GPR88 in D1R-Type and D2R-Type Medium Spiny Neurons Differentially Regulates Affective and Motor Behavior

Visual Abstract


Introduction
Among brain orphan G-protein-coupled receptors (GPCRs), GPR88 shows highest and almost restricted expression in the striatum, a key region in motor control, cognitive functions and motivational processes (Liljeholm and O'Doherty, 2012;Quintana et al., 2012;Ehrlich et al., 2018). Homozygous deleterious mutation of Gpr88 in humans was linked to a familial developmental disorder characterized by a childhood chorea (hyperkinetic movement disorder), learning disabilities and marked speech retardation (Alkufri et al., 2016). Previous reports have shown that mice lacking Gpr88 present hyperlocomotion, increased stereotypies, motor coordination and motor learning deficits (Logue et al., 2009;Quintana et al., 2012;Meirsman et al., 2016b). The total Gpr88 gene deletion in mice also induced failure to habituate to an open field or automated home-cage environment and decreased anxiety-like behaviors (Meirsman et al., 2016b;Maroteaux et al., 2018). Additionally, AAVmediated re-expression of GPR88 in the dorsal striatum [caudate putamen (CPu)] restored the locomotor hyperactivity and motor learning deficits in knock-out (KO) animals, thus providing a direct link between GPR88 loss in the dorsal striatum and the locomotor phenotype of KO mice (Quintana et al., 2012;Meirsman et al., 2016b).
Within the striatum, GPR88 is expressed in the majority of medium spiny neurons (MSNs) of both the direct [coexpressing dopamine D1 receptors (D1Rs) and substance P, D1R-MSNs] and indirect (co-expressing dopamine D2Rs, adenosine A 2A receptor (A 2A R) and Enkephalin, D2R-MSNs] pathways (Quintana et al., 2012). Converging evidence support the opposing influence of these two MSNs populations in motor output systems and motivated behavior. For example, optogenetic depolarization of D2R-MSNs decreased locomotor initiation (Kravitz et al., 2010), while ablation or disruption of these neurons increased motor activity (Durieux et al., 2009(Durieux et al., , 2012Bateup et al., 2010). In contrast, optical stimulation of D1R-MSNs increased locomotion whereas disruption or ablation of these neurons had the opposite effect (Kravitz et al., 2010;Durieux et al., 2012). Also, cell-specific neuron ablation using an inducible diphtheria toxin receptor (DTR)-mediated cell targeting strategy further suggests a differential role of D2R-and D1R-MSNs in acquisition and expression of motor skill learning (Durieux et al., 2012). Ablation of D2R-MSNs neurons delayed the acquisition of a rotarod task but had no effect in a previously acquired motor skill. Contrarily, ablation of D1R-MSNs neurons impaired motor skill learning regardless of the training extension and also disrupted performance of a previously learned motor sequence (Durieux et al., 2012). Further, in recent years, research on MSNs subtypes function has revealed that these two neuronal populations differentially regulate not only motor behaviors but also responses to rewarding and aversive stimuli: while optogenetic activation of the D1R-MSNs was shown to increase reinforcement, activation of D2R-MSNs induced transient punishment and depressive-like behavior (Kravitz et al., 2012;Hikida et al., 2013;Francis et al., 2015).
Despite the established overall function of striatal GPR88 in brain functions and deficits (in humans and mice), no study to date has directly compared the specific role of GPR88 in D1R-and D2R-MSNs. A conditional KO mouse line for GPR88 in D2R-MSNs was developed in a previous study, using a A 2A R-Cre driver line (A 2A R-Gpr88 mice), and mutant mice showed hyperactive behavior, decreased anxiety-like behaviors and increased locomotor response to dopaminergic agonists (Meirsman et al., 2016a. In this study, we have generated conditional Gpr88 KO for D1R-MSNs (D1R-Gpr88 mice), and compared behavioral responses of D1R-Gpr88 with those of A 2A R-Gpr88 mice and total KO (CMV-Gpr88) mice. Results show that GPR88 in D1R neurons regulates locomotor habituation to novel environments and motor skill learning. In contrast, GPR88 in D2R, but not in D1R neurons, control defensive burying and social approach, and also regulate levels of locomotion, stereotypies and initial motor coordination.

Subjects
Mice (male and female) aged 9 -15 weeks where bred in house and grouped-house three to five animals per cage. Animals where maintained on a 12/12 h light/dark cycle at controlled temperature (22 Ϯ 1°C). Food and water were available ad libitum throughout all experiments.
For all experiments, and considering the different genetic background, A 2A R-Gpr88 and D1R-Gpr88 mice were compared to their Gpr88-floxed littermates (A 2A R-CTL and D1R-CTL, respectively) and CMV-Gpr88 mice were compared to their wild-type controls (CMV-CTL). Baseline responses may therefore slightly differ when comparing the three mouse colonies.

Tissue preparation and fluorescent in situ hybridization
RNAscope was used as previously described (Meirsman et al., 2016a). Mice (n ϭ 3 D1R-CTL; n ϭ 3 D1R-Gpr88) were killed by cervical dislocation and fresh brains were extracted and embedded in optimal cutting temperature (OCT) medium (Thermo Scientific) frozen and kept at -80°C. Frozen brains were coronally sliced into 20-m serial sections by using cryostat (CM3050 Leica), placed in superfrost slides (Thermo Scientific) and kept at -80°C until processing. In situ hybridizations were performed using the RNAscope Multiplex Fluorescent Assay. GPR88 and D1R probes were alternatively coupled to FITC or TRITC while D2R probes were coupled with Cy5.

Relative expression of D1R and D2R mRNA in GPR88-positive cells
Image acquisition was performed using the slide scanner Olympus VS120 (Olympus Corporation). Regions of interest (ROIs) were selected using Olyvia software (Olympus) and saved as PNG files. Three brain regions where analyzed: rostral CPu (from 1.42 to 0.98 mm from bregma), caudal CPu (from 0.98 to -0.58 mm from bregma), and nucleus accumbens (Nacc; from 1.42 to 1.10 mm from bregma).
For the CPu (rostral and caudal), at least four ROIs were selected: two for the dorso-lateral striatum (DLS) and two for dorso-median striatum (DMS). Counting was balanced between right and left hemispheres. To evaluate expression of D1R and D2R mRNA in GPR88-expressing cells, counting was performed manually using the FIJI (ImageJ) cell counter. First, cells expressing GPR88 mRNA were marked and counted (Ϯ55 cells/ROI in D1R-CTL mice; Ϯ21 cells/ROI in D1R-Gpr88 mice). For each GPR88positive cell, co-expression of D1R or D2R was verified and counted separately. Relative co-expression (GPR88/ D1R or GPR88/D2R) is represented as a percentage of total GPR88-positive cells counted [(number of GPR88expressing cells co-expressing D1R or D2R ϫ 100)/total number of GPR88-expressing cells]. Statistical analysis where realized with percentages of each ROI calculated using excel. Given the lack of difference in GPR88 expression between lateral and medial CPu, relative percentage of each was pooled for graphical representation and statistical analysis.

[S35]-GTP␥S binding assay
[S35]-GTP␥S assays were performed on membrane preparations as described in previous report (Pradhan et al., 2009). To perform [S35]-GTP␥S assays on whole striatum mice were killed by cervical dislocation and both striatum were rapidly manually removed, frozen in dry ice, and stored at -80°C until use. Two (CMV-Gpr88 and CMV-CTL) and three (D1R-Gpr88 and D1R-CTL) membrane preparations were used. Each membrane preparation was generated using striatum from three animals (males and females). Results are expressed by meaning measures from the three-membrane preparation. All assays were performed on membrane preparations. Membranes were prepared by homogenizing the tissue in icecold 0.25 M sucrose solution 10 vol (ml/g wet weight of tissue). Samples were then centrifuged at 2500 ϫ g for 10 min. Supernatants were collected and diluted 10 times in buffer containing 50 mM TrisHCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA, following which they were centrifuged at 23,000 ϫ g for 30 min. The pellets were homogenized in 800-l ice-cold sucrose solution (0.32 M) and kept at -80°C. For each [35S]GTP␥S binding assay 2 g of protein per well was used. Samples were incubated with and without ligands, for 1 h at 25°C in assay buffer containing 30 mM GDP and 0.1 nM [35S]GTP␥S. Bound radioactivity was quantified using a liquid scintillation counter. B max and K d values were calculated. Non-specific binding was defined as binding in the presence of 10 M GTP␥S and binding in the absence of agonist was defined as the basal binding.

Behavioral experiments
For all behavioral measures, mice from different mouse lines and genotypes were tested in a random order and data were analyzed blind to genotype. However, to avoid order-related variability between mouse lines and genotypes, mouse lines were stratified so that each session included both genotypes from all lines. One mouse cohort (N ϭ 8 CMV-CTL, N ϭ 10 CMV-Gpr88; N ϭ 10 A 2A R-Gpr88, N ϭ 10 A 2A R-CTL; N ϭ 10 D1R-Gpr88, N ϭ 14 D1R-CTL) was used to measure marble burying and social interaction. Independent cohorts of mice (N ϭ 21 CMV-CTL, N ϭ 21 CMV-Gpr88; N ϭ 10 A 2A R-Gpr88, N ϭ 17 A 2A R-CTL; N ϭ 13 D1R-Gpr88, N ϭ 12 D1R-CTL) underwent 5 d of open field locomotion followed by a 48-h resting period before 7 d of rotarod motor skill learning.

Marble burying
Defensive burying was measured as previously described (Meirsman et al., 2016b) using the marble burying test conducted with 20 small glass marbles (15 mm) evenly spaced in a transparent single cage (21 ϫ 11 ϫ 17 cm) over 4-cm sawdust bedding. The cage was covered by a plastic lid in a room illuminated at 40 Lux. Mice were left in the cage for 10 min, and the number of marbles buried more than half in sawdust was counted.

Social interaction test
Social interaction was assessed, as previously described (Meirsman et al., 2016a), in an open field (50 ϫ 50 cm) dimly lit (Ͻ10 Lux) using naive wild-type mice of the same age and weight as interactors. On the first day, all mice were individually placed in the open field arena and left for a 10-min period of habituation. The next day, mice were placed in the open field arena with a wild-type naive interactor and a 10-min session was recorder. Nose contacts were measured manually using video recordings. If an interactor failed to engage in any interaction data from the respective mice were exclude from analysis.

Open field locomotion
To assess basal locomotion and habituation to novel environment mice were placed in a dimly lit open field (Accuscan Instruments) for 30-min daily session. The experiment lasted 5 d, and mice were placed in the same open field for all sessions tested. Open field was cleaned with water and 70% ethanol between trials. Total distance traveled, stereotypy counts, and durations were automatically recorded.

Rotarod
The first day, mice were placed on the rod (30-mm plastic roller, Panlab Harvard) at constant speed of 4 rpm. until achieving 90 s without falling from the rod (habituation; data not shown). On the six next consecutive days, mice were placed on the rod accelerating from 4 to 40 rpm in 5 min and the remaining at maximum speed for the next 5 min for four trials every day. Light intensity in the room was inferior to 10 Lux. Mice rested a minimum of 1 min between trials to avoid fatigue and exhaustion. When mice hang on the rod instead of running, they were left for one complete turn but the timer was stopped if the mice engaged in a second consecutive turn. Animals were scored for their latency to fall (in seconds) in each trial. Mean values of four daily trials were used for statistical analysis.

Statistics
For in situ hybridization cell counting and GPR88 agonist-induced binding assay data were analyzed using two-way ANOVA followed by Sidak's and Tukey's multiple comparisons, respectively. Repeated measures (RM) two-way ANOVA was used to analyze global open field and rotarod results with genotypes as the between-subject factor and time as the RM. One-way ANOVA was used for open field habituation analysis (days 1 and 5), first and last rotarod session analysis. Method of contrasts was used to compare day 1 and day 6 performance on the rotarod. Stereotypies, marble burying and social interaction contacts were analyzed using t test (unpaired with Welch's correction). All statistical analyses were realized using GraphPad Prism 7 (GraphPad Software, Inc) and the accepted level of significance was p Ͻ 0.05. All the statistical methods are summarized in Table 1.

D1R-Gpr88 mice show Gpr88 mRNA deletion in D1Rexpressing neurons
To conditionally delete Gpr88 exon 2 in cells expressing D1R, mice carrying two LoxP sites flanking the second exon of the Gpr88 gene (Meirsman et al., 2016b) were crossed with mice expressing the Cre recombinase under the control of the Drd1a gene promoter (Gensat). We first tested whether GPR88 transcript and protein are reduced in the striatum. We quantified Gpr88 mRNA levels by RT-qPCR for CPu and Nacc from D1R-Gpr88 and their control littermates. As shown in Figure 1A, Gpr88 expression was significantly decreased in striatal regions of conditional KO compared to controls (CPu: t (16) ϭ 3.01, p ϭ 0.008; Nacc: t (13) ϭ 4.19, p ϭ 0.001; Table 1). Testing Gpr88 mRNA levels in the hippocampus and amygdala showed a milder but significant reduction in hippocampus In situ hybridization/ cell counting Open field (all sessions) Open field (sessions 1 and 5)  We then tested whether the genetic deletion was specific to D1R MSNs, using in situ hybridization. As depicted in Figure 2A, we first demonstrate that, in control mice cells expressing Gpr88 mRNA colocalize with both Drd1a (left panel), and Drd2 mRNA-expressing cells (right panel). In D1R-Gpr88 mice, however, cells expressing Gpr88 do not colocalize with Drd1a-expressing cells (left panel), but still colocalize with Drd2-expressing cells (right panel). Quantitative analysis (Fig. 2B) in the CPu and Nacc confirmed that, in control animals, Gpr88 mRNA is found in both Drd1a-positive (CPu: 43.96 Ϯ 1.54% and Nacc: 45.13 Ϯ 3.57%) and Drd2-positive (CPu: 62.11 Ϯ 1.95% and Nacc: 59.03 Ϯ 4.47%) cells whereas in D1R-Gpr88 mice the great majority of cells expressing
These results first confirm that deletion of Gpr88 increases general locomotion and simultaneously abolishes locomotor habituation to a novel environment. Further, our results suggest that deletion of D1R-Gpr88 does not impact general locomotion but abolishes locomotor habituation to a novel environment. In contrast, deletion of Gpr88 in D2R-MSNs increases locomotor activity without altering habituation to a novel environment.
These results show that GPR88 in D2R-expressing but not D1R-expressing neurons regulates motor stereotypies.
These data first confirm that lack of GPR88 abolishes both motor coordination and motor skill learning and further shows that the deletion of A 2A R-Gpr88 alters initial motor coordination while preserving motor skill learning, whereas D1R-Gpr88 deletion selectively impairs motor skill learning.

Discussion
Results from the comparison of total versus conditional mouse lines are summarized in Table 2. In sum, data from marble burying and social interaction tests reveal a D2R cell-specific function of GPR88 in anxiety-related and social behavior (De Boer and Koolhaas, 2003), as modifications are detected in CMV-Gpr88 and A 2A R-Gpr88 KO, but not D1R-Gpr88 KO, mice. With regards to open field results, we observe differential roles of GPR88 in D1Rand D2R-MSNs, suggesting that GPR88 in D1R-MSNs has no role on general locomotion or stereotypies but regulates locomotor habituation to a novel environment, whereas deletion of this receptor in D2R-MSNs increases spontaneous locomotion and stereotypies while preserving locomotor habituation. In the rotarod also, we show differential roles of GPR88 in D1R-and D2R-MSNs, indicating that GPR88 in D1R-MSNs contributes to motor skill learning, whereas the receptor in D2R-MSNs contributes to motor coordination but not learning in the task. Overall therefore, our study demonstrates that GPR88 modulates the function of both D1R-and D2R-MSNs and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior.
We find that specific deletion of Gpr88 in D2R-MSNs, but not in D1R-MSNs, decreases anxiety-like behavior as shown by reduced defensive burying activity (Borsini et al., 2002;De Boer and Koolhaas, 2003;Meirsman et al., 2016a). This result is in line with data showing that blocking D2R-MSNs activity disrupts avoidance behavior and aversive learning (Hikida et al., 2013). We also extend previous data (Meirsman et al., 2016a) by showing that Gpr88 deletion in D2R but not D1R-neurons increases social approach. Reports have shown that dopamine signaling through D1Rs is necessary for mediating pro-social behavior (Gunaydin et al., 2014). Also, it was shown that while D1R-MSNs display reduced mEPSC frequency after chronic social defeat, optical stimulation of D1R-MSNs was sufficient to reverse social avoidance induced by social defeat stress (Francis et al., 2015;Francis and Lobo, 2017). Therefore, although baseline social approach is not affected by D1R-Gpr88 deletion, different results may be obtained after chronic social stress. Knowing that inducible ablation of D1R-MSNs also reduces anxiety behaviors in mice (Révy et al., 2014), our result suggest that D1R-MSNs and D1R's-dependent anxiety and social behaviors was not affected by D1R-Gpr88 ablation. However, to fully understand GPR88 function in affective and social behaviors future studies should compare responses of D1R-Gpr88 and A 2A R -Gpr88 mice in reward and aversive learning paradigms and investigate the role of this orphan receptor in stress-induced social avoidance.
We then show a differential effect of Gpr88 gene KO in D1R-or D2R-MSNs on general locomotion, with hyperlocomotor activity observed after D2R-Gpr88 deletion only. Converging data show that disruption of D2R-MSNs activity results in hyperlocomotor behavior (Durieux et al., 2012;Révy et al., 2014) while ablation of D1R-MSNs decreases locomotion (Durieux et al., 2012;Révy et al., 2014). Therefore, the increased locomotion observed in CMV-Gpr88 and A 2A R-Gpr88 mice could simply result from decreased D2R-MSNs driven inhibition of locomotor output. Although deletion of Gpr88 in D1R-neurons did not alter overall locomotion throughout the five sessions, D1R-Gpr88 mice displayed decreased acute locomotor activity during the first open field session which would suggests impaired D1R-MSNs activity. Overall, locomotion results suggest that lack A 2A R-Gpr88 mimics D2R-MSNs ablation (Durieux et al., 2009(Durieux et al., , 2012Bateup et al., 2010;Révy et al., 2014). The question of how Gpr88 cell-specific deletion affects MSNs firing activity and Another interesting locomotor phenotype in the open field is the lack of intersession habituation to the environment selectively observed in D1R-Gpr88 mice. Open field habituation is described as an adaptive process in which rodents decrease their locomotion with increasing exposure to the same environment and is taken as an index of memory (Tomaz et al., 1990;Cerbone and Sadile, 1994). A previous study showed that total deletion of Gpr88 improved spatial learning and memory tasks performances, thus suggesting that the non-habituating phenotype is not linked to spatial memory functions (Meirsman et al., 2016b). Surprisingly, our results contrast with the lack of open field habituation previously observed after ablation of D2R-MSNs (but not D1R-MSNs; Durieux et al., 2012). Therefore, in opposite to locomotion results, deletion of GPR88 in D1R-MSNs matches results obtained after D2R-MSNs ablation, suggesting either MSNs cross talk or alteration of a common network shaping locomotor habituation. In fact, data show (Sanguedo et al., 2016) that locomotor habituation to novel environments is accompanied by activation of striatal and extra-striatal regions such as amygdala and frontal cortex. Accordingly, CMV-Gpr88 mice have been shown to have altered transcriptional profiles in these structures where both GPR88 and D1R are expressed (Meirsman et al., 2016b). Most importantly, recent studies using CMV-Gpr88 mice have shown impaired multisensory processing (Ehrlich et al., 2018) and sensorimotor gating  that, coupled with altered sensorimotor and corticostriatal functional connectivity (Arefin et al., 2017), suggest a role of this receptor in the integration and processing of sensory information. Interestingly, it has also been suggested that modifications of the striatocortical circuitry may underlie the hyperactivity observed in CMV-Gpr88 mice (Arefin et al., 2017). As such, future studies measuring functional connectivity in D2R-Gpr88 and D1R-Gpr88 mice will elucidate how cell-specific deletion of Gpr88 reshape brain connectome leading to persistent changes in behavior.
Finally, the open field observations also reveal that A 2A R-Gpr88 but not D1R-Gpr88 mice present increased number of stereotypies in the open field. Animal and clinical data indicate that dysregulation of cortico-striatothalamo-cortical circuitry are associated with stereotypies (Lewis and Kim, 2009). Further, one study linked decreased D2R-MSNs activity with enhances stereotypies (Tanimura et al., 2010(Tanimura et al., , 2011) and a recent report indicates that increasing D2R-MSNs activity is sufficient to rescue repetitive behaviors observed in a genetic model of autism (Wang et al., 2017). The increased stereotypies of A 2A R-Gpr88 mice may therefore result from diminished D2R-MSNs inhibitory projection. As for locomotion result, the electrophysiological impact of Gpr88 specific deletion should be assessed in future studies. On the other hand, stereotypies have been linked to dopaminergic overstimulation (Katherine, 2018), which could also cause the phenotype observed in A 2A R-Gpr88 mice. In fact, we have previously reported altered DA levels in the CPu and midbrain nuclei of CMV-Gpr88 mice, and future studies should verify DA levels in conditional Gpr88 KO mice.
As for the open field experiments, rotarod testing also reveals differential D1R-versus D2R-MSNs activities of GPR88. Mutants lacking Gpr88 in D1R-neurons present similar initial rotarod performance than control animals but show absence of motor skill learning throughout 6 d of task. On the contrary, mice lacking Gpr88 in D2R-neurons show decreased latency to fall in the first day but learned the task and increased their motor performances across days. Interestingly, as for the locomotor phenotype, results are comparable to those obtained after inducible ablation of D1R-MSNs and D2R-MSNs (Durieux et al., 2012). Worth noting, previous reports indicate that Gpr88 deletion does not alter striatal cell population or cytoarchitectural organization (Logue et al., 2009;Quintana et al., 2012) but increased levels of striatal pDARPP-32 Thr-34 and the ratio of pDARPP-32 Thr-34/DARPP-32 suggesting compromised MSNs functioning (Logue et al., 2009). Also, mRNA levels of genes encoding neurotransmitter receptors as well as GPCRs activation were found altered in the striatum of CMV-Gpr88 mice (Quintana et al., 2012;Meirsman et al., 2016b). In particular, Gpr88 deletion increased mu opioid and delta opioid receptors activation in the striatum. These receptors are known to activate Gi/o pathways, and could therefore contribute to increase MSNs hyperpolarization in Gpr88 mutant mice (Le Merrer et al., 2013;Pellissier et al., 2018). Interestingly, a previous report showed that chronic administration of DOR antagonist (naltrindole) in CMV-Gpr88 mice reversed their initial motor coordination impairment suggesting that increased DOR activity may underlie the deficit observed in A 2A R-Gpr88. Future studies pharmacologically tackling receptors known to interact with GPR88 will elucidate MSNsspecific GPR88 interactions with other receptors.
In conclusion, the present study demonstrates dissociable roles of GPR88 at the level of MSNs. While GPR88 in D2R-MSNs regulates levels of anxiety, social behavior, stereotypies, locomotion, and motor coordination, this receptor in D1R-MSNs does not seem to impact affective behaviors but regulates habituation to novelty and motor skill learning. It is important to note that in the present study deletion of Gpr88 is not exclusively striatal. Thus, a new approach to restrict D1R-Gpr88 deletion to the striatum will determine if extra-striatal structures are involved in the phenotypes observed in mutants lacking Gpr88 in D1R-neurons. In addition, cellular mechanisms underlying phenotypes observed in this study remain to be clarified. Interesting to note, behavioral analyses show that both the total ablation of D2R MSNs (Durieux et al., 2012) and the deletion of Gpr88 in D2R-neurons (our study) reduce motor coordination and induces hyperlocomotion, suggesting that GPR88 activity normally stimulates D2R-MSNs. This is counterintuitive, as GPR88 has been proposed to be an inhibitory GPCR (Jin et al., 2018). Also, Quintana et al. (2012) have previously shown that total GPR88 ablation reduced tonic GABA current and enhanced glutamatergic signaling in MSNs. They also showed that deletion of Gpr88 similarly affect the re-sponse to cortical excitatory input or the tonic GABA currents in D1R or D2R MSNs. We may, however, consider a strong differential effect of selective versus total deletion of GPR88 on MSNs intrinsic electrical properties. Therefore, electrophysiological studies using cell-specific Gpr88 deletion and also the precise anatomic localization of the receptor at presynaptic or postsynaptic levels should help clarifying how GPR88 modulates D1R-and D2R-MSNs activities. In addition, deficient long distance communication between brain structures observed in CMV-Gpr88 mice (Arefin et al., 2017) may explain some of the present results and upcoming studies should compare respective functional connectivity alterations in the two conditional Gpr88 KO mouse lines. Hence, further dissection of D1R versus D2R specific GPR88 activities is essential to explore the full potential of this receptor as a target for affective and motor disorders.