Introduction

The first pieces of evidence for antagonistic glutamate receptor with dopamine D2 receptor (D2R) interactions were found in 1983–1984 through the ability of glutamate to reduce the affinity of the high-affinity D2R agonist binding sites in striatal membrane preparations. Subsequently, it was observed that mGluR5 agonists alone or combined with an A2AR agonist (CGS-21680) can reduce the affinity of the high-affinity state of D2R for agonist binding sites in the rat striatum [1]. Co-immunoprecipitation experiments also indicated the existence of A2AR-mGluR5 heteroreceptor complexes in HEK293 cells and rat striatal membrane preparations [2]. The colocation of the receptors in striatal neurons was demonstrated [3, 4] as well as their synergistic interactions as studied with in vivo microdialysis and intracellular signalling in striatal preparations [2, 5, 6].

In 1974, the discovery that the methylxanthines caffeine and theophylline could enhance the contralateral turning behaviour induced by levodopa and dopamine receptor agonists in the hemi-Parkinsonian rat model was one early finding leading to the hypothesis that antagonistic adenosine-dopamine interactions existed [7, 8]. Today, a considerable amount of molecular and functional experimental data supports the view that A2AR and D2R form heteroreceptor complexes with antagonistic receptor-receptor interactions on the plasma membrane [9,10,11,12,13,14,15,16].

The existence of A2AR-D2R-mGluR5 higher-order oligomers was postulated, and it was proposed that the receptor-receptor interactions within this high-order complex are important to modulate the dorsal and ventral striatal-pallidal GABA neurons [2, 3, 8]. Years later, it was proposed that combined treatment with A2AR and mGluR5 agonists targeting A2AR-D2R-mGluR5 heteroreceptor complexes in the ventral striatal-pallidal GABA pathway can represent a new strategy for the treatment of schizophrenia [17]. Also, the combine treatment with selective A2A and mGluR5 receptor antagonists represents an alternative therapeutic approach to Parkinson’s disease [18,19,26, 27]. The concentrations of CHPG (500 nM) and MPEP (300 nM) have been selected on the basis of previous studies suggesting that, in this concentration range, the compounds selectively act as agonist or antagonist of mGluR5, respectively [18, 24, 28, 29]. Finally, the dose of haloperidol (1 mg/kg) and raseglurant (1 mg/kg) used in mouse behavioural experiments was previously described [30, 31]. Also, isobutyl-1-methylxanthine (IBMX) and 4-(3-butoxy-4-methoxybenzyl) imidazolidone (Ro 20-1724) were purchased from Tocris Bioscience (Bristol, UK).

Cell Culture and Transfection

Human embryonic kidney 293T (HEK293T cells (American Type Culture Collection, Manassas, VA, USA) cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin/streptomycin and 10% (v/v) foetal bovine serum at 37 °C in an atmosphere of 5% CO2. Cells were plated in 6-well plates (1 × 106cells/well), 96-well plates (1 × 104cells/well) or in 75 cm2 flasks and cultured overnight prior to transfection or experimental procedures. Cells were transiently transfected using linear polyethyleneimines (Polysciences Inc., Warrington, PA, USA) according to the manufacturer’s instructions.

Animals

A2AR−/− and D2R−/− mice generated on a CD-1 genetic background [30, 32] and the corresponding wild-type littermates weighing 20–25 g were used. The animal protocol (no. 7085) was approved by the University of Barcelona Committee on Animal Use and Care. Animals were housed and tested in compliance with the guidelines provided by the Guide for the Care and Use of Laboratory Animals [33] and following the European Union directives (2010/63/EU), the ARRIVE guidelines [34]. Mice were housed in groups of five in standard cages with access to food and water ad libitum while maintained under a 12-h dark/light cycle (starting at 7:30 AM), 22 °C temperature and 66% humidity (standard conditions). All animal experimentation was carried out in a period comprehended between 9:00 AM and 6:00 PM by a researcher blind to drug treatments.

Locomotor Activity Tests

Mice spontaneous or drug-induced locomotor activity was assessed by the open field test. In brief, animals were administered intraperitoneal (i.p.) with raseglurant (1 mg/kg) or vehicle-saline with 5% DMSO and 5% Tween 20 30 min before the testing session. Non-habituated mice were placed in the centre of an activity field arena (30 × 30 cm, surrounded by four 50-cm-high black-painted walls) equipped with a camera above to record activity and connected to the light source. The total distance travelled was analysed using SPOT tracker function from ImageJ (NIH, Bethesda, MD, USA), as previously described [30].

Catalepsy Test

Mouse catalepsy was induced by the administration (i.p.) of haloperidol (1 mg/kg) [30]. After 1 h, haloperidol-induced catalepsy was measured as the duration in seconds of an abnormal upright posture in which the forepaws of the mouse were placed on a horizontal wooden bar (0.6 cm of diameter) that was located 4.5 cm above the floor. Subsequently, mice were administered (i.p.) with either vehicle (i.e. saline with 5% DMSO and 5% Tween) or raseglurant (1 mg/kg). After 20 min, a second haloperidol-induced catalepsy measurement was performed.

The rationale for the use of raseglurant (a mGluR5-negative allosteric modulator) instead of a full antagonist was based on the theoretical advantages that allosteric modulators offer compared with their competitive counterparts. mGluR5 allosteric modulators (negative allosteric modulators (NAM) and positive allosteric modulators (PAM)) have the potential for greater subtype selectivity when compared to orthosteric ligands. Also, mGluR5 NAM and PAM do not possess intrinsic activity and are assumed to be quiescent in the absence of an endogenous agonist and only modulate receptor function when the endogenous agonist is present. In this manner, NAM and PAM have the potential to retain spatial and temporal aspects of endogenous receptor signalling. This is of particular interest for CNS targets where optimal neurotransmission is likely to have an improved therapeutic outcome as opposed to sustained receptor blockade or activation.

Haloperidol-Induced Catalepsy

Mice (n = 10) were randomly assigned to treatment groups, and behavioural testing was performed blind to treatment. The dopamine D2 receptor (D2R) antagonist, haloperidol (1 mg/kg, s.c.), was administered to induce catalepsy. Thirty minutes after the haloperidol administration, mice experienced a full cataleptic response. At this time point, for each mouse, the state of catalepsy was tested by gently placing their front limbs over an 8-cm-high horizontal bar. The intensity of catalepsy was assessed by measuring the time the mice remain in this position being completely immobile for a maximum of 120 s. Only mice that remained cataleptic for the entire 120 s were used for subsequent drug testing. After 30 min of the baseline measurement vehicle (0.5% methylcellulose and 2% DMSO), PBF509 was administered orally via gavage (3, 10 or 30 mg/kg, p.o.), and the catalepsy was then determined at 15, 30 and 60 min PBF509 administration. For each time point, the number of responding mice and the total cataleptic time for each animal were determined.

Membrane Preparation

HEK293T cells or mouse striata were homogenized in ice-cold 10 mM Tris HCl, pH 7.4, 1 mM EDTA and 300 mM KCl buffer containing a protease inhibitor cocktail (Roche, Penzberg, Germany) using a Polytron for three periods of 10 s each. The homogenate was centrifuged for 10 min at 1000 × g. The resulting supernatant was centrifuged for 30 min at 12,000 × g. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl2, washed and resuspended in the same medium. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA).

Bioluminescence Resonance Energy Transfer Saturation Assay

BRET2 saturation curves have been particularly used with the aim to establish the oligomeric order of receptor complexes, as well as the proportion of receptors engaged in dimers or oligomers (BRETmax). In the current work, bioluminescence resonance energy transfer (BRET2) saturation assays were carried out using plasmids encoding for D2RRluc and mGluR5GFP2 according to previously published methods [9, 26, 35, 36]. The netBRET2 ratio was defined as the BRET ratio for co-expressed Rluc and GFP2 constructs normalized against the BRET ratio for the Rluc expression construct alone: netBRET2 ratio = [(GFP2 emission at 515 ± 30 nm)/(Rluc emission 410 ± 80 nm)]-cf. The correction factor, cf, corresponds to (emission at 515 ± 30 nm)/(emission at 410 ± 80 nm) found with the receptor-Rluc construct expressed alone in the same experiment. The maximal value of BRET (netBRET2max) corresponds to the situation when all available donor molecules are paired up with acceptor molecules [8]. Also, saturation assay was used to compare the relative affinity of receptors for each other and their probability to form a complex, the so-called BRET50, which represents the acceptor/donor ratio giving 50% of the maximal signal. The ratio is calculated from fluorescence and bioluminescence values expressed as arbitrary units. BRET50 values should not be regarded as a common or classical value to expressed affinities as Molar units. Pairs with low BRET50 value thought to form oligomers or an increased tendency to dimerize, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors. The specificity of D2RRluc-mGluR5GFP2 interactions was assessed by comparison with co-expression of A1RGFP2 and D2RRluc.

In Situ PLA in Cultured Cells

In situ proximity ligation assay (PLA) in cultured cells was performed using the Duolink in situ PLA detection kit (Sigma-Aldrich, St. Louis, MO, USA), following the protocol described previously [11, 37, 38] using mouse monoclonal anti-D2R (2 μg/ml, MABN53; Millipore, Billerica, MA, USA) and rabbit polyclonal anti-mGluR5 (2 μg/ml, AB5675; Millipore) primary antibodies. PLA control experiments employed only one primary antibody. The PLA signal was visualized and quantified by using a TCS-SL confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) and the Duolink ImageTool software. High magnifications of the microphotograph were taken and visualized using multiple z-scan projections.

The background signal was estimated from both PLA control experiments and from PLA experiments performed on non-transfected HEK293T cells (HEK293T cell line expresses endogenously small amount of D2R, A2AR and mGluR5). In general, the positive PLA values obtained in these experiments were residuals. The assay cut-off value was set to two standard deviations over the background signal. Therefore, samples with values below this cut-off were negative for the interaction of interest, while samples with values higher than the threshold were positive.

Immunohistofluorescence and In Situ PLA in Mouse Brain

Mice were anaesthetized and intracardially perfused with 50–200 ml of ice-cold 4% formaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS; 1.47 mM KH2PO4, 8.07 mM Na2HPO4, 137 mM NaCl, 0.27 mM KCl with pH 7.2). The brains were post-fixed overnight in the same 4% formaldehyde solution at 4 °C. The vibratome (Leica Lasertechnik GmbH, Heidelberg, Germany) was used to make coronal section (50 μm). Slices were collected and kept in Walter’s antifreezing solution (30% glycerol, 30% ethylene glycol in PBS with pH 7.2) at −20 °C until further processing [39].

For immunohistofluorescence (IHF), experiments coronal brain slices were washed three times with PBS for 10 min at 22 C, then permeabilized with 0.3% Triton X-100 in PBS (2 h at 22 °C) and rinsed (3×) with washing solution (PBS containing 0.05% Triton X-100, 10 min, at 22 °C). Blocking of the slices was performed with washing solution containing 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h at 22 °C. To avoid unspecific binding, the slices were incubated with secondary anti-mouse IgG (no. 715-005-150; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in washing solution (2 h at 22 °C). Then, the slices were incubated with mouse anti-mGluR5 monoclonal (20 μg/ml, MABN540; Millipore) and rabbit anti-D2R polyclonal (1 μg/ml, D2R-Rb-Af960; Frontier Institute Co. Ltd, Shinko-nishi, Ishikari, Hokkaido, Japan) in washing solution with 5% NDS overnight at 4 °C. Subsequently, the slices were washed twice with a washing solution containing 1% NDS (10 min at 22 °C). Next, the slices were incubated with anti-Cy2 donkey anti-rabbit (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and anti-Cy3 donkey anti-mouse (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in washing solution with 1% NDS for 2 h at 22 °C. Finally, slices were washed two times with washing solution containing 1% NDS (10 min at 22 °C), two times with PBS (10 min at 22 °C) and then mounted with Duolink® in situ mounting medium with DAPI (Sigma-Aldrich). The Leica TCS 4D confocal scanning laser microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) was used to capture the fluorescence striatal images.

For in situ PLA in mouse brain, the Duolink in situ PLA detection kit (Sigma-Aldrich) was used as previously described [37, 39, 40]. Thus, the experimental procedure until the secondary antibody incubation step was the same as the IHF (see above). Subsequently, the following steps were performed according to the manufacturer’s protocol. Images were acquired and analysed as previously described [39]. The background signal was estimated from PLA control experiments, and the assay cut-off value was performed as described above.

Radioligand Competition Binding Experiments

For the binding experiments, membrane preparations (60 μg protein/ml) were obtained from HEK293T cells expressing either D2R and mGluR5 or A2AR, D2R and mGluR5, and [3H]-raclopride (Novandi Chemistry AB, Södertälje, Sweden) competition assays with minor modifications were performed according to previously published methods [26, 27, 41]. [3H]-raclopride (75 Ci/mmol), a D2-like receptor antagonist competing [42] with quinpirole for binding to D2-like receptors in HEK293T membrane preparations, was used to determine the D2R high-affinity (Ki, High) and D2R low-affinity (Ki, Low) values. (+)-Butaclamol, a selective D2R antagonist (100 μM, Sigma-Aldrich), was used to determine the non-specific binding. The amount of bound [3H]-raclopride was determined by liquid scintillation spectrometry.

cAMP Functional Assay

Intracellular cAMP levels were determined using a cAMP-Glo™ assay detection kit (Promega, Madison, WI, USA). HEK293T cells expressing either D2R and mGluR5 or A2AR, D2R and mGluR5 were plated at a density of 10,000 cells/well in 96-well microtiter plates coated with poly-L-lysine (Sigma-Aldrich) and incubated overnight. Culture medium was then removed; cells were washed with 1 × PBS before the induction buffer (red phenol/serum-free DMEM containing 500 μM IBMX and 100 μM Ro 20-1724) was added. The cells were incubated for 1 h prior to drug incubation. To examine the Gi protein-mediated inhibition of adenylyl cyclase, the levels of cAMP were first raised with 5 µM forskolin for 10 min. Drug dilutions were prepared in the induction buffer, and the temperature- and carbon dioxide-equilibrated drug dilutions (37 °C cell culture incubator for 30 min) were added as indicated, and cells were then incubated at 37 °C for 30 min. The assay was performed accordingly to the manufacturer’s specifications (Promega, Sweden). Readings of luminescence intensity were performed using the POLARstar Optima plate reader (BMG Lab Technologies, Offenburg, Germany). cAMP levels in non-transfected, non-treated cells and non-transfected cells treated only with forskolin were defined as basal and control, respectively.

Gel Electrophoresis and Immunoblotting

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 7% polyacrylamide gels. Proteins were transferred to Hybond-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot Turbo™ transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline (PBS; 8.07 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) during 1 h at 20 °C before being immunoblotted with the indicated antibody in blocking solution overnight at 4 °C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in blocking solution at 20 °C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain).

Statistical Analysis

The number of independent experiments (n) in each group is indicated in figure legends. Data are represented as mean ± standard error of mean (SEM). Outliers were assessed by the ROUT method [43]; thus, subjects were excluded assuming a Q-value of 1% in GraphPad Prism 9 (San Diego, CA, USA). Data normality was assessed by the Shapiro-Wilk normality test (p < 0.05). When two groups were evaluated, unpaired Student’s t-test or Mann-Whitney U-test was used. Comparisons among more than two experimental groups were performed by one-, two- or three-way factor analysis of variance (ANOVA) followed by either Dunnett’s, Šídák’s or Tukey post hoc test using GraphPad Prism 9, as indicated in the figure legends. A p-value ≤ 0.05 was considered significant.

Results

BRET.2 Experiments : Transient Co-expression of A 2A R with D 2 R and mGluR 5 Had a Significant Impact on D 2 R-mGluR 5 Heteroreceptor Complex Formation

HEK293T cells were transiently transfected with constant amounts of D2RRluc and increasing amounts of plasmids encoding for mGluR5GFP2 with/without transient co-expression of A2AR. The transient co-expression of A2AR with D2RRluc and mGluR5GFP2 had a significant impact on D2RRluc-mGluR5GFP2 heteroreceptor complex formation (Fig. 1A). Transient co-expression of A2AR promoted a significant increase of netBRET2max ratio value (0.084 ± 0.003 AU) compared to that found in cells without transient co-expression of A2AR (0.043 ± 0.002 AU) (Fig. 1B). When the A2AR was coexpressed with D2RRluc and mGluR5GFP2, these receptors hence showed an increased ability to heteromerize.

Fig. 1
figure 1

Effect of A2AR expression in D2R-mGluR5 heteromer formation assessed by BRET2 assay in HEK293T cells. Cells were transiently transfected with plasmids encoding the D2R tagged with Rluc (i.e. D2RRluc) and mGluR5 with GFP2 (i.e. mGluR5GFP2) in the absence (blue squares) or presence (red circles) of A2AR expression. The A1RGFP2–D2RRluc pair was used as a control (grey triangles). BRET2 saturation curves (A) were constructed by co-transfecting a constant amount of the plasmid for D2RRluc and increasing amounts of the mGluR5GFP2 plasmid or A1RGFP2 plasmid. Curves are based on mean values of four independent experiments performed in quadruplicates. The netBRET2max (B) and netBRET250 (C) values from the BRET.2 saturation curves shown in A are represented. BRET ratio is calculated from fluorescence and bioluminescence values expressed as arbitrary units. Results are expressed as mean ± SEM (n = 4, each determination performed in quadruplicates). ****p < 0.0001 and **p < 0.01, Student’s t-test

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Also, saturation assay was used to compare the relative affinity of receptors for each other and their probability to form a complex, the so-called BRET50, which represents the acceptor/donor ratio giving 50% of the maximal signal. The netBRET250 ratio value for D2RRluc-mGluR5GFP2 heteromerization was significantly reduced by transient co-expression of A2AR from (1.58 ± 0.09 AU) to (0.94 ± 0.11 AU) (Fig. 1C) indicating increased affinity of the two receptor protomers for each other. Pairs with low BRET50 value thought to form oligomers or an increased tendency to dimerize, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors.

Proximity Ligation Assay Experiments : Transient Co-expression of A 2A R Promoted the Formation D 2 R-mGluR 5 Heteroreceptor Complexes in HEK Cells

The role of A2AR in the dynamics of the D2R-mGluR5 heteromers was also evaluated by in situ proximity ligation assays (PLA) in transiently co-transfected HEK293T cells. The PLA results were in line with the results from the BRET2 assays. The in situ PLA demonstrated the existence of D2R-mGluR5 heteroreceptor complexes in cells to a low degree without transient co-expression of A2AR (Fig. 2A). Furthermore, the transient co-expression of A2AR highly significantly promoted the formation D2R-mGluR5 heteroreceptor complexes as shown by the marked increase in the number of PLA-positive D2R-mGluR5 complexes, while this was significantly reduced in HEK293T cells without co-expressing A2AR (Fig. 2 B and D). Few and weak PLA clusters were detected in the PLA-negative controls (lack of D2R antibodies) representing background labelling (Fig. 2C).

Fig. 2
figure 2

In situ PLA assessment of D2R-mGluR5 heteromer formation in the absence (A) or presence (B) of A2AR (see “Methods”). The in situ PLA-positive D2R-mGluR5 heteroreceptor complexes were shown as red blobs (arrows) and nuclei in blue (DAPI staining). A negative in situ PLA control (C) was included by incubating the cells in the absence of the primary anti-D2R antibody. D Quantification of D2R-mGluR5 complexes. The number of PLA blobs (red clusters) per positive cell (n = 4 × 50 cells) was assessed as described in Methods. Results were expressed as mean ± SEM (n = 4 independent experiments). ****p < 0.0001 and **p < 0.01, Student’s t-test

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The specificity of the PLA-positive D2R-mGluR5 complexes, shown as red blobs in the mouse dorsal striatum (Fig. 3A), was demonstrated using D2R−/− mice (Fig. 3C). In the sections from the mouse striatum, the appearance of the red PLA-positive D2R-GluR5 complexes, shown as mean number of red blobs/Nucleus, was markedly and highly significantly reduced (Fig. 3D). Furthermore, the loss of the red D2R-mGluR5 blobs to the same high degree in the A2AR−/− mice (Fig. 3 B, D) likely reflects the requirement of D2R-mGluR5 heterocomplexes to be part of an A2AR-D2R-mGluR5 to be expressed in the mouse striatum, probably by dorsal striatal-pallidal GABAergic neurons. In this way, it forms D2R-mGluR5 complexes that are close enough to be visualized by PLA.

Fig. 3
figure 3

Assessment of D2R-mGluR5 heteromer formation in mouse dorsal striatum by in situ PLA. Photomicrographs showing PLA recognition of D2R-mGluR5 heteromers in the dorsal striatum of wild type (A), A2AR–/– (B) and D2R–/– (C) mice. The in situ PLA-positive D2R-mGluR5 heteroreceptor complexes are shown as red blobs (arrows) and nuclei in blue (DAPI staining). D Quantification of D2R-mGluR5 complexes showing a highly significant reduction of D2R-mGluR5-positive red blobs in the absence of A2AR–/– or D2R–/.–. The number of PLA blobs (red clusters) per nucleus was assessed as described in Methods. Results were expressed as mean ± SEM (n = 5 animals). ***p < 0.001, one-way ANOVA followed by Dunnett’s post hoc test when compared with wild-type animals

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[.3 H]-Raclopride/Quinpirole Competition Experiments: the A 2A R and mGluR 5 Protomers Interact and Modulate D 2 R Protomer Recognition

In HEK293T cells expressing D2R and mGluR5, the mGluR5 agonist CHPG (500 nM) reduced the affinity of the high-affinity state (Ki, High) of the D2R for the agonist quinpirole with no effects on its low-affinity state (Ki, Low). Co-treatment with A2AR agonist CGS-21680 (100 nM) did not significantly alter the D2R Ki, High and Ki, Low values obtained when the cells were treated only with CHPG (500 nM) (Fig. 4A and Table 1). In HEK293T cells expressing A2AR, D2R and mGluR5, mGluR5 agonist stimulation also reduced the affinity of the high-affinity state (Ki, High) of the D2R for the agonist quinpirole with no statistically significant effects on its low-affinity state (Ki, Low) (Fig. 4A and Table 2). However, the transient co-expression of A2AR by itself (without agonist stimulation) potentiates mGluR5 agonist effects on the high-affinity D2R agonist binding sites (Fig. 4B, Tables 1 and 2). Finally, the co-stimulation of A2AR and mGluR5 synergistically increased in the Ki, High values of the D2R protomer upon co-expression of the A2AR (Table 2). Nevertheless, in cells expressing A2AR, D2R and mGluR5, further analysis should be performed to test the effect of combine treatment of A2AR (ZM-241385) and mGluR5 (CHPG) to figure out if the expression of A2AR, without agonist stimulation and its corresponding constitutive activity, is responsible for increased in the Ki, High values of the D2R protomer upon co-expression of the A2AR.

Fig. 4
figure 4

Assessing A2AR-dependent allosteric modulation of D2R-GluR5 heteromer by [3H]-raclopride/quinpirole competition binding experiments. A Competition assays were performed in HEK293T cells transiently expressing D2R and mGluR5 (blue squares) or A2AR, D2R and mGluR5 (red circles) with/without agonist(s)/antagonist(s) for adenosine A2AR or mGluR5 either alone or in combination as indicated. ( +)-Butaclamol (100 μM) was used to determine the non-specific binding, and the specific binding at the lowest concentration of the [3H]-raclopride employed was defined as 100%. Results are expressed as percentage of specific binding (mean ± SEM; n = 4 independent experiments performed in triplicate). B Percentage of change comparing CHPG alone or CHPG plus CGS-21680-induced changes in the D2R high affinity values ((Ki, High (nM)) with/without transient co-expression of A2AR. Results are expressed as means ± S.E.M.; n = 4, each determination performed in triplicate. *p < 0.05, Mann–Whitney U-test

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Table 1 Values for quinpirole binding site affinities to the D2-likeR by [.3H]-raclopride/quinpirole competition assays in HEK293T cells transiently expressing D2R and mGluR5 incubated with agonist(s) or/and antagonist(s) as indicated
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Table 2 Values for quinpirole binding site affinities to the D2-likeR by [.3H]-raclopride/quinpirole competition assays in HEK293T cells transiently expressing A2AR, D2R and mGluR5 incubated with agonist(s) or/and antagonist(s) as indicated
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In both HEK293T cells expressing D2R and mGluR5 or A2AR, D2R and mGluR5, the incubation with A2AR antagonist ZM-241385 (1 μM) and mGluR5 antagonist MPEP (300 μM) alone or in combination resulted in an almost complete blockade of the mGluR5 increase of the D2R Ki, High values and A2AR agonist-induced increase of mGluR5 agonist effects on the high-affinity D2R agonist binding sites (Tables 1 and 2).

cAMP Functional Experiments : the A 2A R and mGluR 5 Protomers Interact and Modulate D 2 R Protomer Signalling

In cells expressing D2R and mGluR5 forming D2R-mGluR5 heterocomplexes (Fig. 2), the D2R agonist activation with quinpirole (100 nM) induced a Gi protein-mediated inhibition of adenylyl cyclase that first was raised with 5 µM forskolin (Fig. 5A). This effect was highly significantly blocked by the D2R antagonist raclopride (1 μM). In these cells, the mGluR5 agonist CHPG stimulation significantly counteracted the D2R agonist-induced reduction of cAMP accumulation (Fig. 5A). The significant effect of CHPG (500 nM) was significantly reduced by the mGluR5 antagonist MPEP (300 μM). The co-treatment with the A2AR agonist did not enhance the counteraction of the inhibitory D2R signalling by CHPG (Fig. 5A).

Fig. 5
figure 5

Functional evaluation of A2AR-mediated modulation of D2R-GluR5 heteromer. HEK293T cells transiently expressing D2R and mGluR5 (A) or A2AR, D2R and mGluR5 (B) were treated with forskolin before incubation with quinpirole (Quinp, X nM), raclopride (Raclop, X nM), CHPG (X nM), MPEP (X nM), CGS-21680 (CGS, X nM) and ZM-241385 (ZM, X nM). The cAMP levels were determined using a cAMP-Glo™ Assay detection kit (see Methods). Intracellular cAMP levels are given in luminescence intensity (AU, arbitrary units) after subtracting background basal luminescence (cAMP levels measured in non-transfected, non-treated cells). Results are expressed as means ± SEM; n = 4 independent experiments, each determination performed in quadruplicates. ***p < 0.001, **p < 0.01 and *p < 0.05, one-way ANOVA followed by Tukey’s post hoc test compared with cells treated only with forskolin (control). †††p < 0.001, ††p < 0.01 and †p < 0.05, one-way ANOVA followed by Tukey’s post hoc test when compared with cells treated only with quinpirole; §§§p < 0.001, §§p < 0.01 and §p < 0.05, when compared with cells treated only with quinpirole plus raclopride; δp < 0.05, when compared to cells treated with quinpirole and CHPG. C CHPG and CGS21680-induced cAMP levels. HEK293T cells transiently expressing D2R and mGluR5 (top inset box) or A2AR, D2R and mGluR5 (Bottom inset box) were treated with and without forskolin before incubation with ligands. Results are expressed as means ± S.E.M.; n = 4 independent experiments, each determination performed in quadruplicates. Statistical analysis performed using one-way ANOVA followed by Tukey’s post hoc test (top inset box). CHPG versus CHPG + forskolin (p < 0.001), CHPG versus CHPG + forskolin + MPEP (p < 0.05); CGS-21680 versus CGS-21680 + forskolin (p < 0.001), CGS-21680 versus CGS-21680 + forskolin + ZM-241385 (p < 0.001) (bottom inset box). CHPG versus CHPG + forskolin (p < 0.01), CHPG versus CHPG + forskolin + MPEP (ns); CGS-21680 versus CGS-21680 + forskolin (p < 0.001), CGS-21680 versus CGS-21680 + forskolin + ZM-241385 (p < 0.001). Concentrations for ligands used: quinpirole (100 nM), forskolin (5 µM), raclopride (1 μM), CHPG (500 nM), MPEP (300 μM), CGS-21680 (100 nM) and ZM-241385(1 μM)

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Likewise, quinpirole significantly reduced the cAMP level in cells expressing A2AR, D2R and mGluR5 (Fig. 5B). The mGluR5 agonist CHPG had an improved ability to counteract the adenylyl cyclase inhibition produced by the D2R agonist in these cells, yielding cAMP levels similar to those obtained after blocking D2R signalling with raclopride (Fig. 5B). Upon A2AR and mGluR5 agonist co-activation, a larger counteraction of the D2R agonist action was found compared to that obtained with such a co-treatment performed in cells expressing only D2R and mGluR5. These results suggest a synergistic and significant counteraction by A2AR and mGluR5 agonists of the D2R agonist-induced decrease of cAMP accumulation (Fig. 5B). Such effects of the combined agonist treatment were only weakly reduced by the A2AR antagonist (ZM-241385). In cells not expressing the A2AR, the A2AR antagonist failed to produce any changes in the cAMP accumulation under such co-agonist treatments.

It should be noted that CHPG agonist produces similar increases in cAMP levels as found after the A2AR agonist GGS in HEK293T cells co-expressing D2R, A2AR and mGluR5 (Fig. 5C). Therefore, we should consider also that mGluR5 might simply activate Gs, inducing cAMP accumulation, independently of D2-Gi-induced inhibition of adenylate cyclase (Fig. 5 A−C).

Experiments on Haloperidol-Induced Catalepsy

Catalepsy is a nervous condition characterized by loss of muscle control and fixity of posture. It is considered a symptom of certain nervous disorders such as Parkinson’s diseases and epilepsy [44]. It is also a characteristic symptom of cocaine withdrawal, as well as one of the features of catatonia. The catalepsy is mainly produced by haloperidol induced blockade of D2R complexes in the dorsal striatal-pallidal GABA neurons within the dorsal striatum [44,45,46]. These GABA neurons mediate motor inhibition, counteracted by D2R agonist-induced activation of the D2R homo- and heterocomplexes like the D2R-A2AR or the D2R-mGluR5 heterocomplexes [24, 47,48,49,50]. The D2R activation of the dorsal striatal-pallidal GABA neurons is also essential for maintenance of normal locomotor activity.

The catalepsy induced by the D2R antagonist haloperidol was evaluated in 10-min time intervals from 60 to 90 min after the injection of haloperidol (Fig. 6). In wild-type mice, the mGluR5-negative allosteric modulator raseglurant produced in this time period a significant reduction of the catalepsy time which was in the order of 25% (Fig. 6). In contrast, such a reduction of catalepsy was not observed by raseglurant treatment of A2AR−/− mice. Furthermore, in vehicle-treated A2AR−/− animals, the haloperidol-induced catalepsy was markedly reduced compared to that obtained in vehicle treated wild-type mice (Fig. 6).

Fig. 6
figure 6

A2AR expression is needed for mGluR5 modulation of D2R-dependent behaviour in mice. Raseglurant reverses haloperidol-induced catalepsy. Haloperidol-induced cataleptic behaviour was measured as the time spent with both front paws resting on the bar (see Methods). Wild-type (circles), A2AR.−/− (squares) animals were pretreated with haloperidol (i.p., 1 mg/kg, i.p.) at time 0 (green arrow). Subsequently, 1 h later, animals were administered (i.p.; black arrow) with either vehicle (20% DMSO in saline) or raseglurant (Ras, 1 mg/kg). The time spent in a cataleptic position was measured after 5, 15 and 30 min after raseglurant (or vehicle) administration. Results are expressed as the mean time spent cataleptic ± SEM over a period of 120-s measurement (n = 6 animals per group). The cataleptic behaviour was calculated and compared within groups by a multiple t-test statistical analysis. *p < 0.05 and **p < 0.01, two-way ANOVA followed by Šídák’s post hoc test. ††††p < 0.0001, three-way ANOVA (phenotype, F(1, 100) = 279.6)

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Discussion

The field of dopamine D2Rs changed markedly with the discovery of many types of D2R homo- and heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum [4, 16, 40]. The results indicate that the D2R is a hub receptor [51] which interacts not only with many other GPCRs including dopamine isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and dopamine transporters [24, 52, 53]. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of brain disorders through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the dopamine signal, especially to the ventral striato-pallidal GABA pathway [37, 52, 54]. Of high relevance was the discovery of A2AR-D2R and A2AR-mGluR5 heteroreceptor complexes in native tissue [4, 16, 40, 55, 56]. Furthermore, the existence of the D2R-mGluR5 heterodimers in the biomembranes of living cells was demonstrated by bimolecular fluorescence complementation experiments in cellular models [21]. Although when tested by FRET microscopy in tsA 201 cells, D2R did not associate with mGluR5 [57]. Nevertheless, by combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, the occurrence of an A2AR-D2R-mGluR5 heteroreceptor complexes was observed in living cells. Furthermore, by co-immunoprecipitation, experiments validated the existence of an association of mGluR5, D2R and A2AR in rat striatum homogenates [21].

Herein, we present new findings that further expand the understanding of A2AR-D2R-mGluR5 heteroreceptor complexes. Also, strong evidences which support that the expression of the A2AR is necessary to facilitate the association of D2R and mGluR5 in a complex.

Our new findings are that transient co-expression of A2AR in HEK293T cells together with D2RRluc and mGluR5GFP2 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heterodimer, a component of the A2AR-D2R-mGluR5 heterocomplex, based on the increase in the BRET2 max values. Such an increase could be related to the development of an increased affinity of the two D2R and mGluR5 protomers for each other due to allosteric changes related to the formation of the A2AR-D2R-mGluR5 complex. In line with this hypothesis, the BRET250 values were significantly reduced for the D2R-mGluR5 heteromeric component of this trimeric heteroreceptor complex.

These results are also supported by the demonstration with PLA that an increased density of PLA-positive D2R-mGluR5 clusters was observed when A2AR expression had been added to the cells compared to cells only expressing D2R and mGluR5. In agreement, in the mouse dorsal striatum, the D2R-mGluR5 complexes were significantly reduced in the A2AR−/− mice. Thus, it becomes clear that the expression of the A2AR in the mouse dorsal striatum is necessary to facilitate that the D2R and mGluR5 form a complex. It underlines that the multiple receptor protomers in the high-order heteroreceptor complexes are dependent on each other to improve or facilitate the formation of such complexes in the dorsal striatum.

The different results obtained on haloperidol-induced catalepsy in wild-type mice vs A2AR−/− mice are of substantial interest since they can indicate a functional role of the A2AR-D2R-mGluR5 heteroreceptor complexes in the dorsal striatum as previously discussed [8, 17]. There was a marked reduction in the haloperidol-induced catalepsy in the A2AR−/− mice compared to wild-type mice. Thus, in the absence of the A2AR, the D2R antagonist haloperidol appears to have a substantially reduced potency to block the D2R which can be caused by the loss of the antagonistic A2AR-D2R interaction [9, 58]. According to the current findings in cell lines, the D2R-mGluR5 heterocomplexes should be also formed to a much lower degree in the absence of A2AR in view of their dependency of A2AR according to the PLA experiments performed. The counteraction of the D2R-mediated inhibitory actions on cAMP signalling by CHPG, a mGluR5 agonist, was in our cell line also more effective in cells co-expressing beside D2R and mGluR5, also A2AR.

It seems likely that the formation of the A2AR-D2R-mGluR5 complex enhances the affinity of the D2R and mGluR5 protomers for each other in this complex. It is of high interest that the biochemical binding experiments reveal that the mGluR5 CHPG agonist-induced increase in D2R Ki, High values becomes significantly higher in the A2AR-D2R-mGluR5 complex compared to the D2R-mGluR5 complex despite the absence of A2AR agonist exposure. Thus, although agonist activation of the A2AR seems necessary to exert negative allosteric modulation of the D2R protomer agonist binding via heteroreceptor complexes, an increased constitutive activity of the A2AR protomer could explain the above results.

As expected, the combined incubation with CHPG and CGS-21680 led to an even stronger increase in the D2R Ki, High values of the A2AR-D2R-mGluR5 complex, demonstrating the impact of the A2AR protomer on the D2R-mGluR5 allosteric interactions, which can involve both constitutive and A2AR agonist-induced inhibition of D2R agonist binding. Our findings represent one of the first examples of integrative activity within a higher-order heteroreceptor complex and show how one receptor (A2AR) can substantially modulate the structure and recognition of a participating receptor heterodimer (D2R-mGluR5) in such a trimeric receptor complex.

The pharmacological analysis of the A2AR-D2R-mGluR5 complex and its impact on cAMP levels indicated that the A2AR can modulate the effects of the D2R-mGluR5 interactions on cAMP signalling. It was found that when the A2AR-D2R-mGluR5 complex was likely to be formed through the expression also of the A2AR, the mGluR5 agonist had an increased ability to counteract the D2 agonist-induced Gi/o-mediated inhibition of the cAMP levels in comparison with the counteraction observed in the absence of A2AR expression. The same was also true for the combined treatment with the mGluR5 agonist CHPG and the A2AR agonist CGS-21680 when the A2AR was coexpressed. A stronger counteraction of the D2R-induced inhibition of the cAMP levels was observed when A2AR expression was present.

Taken together, our work on cell lines gives strong indications that, in the A2AR-D2R-mGluR5 complex, the A2AR protomer enhances the formation of the D2R-mGluR5 component of the complex with enhanced inhibition of D2R agonist binding recognition and its Gi/o-mediated cAMP signalling. The inhibitory effects by A2AR and mGluR5 on D2R recognition and signalling reveal a significant molecular integration in A2AR-D2R-mGluR5 complexes, likely formed also in the dorsal striatum. The A2AR and mGluR5 antagonists targeting the A2AR-D2R-mGluR5 complexes in dorsal striatum may reduce the haloperidol-induced catalepsy by removal of the A2AR and mGluR5 protomer-mediated allosteric inhibition of the D2R protomer. Understanding of the trimeric complexes formed by these GPCRs could provide novel strategies for development of drugs against neuropsychiatric and neurodegenerative diseases by targeting their antagonistic receptor-receptor interactions.