The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A₁receptors (A₁Rs) and A_2Areceptors (A_2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A₁R–A_2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A₁R and A_2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A₁R–A_2AR heteromer is the ability of A_2AR activation to reduce the affinity of the A₁R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A₁R–A_2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A₁R–A_2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R) as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR), which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection.

Despite the profound effect of cannabinoids on motor function, and their therapeutic potential in Parkinson's and Huntington's diseases, the cellular and subcellular distributions of striatal CB₁receptors are not well defined. Here, we show that CB₁receptors are primarily located on GABAergic (vesicular GABA transporter-positive) and glutamatergic [vesicular glutamate transporter-1 (VGLUT-1)- and VGLUT-2-positive] striatal nerve terminals and are present in the presynaptic active zone, in the postsynaptic density, as well as in the extrasynaptic membrane. Both the nonselective agonistWIN55212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt] (EC₅₀, 32 nm) and the CB₁-selective agonist ACEA [N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] inhibited [³H]GABA release from rat striatal slices. The effect of these agonists was prevented by the CB₁-selective antagonists SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (1 μm) and AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt] (1 μm), indicating that cannabinoids inhibit the release of GABA via activation of presynaptic CB₁receptors. Cannabinoids modulated glutamate release via both CB₁and non-CB₁mechanisms. Cannabinoid agonists and antagonists inhibited 25 mmK⁺-evoked [³H]glutamate release and sodium-dependent [³H]glutamate uptake. Partial involvement of CB₁receptors is suggested because low concentrations of SR141716A partly and AM251 fully prevented the effect ofWIN55212-2 and CP55940 [5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol]. However, the effect of CB₁agonists and antagonists persisted in CB₁knock-out mice, indicating the involvement of non-CB₁,CB₁-like receptors. In contrast, cannabinoids did not modulate [³H]dopamine release or [³H]dopamine and [³H]GABA uptake. Our results indicate distinct modulation of striatal GABAergic and glutamatergic transmission by cannabinoids and will facilitate the understanding of the role and importance of the cannabinoid system in normal and pathological motor function.

ATP is released in a vesicular manner from nerve terminals mainly at higher stimulation frequencies. There is a robust expression of ATP (P2) receptors in the brain, but their role is primarily unknown. We report that ATP analogs biphasically modulate the evoked release of glutamate from purified nerve terminals of the rat hippocampus, the facilitation being mediated by P2X₁, P2X_2/3, and P2X₃[antagonized by 8-(benzamido)naphthalene-1,3,5-trisulfonate and 2′,3′-O-(2,4,6-trinitrophenyl)-ATP] and the inhibition by P2Y_1,P2Y₂, and/or P2Y₄[antagonized by reactive blue 2 and 2′deoxy-N⁶-methyladenosine-3′,5′-bisphosphate and mimicked by P1-(urinine 5′-),P4-(inosine 5′-) tetraphosphate and 2-methylthio-ADP] receptors. The combination of single-cell PCR analysis of rat hippocampal pyramidal neurons, Western blot analysis of purified presynaptic active zone fraction, and immunocytochemical analysis of hippocampal glutamatergic terminals revealed that the P2 receptors expressed in glutamatergic neurons, located in the active zone and in glutamatergic terminals, were precisely P2X_1,P2X₂, and P2X₃subunits and P2Y_1,P2Y₂, and P2Y₄receptors. This provides coincident functional and molecular evidence that P2 receptors are present and act presynaptically as a modulatory system controlling hippocampal glutamate release.

The anti‐Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A_2A receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinson's disease. mGluR5 and A_2A antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the anti‐Parkinsonian and neuroprotective effects of A_2A and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A_2A agonist, CGS21680 (1–30 nm) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A_2A antagonist, SCH58261 (50 nm). The mGluR5 agonist, CHPG (300–600 μm) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 μm). Both mGluR5 and A_2A receptors were located in the active zone and 57 ± 6% of striatal glutamatergic nerve terminals possessed both A_2A and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nm) and CHPG (100 μm) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nm CGS21680 was prevented by 10 μm MPEP, whereas facilitation by 300 μm CHPG was prevented by 10 nm SCH58261. These results provide the first direct evidence that A_2A and mGluR5 receptors are co‐located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinson's disease.