Effect of A₁-A_2A double heterozygosity on the number of adenosine receptors and on the response to adenosine. A and B: autoradiographic quantitation of A₁ (A₁R) and A_2A adenosine receptors (A_2AR) in mice with different genotypes. Graph in A shows a binding isotherm for A₁R binding in cortex using 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) as the radioligand. Binding was not detectable in A₁R knockout (KO) mice. K_d values (mean and 95% confidence intervals) for mice were as follows: wild-type (WT), 4.5 (3.1–5.9); A₁R heterozygous (Hz), ±4.0 (3.0–5.0); A_2AR KO, 3.6 (2.5–4.7); A_2AR Hz, 3.7 (2.6–4.7); and A₁R-A_2AR double heterozygous (dHz), 4.0 (2.8–2.3). Maximum binding (B_max) values were as follows: WT, 148 (130–165); A₁R Hz, 86 (77–94); A_2AR KO, 135 (119–150); A_2AR Hz, 138 (124–153); and A₁R-A_2AR dHz, 85 (75–95). Two to three sections were analyzed at each concentration of radioligand. Graph in B shows a binding isotherm for A_2AR binding in caudate-putamen using SCH-58261 as the radioligand. Binding was not detectable in A_2AR KO mice. K_d values (mean and 95% confidence intervals) were as follows: WT, 0.7 (0.5–0.9); A₁R Hz, 0.7 (0.4–0.9); A₁R KO, 0.6 (0.4–0.8); A_2AR Hz, 0.8 (0.4–1.1); and A₁R-A_2AR dHz, 0.9 (0.6–1.2). B_max values were as follows: WT, 159 (144–174); A₁R Hz, 162 (141–184); A₁R KO, 171 (157–185); A_2AR Hz, 115 (101–130); and A₁R-A_2AR dHz, 113 (100–126). Three sections were analyzed at each concentration of radioligand. Graph in C shows that adenosine responses were shifted to the right in mice with one-half the receptor number, approximately to the same extent as by caffeine in commonly used doses. Adenosine responses are shown as the inhibition of lipolysis by a stable adenosine analog, 2-chloroadenosine (2-CADO), in adipocytes from WT (•) or A₁R-A_2AR dHz mice (○). The cells were incubated in the presence of adenosine deaminase and 10 nM norepinephrine. In some experiments, 30 (WT caff 30) or 100 μM caffeine (WT caff 100) was added in addition. Increasing concentrations of 2-CADO were then added, and glycerol release was measured after the end of incubation. Results are from triplicate determinations from 3 (WT) or 2 (dHz) batches of cells prepared from 4 mice.

Increased activity in mice receiving oral caffeine ingestion. The locomotor activity (LA) of WT female (A) and male mice (B) mice was recorded using telemetry. LA data before caffeine injection are presented as 5-day averages in 30-min intervals, shown as control (n = 13). LA data during the second period are presented as 3-day averages (the 5th, 6th, and 7th days after start of the period); mice drank water containing 0.3 g/l caffeine (caffeine, n = 8) or drank only tap water (water, n = 5), as described in materials and methods. The dark period (shaded) is from 7:00 PM to 7:00 AM. Values are means ± SE. *P < 0.05 compared with control. #P < 0.05 compared with water. C: the protocol of telemetry recording: 5 days were recorded for baseline HR, body temperature (Temp), and LA (control); 7 days were recorded during mouse ingestion of caffeine or tap water, for which the average of the 5th, 6th, and 7th days is shown as second period data (caffeine or water).

The stimulatory effect of caffeine injection is reduced in A₁R-A_2AR dHz male mice. Saline, 7.5 mg/kg caffeine, and then 30 mg/kg caffeine were administrated at 2-h intervals to WT (•) and A₁R-A_2AR dHz (○) male mice (n = 8). The LA was measured at 30-min intervals. Values are means ± SE. *P < 0.05, WT vs. dHz mice.

Effect of caffeine ingestion on heart rate (HR). HR was recorded using telemetry for consecutive 10-min periods in WT and A₁R-A_2AR dHz mice. In the top, and middle panels the dark period (shaded) is from 7:00 PM to 7:00 AM. Basal HR data are presented as 5-day averages, shown as WT control (n = 8 females, n = 8 males) and A₁R-A_2AR dHz control (n = 8 females, n = 9 males). HR data during caffeine ingestion are presented as 3-day averages (the 5th, 6th, and 7th days after start of the period), shown as WT caffeine and A₁R-A_2AR dHz caffeine. Bottom panel: summary of the data presented in top and middle. Values show the summarized HR from 12:00 AM to 12:00 PM (beats/day). Data are means (top and middle) or means ± SE (bottom). *P < 0.05 compared with WT control mice. #P < 0.05 compared with WT caffeine.

LA measured in a locomotor box. A and B: the LA of tap water-treated WT (WT H₂O) (n = 15 females, n = 16 males), long-term caffeine-treated WT (WT chr caff; n = 7 females, n = 10 males), A₁R-A_2AR dHz (n = 11 females, n = 11 males), A₁R-A_2AR double knockout (A₁R-A_2AR dKO; n = 7 females, n = 8 males), A₁R KO (n = 16 females, n = 15 males), and A_2AR KO mice (n = 6 females, n = 12 males) was measured in an open field as described in materials and methods. LA was increased in WT chr caff, A₁R-A_2AR dHz, and A₁R KO mice (*P < 0.05) but not in A₁R-A_2AR dKO and A_2AR KO mice, including both sexes.

Responses to caffeine in genetically modified mice. Reduced response to caffeine injection in A₁R, A_2AR KO, A₁R-A_2AR dKO, and A₁R-A_2AR dHz mice. After acute administration of 15 mg/kg caffeine, the accumulated LA was measured between 46 and 135 min in WT H₂O (n = 10 females, n = 4 males), WT chr caff (n = 4 females, n = 5 males), A₁R-A_2AR dHz (n = 7 females, n = 8 males), A₁R-A_2AR dKO (n = 6 females, n = 6 males), A₁R KO (n = 6 females, n = 6 males), and A_2AR KO mice (n = 4 females, n = 6 males) using a locomotor box as described in materials and methods. *P < 0.05 compared with WT H₂O.