All animals were handled in accordance with the protocols approved by the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou Medical University, China. Eighteen adult C57B6/J (n = 6/experimental group), three A_2AR knockout mice (from Chen's laboratory at Boston University School of Medicine) and three D₂R knockout mice (from The Jackson Laboratory, USA, Drd2^tm1Low, stock No. 003190) were used for the experiments.

Instrumental training and behavioral testing schedules were performed following the procedure by Rossi et al. (Rossi and Yin, 2012). In brief, mice first underwent a 5-day food deprivation schedule to reach 80–85% of their free-feeding weight before instrumental training sessions. Mice were then given one 30-min magazine training session during which one drop of 20 μl 20% sucrose solution as reward was delivered on a random time 60-s schedule. During continuous reinforcement (CRF) sessions, each lever press resulted in delivery of the sucrose reward. Sessions ended after 60 min or 50 rewards had been earned, whichever came first. After CRF, mice underwent a random interval (RI) schedule to promote habit formation or a random ratio (RR) schedule to promote goal-directed behavior. Mice underwent the RI schedule were trained for 2 days on random interval 30 s (RI30), with a 0.1 probability of reward availability every 3 s contingent upon lever pressing, followed by 4 days on RI60 schedule. Progressively leaner schedules of reinforcement were used for the RR training procedure: RR5 (each response was rewarded at a probability of 0.2 on average), RR10, RR20 each for 2 days.

Following the RI and RR training sessions, a 2-day devaluation test was conducted. A specific satiety procedure was applied to alter the current value of a specific reward. On each day the mice were allowed to have free access to home chows which were used for maintaining their weights (i.e., valued condition, the sucrose solution was still valued) or sucrose solution which was earned by their lever pressing in the training sessions (i.e., devalued condition, the sucrose solution was devalued) for at least an hour to achieve sensory-specific satiety. Immediately after the unlimited pre-feeding session, mice were given a 5-min extinction test during which the lever was inserted and pressing times was recorded but without reward delivery. The orders of the valued and devalued condition tests (day 1 or 2) were counterbalanced across each group. Mice insensitive to manipulation of outcome value, that is habit, would mildly change lever presses on the devalued condition compared to the valued condition, whereas goal-directed mice that performed sensitively to outcome value would significantly reduce their lever presses on the devalued condition. The control mice underwent food deprivation schedule and were handled exactly the same way every day just as the RR and RI training group before instrumental training. During the training sessions, the control mice were also placed in the operant chambers in which the sucrose reward was delivered in a random 30/60 s (corresponding to RI30/RI60) manner but without lever stretched. In the devaluation test, the control mice were also exposed to the operant chamber for 5 min with no lever stretched out and no reward available. In the present study, three groups (n = 6 for each group) were examined for the A_2AR-D₂R/A_2AR-mGLuR5 heterodimers in the striatum after instrumental learning: (a) mice without instrumental training as “control group,” (b) mice underwent RI/RR training sessions as “RI group,” and (c) mice underwent RR training sessions as “RR group.”

After two additional RI60 or RR20 training sessions following devaluation test, mice were sacrificed for PLA detection of the A_2AR-D₂R and A_2AR-mGLuR5 heterodimers in the striatum. We performed PLA analysis according to the procedure described recently (Augusto et al., 2013; Pinna et al., 2014). Three sections from one brain (from anterior, middle, and posterior parts of the striatum, respectively) were rinsed in TBS at room temperature. The sections were incubated with 1% BSA and 0.5% Triton X-100 for 2 h at room temperature for blocking and permeabilization. The mouse anti-A_2AR (1:300; millipore) and rabbit anti-D₂R (1:300; millipore)/rabbit anti-mGluR5 (1:300; millipore) were incubated with sections overnight at room temperature. Sections were then rinsed for four times (30 min each time) in TBS with 0.2% Triton X-100 following the manufacturer's protocol. Slices were then incubated at 37°C with the PLA secondary probes (1:5; Olink Bioscience) for 2 h. After rinsing with “Duolink II” Wash Buffer A, the slices were then incubated with the ligation-ligase solution for 30 min at 37°C followed by rinsing with Duolink II Wash Buffer A. Sections were ready for amplification with polymerase (1:40; Olink Bioscience). Then the sections were washed in decreasing concentrations SSC buffers (Olink Bioscience) and mounted on slides. Fluorescent mounting medium (containing DAPI) were applied on the sections. The fluorescence images (three non-overlapping and random microscopic fields respective from DMS and DLS of each brain section) were acquired by confocal microscope (Figure 2A). The quantitative analysis was done following the procedure by Bonaventura et al. (2014). The cells surrounded by the red puncta were defined as positive cells (white arrows in Figure 1). The cell number was counted by software “Image J.” Each microscopic field image was quantified as “positive cell number/total cell number.” The quantified value of the experimental mice was normalized to that of A_2AR KO mice (as the background).

Mice were deeply anesthetized and then transcardially perfused with 0.01 M PBS (pH = 7.4) followed by ice-cold 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 4–6 h at 4°C, and then allowed to equilibrate using gradient sucrose solution (10, 20, and 30%). Immunofluorescence were performed on 30 μm free-floating sections. Free-floating sections were washed in PBS and incubated for 60 min in 0.3% Triton X-100 and 10% normal donkey serum and then incubated with mouse anti-A_2AR antibody (Millipore, 1:200) and rabbit anti-D₂R antibody (Millipore, 1:200) at 4°C overnight. Brain sections were incubated with Alexa 488-conjugated secondary antibodies (Invitrogen, 1:1000). The sections were washed and mounted. Fluorescent mounting medium were applied on the sections. Images were acquired by a fluorescence microscope.

Instrumental behavior training and test processes were analyzed using two-way ANOVA for repeated measurements with the training or test sessions as within-subjects effect and the different training procedures as between-subjects effect. In the PLA assay, two-way ANOVA was used with striatal subregions and training procedures as main effects. Paired t-test was conducted to compare the distribution difference of A_2AR-D₂R and A_2AR-mGluR5 heterodimers between the DMS and DLS. One-way ANOVA with LSD post-hoc was used to compare the distribution variation of A_2AR-D₂R heterodimers on the anterior-posterior axes in both DMS and DLS after instrumental learning.

To detect the striatal A_2AR-D₂R and A_2AR-mGluR5 heterodimers by PLA assay, we first confirmed the specificity of the PLA detection of the A_2AR-D₂R and A_2A-mGluR5 heterodimers using the A_2AR KO and D₂R KO mice. The specificity of the A_2AR and D₂R antibody was evident with highly enriched expression pattern of the A_2ARs or D₂Rs in the striatum by immunofluorescence, which was absent in the A_2AR or D₂R KO mice (Figure 1A). Moreover, the specific labeling of the A_2AR-D₂R heterodimer signals (cellular membrane) were detected in ~15% of striatal neurons of wild-type mice in close association of DAPI (nuclei; as indicated by white arrow; Figures 1B,C). Importantly, these signals for the A_2AR-D₂R protein complexes were essentially absent in the striatum of the A_2AR KO or D₂R KO mice (Figures 1B,C), confirming the specificity of the PLA detection of the A_2AR-D₂R heterodimers. Similarly, the A_2AR-mGluR5 heterodimer signals were detected by PLA in the striatum of wild-type mice but not in A_2AR KO mice, supporting the specificity of PLA detection of the A_2AR-mGluR5 heterodimers (Figures 1D,E).

Following the confirmation of the specificity of the PLA detection of the A_2AR-D₂R and A_2AR-mGluR5 heterodimers, we examined the heterogeneous distribution of these heterodimers in the DMS and DLS in normal mice. PLA analysis showed that the A_2AR-D₂R complexes were more prominent in the DLS than DMS (Figure 2B). Quantitative analysis confirmed the heterogeneous distribution of A_2AR-D₂R heterodimers in the striatum (i.e., DLS > DMS; Figure 2B). By contrast, there was no heterogeneous distribution of the A_2AR-mGluR5 heterodimers in the DMS and DLS by PLA (Figure 2C).

Following RI training sessions, mice gradually, and steadily increased their lever presses and reached plateau at RI60 schedule (Figure 3A). Consistent with the previous studies (Dickinson et al., 1983; Yin and Knowlton, 2006), devaluation test (Figure 3B) showed that mice trained by RI procedure showed insensitive to outcome devaluation, indicating their habitual action. In association with habit formation after the RI training sessions, we detected the increased formation of the A_2AR-D₂R heterodimers compared to mice without training (“control;” Figures 3C,D). Moreover, the A_2AR-D₂R heterodimers increased in both DMS and DLS, and accordingly, the heterogeneous pattern of this heterodimers in DMS and DLS persisted after the trainings.

Over the RR training sessions, mice also gradually and steadily increased their lever presses and reached plateau at RR20 schedule (Figure 4A). Consistent with the previous studies (Dickinson et al., 1983; Yin and Knowlton, 2006), devaluation test showed that mice trained by RR schedule markedly reduced their lever presses in the devalued condition, indicating their goal-directed behavior (Figure 4B). In association with goal-directed behavior after RR training sessions, we did not detect any significant change in the A_2AR-D₂R heterodimers compared to mice without training (“control;” Figures 4C,D). Furthermore, the striatal A_2AR-D₂R heterodimers underwent RI schedule were significantly increased in both DMS and DLS compared to the RR group [DMS: F_{(2, 17)} = 6.351, p = 0.010, DLS: F_{(2, 17)} = 9.605, p = 0.002; ^*p < 0.05, ^**p < 0.01; one-way ANOVA with LSD post-hoc test, n = 6/group]. Thus, the increased association of striatal A_2AR-D₂R heterodimers is selectively induced by the RI training schedule in association with habit formation, but not affected by RR training schedule which produced goal-directed behavior.

We have also performed detailed analysis of the A_2AR-D₂R hetrerodimers in three subregions of the DMS and DLS on anterior-posterior axes (i.e., anterior, middle, and posterior, Figure 5). In the DMS, the A_2AR-D₂R heterodimers in both the anterior and posterior parts were increased after RI training compared to the control or RR group (Figure 5A). In the DLS, the change in the A_2AR-D₂R heterodimers was relatively less pronounced such that the increase was observed only in the middle DLS after RI training compared to the control (Figure 5B). There was no difference in the A_2AR-D₂R heterodimers in the anterior, middle and posterior part of the DMS and DLS between the RR or control groups, with exception of apparently a small increase in the middle part of the DMS in the RR group (Figures 5A,B).

We also examined the subregional distribution and adaptive changes of the A_2AR-mGluR5 heterodimers after the RI and RR training schedules (Figure 6). The A_2AR-mGluR5 heterodimers displayed neither heterogeneous distribution between DMS and DLS nor adaptive changes after the RI training (leading to habit formation) or RR training (leading to goal-directed behavior) sessions (Figures 6A,B). These indicated that such heterogeneous distribution (DLS vs. DMS) and adaptive changes over the instrumental learnings (i.e., increased formation of the heterodimers) were specific for the A_2AR-D₂R heterodimers.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.