Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A_2A receptors (A_2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A_2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A_2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A_2ARs were focally removed from the NAc core or other A_2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

Adenosine is a neuromodulator acting through inhibitory A₁ receptors (A₁Rs) and facilitatory A_2ARs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A₁Rs, but the source of the adenosine activating A_2ARs is unknown. We now show that ecto-5′-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A_2ARs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A_2ARs and proximity ligation assays confirmed the close proximity of CD73 and A_2ARs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A_2AR prodrug that requires CD73 metabolization to activate A_2ARs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A_2AR KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A_2AR KO mice, as well as upon pharmacological A_2AR blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A_2AR function. This study points to CD73 as a new target that can fine-tune A_2AR activity, and a novel therapeutic target to manipulate A_2AR-mediated control of striatal function and neurodegeneration.

The function of striatal adenosine A_2Areceptors (A_2ARs) is well recognized because of their high expression levels and the documented antagonistic interaction between A_2ARs and dopamine D₂receptors in the striatum. However, the role of extrastriatal A_2ARs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A_2ARs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A_2ARs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A_2AR knock-out (st-A_2AR KO) mice in comparison with forebrain-specific A_2AR KO (fb-A_2AR KO) mice. In contrast to fb-A_2AR KO (deleting A_2ARs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A_2AR KO mice exhibited Cre-mediated selective deletion of the A_2AR gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A_2AR KO but attenuated in fb-A_2AR KO mice. Furthermore, selective inactivation of the A_2ARs in extrastriatal cells by administering the A_2AR antagonist KW6002 into st-A_2AR KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A_2ARs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A_2ARs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A_2ARs.