Fig. 1.

Generation of A_2A KO mice with target inactivation of the A_2A receptor.A, Schematic diagram of the A_2A receptor targeting vector; a standard replacement-type vector was constructed with 5 and 4.5 kb A_2A receptor genomic fragments split by a positive selection marker (Neo cassette), which replaced the 3′ end of exon 2 (E2) and the adjacent 5′ splice junction and intron sequences. Digestion of wild-type and mutant A_2A receptor genes with BamHI (at sites labeled B) generates 7.5 and 5.0 kb fragments, respectively, that can be distinguished using a nonoverlapping 3′ probe (as in B). B, Genomic Southern analysis of WT (+/+), heterozygous (+/−), and homozygous (−/−) mice with respect to the A_2A receptor gene was performed as described in Materials and Methods, using the 3′ nonoverlapping probe illustrated in A. WT mice displayed a single 7.5 kb band, whereas homozygous A_2A KO mice showed a single 5.0 kb band corresponding to the restriction fragments for WT and mutant alleles, respectively. Heterozygous mice showed both 7.5 and 5.0 kb bands.C, Homozygous A_2A receptor KO mice are defi-cient in A_2A receptors detected by receptor autoradiography; A_2A receptor binding was determined using³H-CGS 21680 as a ligand. A representative coronal brain section from a WT mouse shows specific labeling of A_2Areceptors in striatum (caudate putamen, CP; nucleus accumbens, NA) and olfactory tubercle (OT), whereas that from a homozygous mouse shows no ³H-CGS 21680 binding. D, Behavioral responses to the A_2A agonist CGS 21680 in WT and A_2A KO mice; ambulation was measured in WT and A_2A KO mice (n = 14–16) before and after challenge with CGS 21680 (0.2 mg/kg, i.p.) by recording contiguous photobeam interruptions (ambulation) for 60 min. Error bars represent the mean ± SEM. *p < 0.05 (Student's t test) when compared with ambulation in the WT mice before treatment.