Article Figures & Data
Figures
- Figure 1.
Molecular characterization of conditional A2AR-Gpr88 KO mice. A, Triple-fluorescent in situ hybridization probing Gpr88 (Aa, Ab, Ae, Af, Ai, and Aj; probe labeled in green), Drd2 (Ac–Af; probe labeled in orange), and Drd1 (Ag–Aj; probe labeled in red). Representative images are shown. In Gpr88flx/flx control animals, Gpr88 mRNA colocalizes with both Drd2 (Ae: merge GPR88/Drd2, white arrows) and Drd1 mRNA (Ai: merge GPR88/Drd1, yellow arrows). In contrast, Gpr88A2A-Cre conditional mice show almost no colocalization with Drd2 (Af: merge GPR88/D2R), while colocalization with Drd1 remains (Aj: merge GPR88/Drd1, yellow arrows). DAPI staining (blue) was used to label all cell nuclei. Scale bar, 25 µm. B, Quantification shows a strong decrease of Gpr88/Drd2 double-positive neurons (red), but not GPR88/Drd1 double-positive (blue) neurons, in the CPu, Nac, and CeA of Gpr88A2A-Cre conditional mice. Percentage of Gpr88 expression was calculated based on the total number of Drd1- or Drd2-positive cells counted [(number Drd1- or Drd2-expressing cells coexpressing Gpr88 × 100)/total number of Drd1- or Drd2-expressing cells]. Data are presented as the mean ± SEM. n = 4, Gpr88A2A-Cre; n = 4, Gpr88flx/flx; solid asterisks). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).
- Figure 2.
Agonist-induced GPR88 activation in conditional A2AR-Gpr88 KO mice. Introduction of LoxP sites does not alter GPR88 activation. A, GPR88-mediated [35S]-GTPγS was totally and partially abolished in the striatum of Gpr88 −/− and Gpr88A2A-Cre mice, respectively. Gpr88+/+ and Gpr88flx/flx mice present similar GPR88 agonist-induced receptor activation. Two membrane preparations were used per genotype, with each membrane preparation gathering tissue from three animals (1 male/2 females or 2 males/1 female). B–F, Decreased activation of GPR88 in several brain regions of Gpr88A2A-Cre mice: GPR88-mediated [35S]-GTPγS binding is decreased in the CPu, Nac, and central amygdala of mutant animals. Data are represented as the mean ± SEM. A, Text: ***p < 0.001 (post hoc Tukey’s multiple-comparisons test of Gpr88flx/flx or Gpr88+/+ vs Gpr88A2A-Cre and Gpr88 −/−; B–F); n = 6, Gpr88A2A-Cre ; n = 6, Gpr88 −/−; n = 6, Gpr88flx/flx ; text: ***p < 0.001 (post hoc Tukey's multiple comparisons test).
- Figure 3.
Locomotor activity is similarly increased in A2AR-Gpr88 KO and total KO mice. A, B, When placed individually in a dimly lit open field for 30 min, both A2AR-Gpr88 KO mice (A) and total KO mice (B) traveled a longer distance than their control littermates. Line graphs show the distance traveled (in centimeters) in 5 min bins over a 30 min session. Bar graphs show the average total distance traveled (in centimeters) over the 30 min sessions. Data are represented as the mean ± SEM. n = 10, Gpr88A2A-Cre ; n = 10, Gpr88flx/flx (A); n = 12, Gpr88−/−; n = 12, Gpr88+/+ (B). Open asterisks: ***p <0.001 (repeated-measures ANOVA); solid asterisks: ***p < 0.001 (Student’s t test).
- Figure 4.
Anxiety-related responses are similarly increased in A2AR-Gpr88 KO and total KO mice. A–D, A2AR-Gpr88 mice (A, B) and total KO mice (C, D) enter more frequently and spent more time in the light compartment of the light/dark test. E–J, In the elevated plus maze, Gpr88A2A-Cre and Gpr88 −/− mice present higher open arms exploration ratios (E, H, respectively), more frequent total and distal head dips (F, I), and increased time spent in the distal zone of the open arms when compared with their control littermates (G, J). K–R, Social interactions were evaluated in a dimly lit open field with wild-type naive mice of the same age and gender. Both mutant animals display increased number of nose and paw contacts, as well as increased following behaviors. Gpr88−/− mice, but not Gpr88A2A-Cre mice, engaged less frequently in grooming episodes than control animals Data are presented as the mean ± SEM. A, B: n = 11, Gpr88A2A-Cre ; n = 9 Gpr88flx/flx ; C, D: n = 10, Gpr88 −/−; n = 11, Gpr88+/+ ; E–G: n = 12, Gpr88A2A-Cre ; n = 13, Gpr88flx/flx ; H–J: n = 11, Gpr88 −/−; n = 9, Gpr88+/+ ; K–N: n = 10, Gpr88A2A-Cre ; n = 9, Gpr88flx/flx ; O–R: n = 6, Gpr88 −/−; n = 6, Gpr88+/+ . Solid asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).
- Figure 5.
A2AR-Gpr88 gene deletion increases avoidance but does not regulate approach behaviors. A, B, In the marble-burying test (Gpr88A2A-Cre , A; for Gpr8 −/−, B), both KO mice buried fewer marbles than controls. C, F, When assessing novelty preference, total KO mice (F), but not A2AR-Gpr88 KO mice (C), spent more time in the novel compartment when compared with their littermates. D, E, G, In the novelty-suppressed feeding test, Gpr88A2A-Cre mice exhibit similar latencies to start eating and home cage food intake (D) than Gpr88flx/flx mice. Gpr88−/− mice display shorter latencies to start eating in the center of the arena compared with Gpr88+/+ mice (G), eating normally when placed back in their home cage (E). Data are presented as the mean ± SEM. n = 11, Gpr88A2A-Cre ; n = 9, Gpr88flx/flx (A); n = 7, Gpr88 −/−; n = 7, Gpr88+/+ (B); n = 8, Gpr88A2A-Cre ; n = 6, Gpr88flx/flx (C); n = 11, Gpr88A2A-Cre ; n = 11, Gpr88flx/flx (D, E); n = 7, Gpr88 −/−; n = 7, Gpr88+/+ (F); n = 7, Gpr88 −/−; n = 7, Gpr88+/+ (G, H). Solid asterisk: *p < 0.05, **p < 0.01 (Student’s t test).
- Figure 6.
Total but not A2AR-Gpr88 gene deletion impairs fear conditioning. To assess whether Gpr88 deletion affects fear responses, we tested mice in a fear-conditioning test. A, D, During the conditioning session, mutant and control animals displayed similar levels of immobility before and after tone–shock pairing when compared with control mice. E, Twenty-four hours later, Gpr88 −/− mice displayed significantly lower context fear than Gpr88+/+ mice. F, The percentage of immobility of Gpr88 −/− was also decreased when tested for cued fear memory. Deletion of Gpr88 in A2AR-expressing neurons did not affect context (B) or cued (C) fear memories. n = 11, Gpr88A2A-Cre ; n = 11, Gpr88flx/flx (A–C); n = 10, Gpr88 −/−; n = 10, Gpr88+/+ (D–F). Solid asterisks: *p < 0.05 (Student’s t test); text asterisks: *p < 0.05, **p < 0.01 (post hoc Sidak’s multiple comparisons test).
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