve terminal preparations (see Table 1 and text).

Extracellular levels of glutamate (a) and dopamine (b) in the striatum after cortical electrical stimulation. Cortical stimulation in the orofacial area of the motor cortex produced a significant increase in the extracellular concentrations of glutamate (Glu) and dopamine (DA) in the lateral but not the medial striatum. Perfusion of the A_2A receptor antagonist MSX-3, but not the D₁ receptor antagonist SCH23390, significantly counteracted the increase in glutamate and dopamine extracellular levels in the lateral striatum induced by cortical electrical stimulation (repeated measures ANOVA: p<0.05). Results represent means±SEM of percentage of the mean of the three values before the stimulation; n=5–10 per group. Time ‘0’ represents the values of the samples previous to the stimulation. The train of vertical lines represents the period of cortical stimulation. * and **: significantly different (p<0.05 and p<0.01, respectively; Newman-Keuls post-hoc tests) compared to value of the last sample before the stimulation.

(a) The A_2A receptor antagonist MSX-3 (3 mg/kg), but not the D₁ receptor antagonist SCH23390 (0.3 mg/kg) counteracted striatal ERK1/2 phosphorylation induced by cortical stimulation. (b) Both MSX-3 and SCH23390 counteracted striatal PKA-dependent phosphorylation of GluR₁ subunit of AMPA receptor. Results are shown in means±SEM (n=6–13 per group) and representative Western blots. *,***: significantly different compared to saline-treated group (p<0.05 and p<0.001, respectively). “S” refers to cortical stimulation.