A critical role of the adenosine A2A receptor in extrastriatal neurons in modulating psychomotor activity as revealed by opposite phenotypes of striatum and forebrain A2A receptor knock-outs

Abstract

The function of striatal adenosine A(2A) receptors (A(2A)Rs) is well recognized because of their high expression levels and the documented antagonistic interaction between A(2A)Rs and dopamine D(2) receptors in the striatum. However, the role of extrastriatal A(2A)Rs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A(2A)Rs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A(2A)Rs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A(2A)R knock-out (st-A(2A)R KO) mice in comparison with forebrain-specific A(2A)R KO (fb-A(2A)R KO) mice. In contrast to fb-A(2A)R KO (deleting A(2A)Rs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A(2A)R KO mice exhibited Cre-mediated selective deletion of the A(2A)R 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(2A)R KO but attenuated in fb-A(2A)R KO mice. Furthermore, selective inactivation of the A(2A)Rs in extrastriatal cells by administering the A(2A)R antagonist KW6002 into st-A(2A)R KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A(2A)Rs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A(2A)Rs 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(2A)Rs.

Figure 1.

Characterization of striatum A_2AR KO mice with selective deletion of A_2AR gene and proteins in striatal neurons. A, Brain region specificity of Cre-mediated A_2AR gene deletion in st-A_2AR KO and fb-A_2AR KO mice. Floxed alleles of the A_2AR gene (“Flox” band) or Cre-mediated deletion of the A_2AR gene (“KO” band) were detected by PCR analysis using a three-primer set as described previously (Bastia et al., 2005). Genomic DNAs were isolated from OB, ST, HIP, CTX, MB, HYP, CB, BS, thymus (THY), heart (HRT), lung (LG), kidney (KID), liver (LIV), and spleen (SPL). KO cont. and WT cont. are PCR products of genomic DNA isolated from the tail of fb-KO and fb-WT mice, respectively. B, Cell-type specificity of Cre-mediated A_2AR gene deletion in st-A_2AR KO mice by flow cytometric sorting and PCR analyses. Striatal neurons (β-tubulin III+ cells) and astrocytes (GFAP+ cells) of st-A_2AR KO (i.e., Cre+) and their WT littermates (i.e., Cre−) were separated by flow cytometric sorting, followed by PCR analysis of genomic DNAs in the sorted cells. C, In situ hybridization of A_2AR mRNA in the st-A_2AR KO and gb-A_2AR KO mice and their corresponding WT littermates. D, Quantitative analysis of ³H-ZM241385 binding in total membrane preparations of the striatum and cerebral cortex from mice of each of the six different genotypes. The data were presented as mean ± SEM (fmol/mg protein; n = 3–4 per group). *p < 0.05 (1-way ANOVA, post hoc Bonferroni test), comparing gb-A_2AR KO, fb-A_2AR KO, and st-A_2AR KO groups to their corresponding WT group.

Figure 2.

Cocaine- or PCP-induced psychomotor activity is attenuated in forebrain A_2AR KO mice but enhanced in striatum A_2AR KO mice. Ambulation was recorded in KO and WT mice for 120–180 min after injection of cocaine (25 mg/kg, i.p.), KW6002 (3.3 mg/kg, i.p.), PCP (10 mg/kg, i.p.), or vehicle. The arrows indicate time of injection. A, KW6002-induced motor activity in fb-A_2AR KO (n = 8) and fb-WT (n = 8) mice. B, KW6002-induced motor activity in st-A_2AR KO (n = 9) and st-WT (n = 15) mice. C, Cocaine-induced psychomotor activity in fb-A_2AR KO (n = 11) and fb-WT (n = 12) mice. D, Cocaine-induced psychomotor activity in st-A_2AR KO (n = 13) and st-WT (n = 13) mice. E, PCP-induced psychomotor activity in fb-A_2AR KO (n = 8) and fb-WT (n = 8) mice. F, PCP-induced psychomotor activity in st-A_2AR KO (n = 8) and st-WT (n = 8) mice. ^#p < 0.05 (1-way ANOVA, post hoc Bonferroni test), comparing fb-A_2AR KO and st-A_2AR KO groups to their corresponding WT group.

Figure 3.

KW6002 effect on cocaine-induced psychomotor activity in forebrain A_2AR KO and striatum A_2AR KO mice. A–D, The fb-A_2AR KO and st-A_2AR KO mice and their corresponding WT littermates were treated with KW6002 (3.3 mg/kg, i.p.) or vehicle 10 min before cocaine (25 mg/kg, i.p.) administration. Ambulation was recorded for 120 min after cocaine injection. KW6002 increases cocaine-induced ambulation in st-WT mice (n = 12; A) and fb-WT mice (n = 8; C). KW6002 attenuates cocaine-induced ambulation in st-A_2AR KO mice (n = 8; C) and shows no additional effect on cocaine-induced ambulation in fb-A_2AR KO mice (n = 8; D). ^#p < 0.05, comparing cocaine plus KW6002 to cocaine plus vehicle.