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Parkinson's disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A1 and A2A receptors (A1R and A2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A2AR, thus suggesting an A2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A1R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A1R dependent but A2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects. [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A2A receptors (A2AR) and dopamine D2 receptors (D2R) predominantly form A2AR‐D2R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A2AR and D2R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain‐related differences, a new D2R‐deficient mouse with the same genetic background (CD‐1) than the A2AR knock‐out mouse was generated. Locomotor activity, pre‐pulse inhibition (PPI) and drug‐induced catalepsy were then evaluated in wild‐type, A2AR and D2R knock‐out mice, with and without the concomitant administration of either the D2R agonist sumanirole or the A2AR antagonist SCH442416. SCH442416‐mediated locomotor effects were demonstrated to be dependent on D2R signaling. Similarly, a significant dependence on A2AR signaling was observed for PPI and for haloperidol‐induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A2AR‐D2R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Introduction: Chronic pain is a debilitating disease that is usually comorbid to anxiety and depression. Current treatment approaches mainly rely on analgesics but often neglect emotional aspects. Nonpharmacological interventions, such as listening to music, have been incorporated into clinics to provide a more comprehensive management of chronic pain. However, the underlying mechanisms of music-mediated pain relief are not fully understood., Objectives: Our aim was to evaluate the effects and mechanisms of music exposure in an animal model of chronic pain., Methods: We injected mice with the complete Freund adjuvant (CFA) inflammatory agent into the hind paw and housed them for 14 days with background music, or ambient noise, during their active period (Mozart K.205, overnight). The effect of music exposure on nociception, anxiety-like behaviors, and depression-like behaviors was evaluated through different paradigms, including the hot plate, Von Frey, elevated plus maze, splash, and tail suspension tests. In addition, we conducted fiber photometry experiments to investigate whether music influences dopamine dynamics in the nucleus accumbens (NAcc), a crucial region involved in pain processing, anhedonia, and reward., Results: Our findings indicate that music exposure prevents the decrease in NAcc activity observed in CFA-injected mice, linking with a sex-dependent reduction in allodynia, anxiety-like behaviors, and depression-like behaviors. Accordingly, female mice were more sensitive to music exposure than male mice., Conclusion: Collectively, our findings provide compelling evidence for the integration of music as a nonpharmacological intervention in chronic pain conditions. Moreover, the observed effect on NAcc suggests its potential as a therapeutic target for addressing chronic pain and its associated symptoms., Competing Interests: The authors have no conflicts of interest to declare.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

Objectives: Cannabidiol (CBD) is a phytocannabinoid with great potential in clinical applications. The mechanism(s) of action of CBD require further investigation. Previous studies suggested that adenosine A 2A receptors (A 2A Rs) could play a role in CBD-induced effects. Here, we evaluated the ability of CBD to modify the function of A 2A R., Methods: We used HEK-293T cells transfected with the cDNA encoding the human A 2A R and Gαs protein, both modified to perform bioluminescence-based assays. We first assessed the effect of CBD on A 2A R ligand binding using an A 2A R NanoLuciferase sensor. Next, we evaluated whether CBD modified A 2A R coupling to mini-Gαs proteins using the NanoBiT™ assay. Finally, we further assessed CBD effects on A 2A R intrinsic activity by recording agonist-induced cAMP accumulation., Results: CBD did not bind orthosterically to A 2A R but reduced the coupling of A 2A R to Gαs protein and the subsequent generation of cAMP., Conclusion: CBD negatively modulates A 2A R functioning.

The orphan G protein-coupled receptor 37 (GPR37), widely associated with Parkinson's disease (PD), undergoes proteolytic processing under physiological conditions. The N-terminus domain is proteolyzed by a disintegrin and metalloproteinase 10 (ADAM-10), which generates various membrane receptor forms and ectodomain shedding (ecto-GPR37) in the extracellular environment. We investigated the processing and density of GPR37 in several neurodegenerative conditions, including Lewy body disease (LBD), multiple system atrophy (MSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease (AD). The presence of ecto-GPR37 peptides in the cerebrospinal fluid (CSF) of PD, MSA, CBD and PSP patients was assessed through an in-house nanoluciferase-based immunoassay. This study identified increased receptor processing in early-stage LBD within the PFC and striatum, key brain areas in neurodegeneration. In MSA only the 52 kDa form of GPR37 appeared in the striatum. This form was also significantly elevated in the striatum of AD necropsies. On the contrary, GPR37 processing remained unchanged in the brains of CBD and PSP patients. Furthermore, while CSF ecto-GPR37 increased in PD patients, its levels remained unchanged in MSA, CBD, and PSP subjects. Importantly, patients with PD with rapid progression of the disease did not have elevated ecto-GPR37 in the CSF, while those with slow progression showed a significant increase, suggesting a possible prognostic use of ecto-GPR37 in PD. This research underscores the distinctive processing and density patterns of GPR37 in neurodegenerative diseases, providing crucial insights into its potential role as an indicator of PD progression rates., (© 2024. The Author(s).)

Chronic pain and depression are highly prevalent pathologies and cause a major socioeconomic burden to society. Chronic pain affects the emotional state of the individuals suffering from it, while depression worsens the prognosis of chronic pain patients and may diminish the effectiveness of pain treatments. There is a high comorbidity rate between both pathologies, which might share overlapping mechanisms. This review explores the evidence pinpointing a role for the ventral tegmental area (VTA) as a hub where both pain and emotional processing might converge. In addition, the feasibility of using the VTA as a possible therapeutic target is discussed. The role of the VTA, and the dopaminergic system in general, is highly studied in mood disorders, especially in deficits in reward-processing and motivation. Conversely, the VTA is less regarded where it concerns the study of central mechanisms of pain and its mood-associated consequences. Here, we first outline the brain circuits involving central processing of pain and mood disorders, focusing on the often-understudied role of the dopaminergic system and the VTA. Next, we highlight the state-of-the-art findings supporting the emergence of the VTA as a link where both pathways converge. Thus, we envision a promising part for the VTA as a putative target for innovative therapeutic approaches to treat chronic pain and its effects on mood. Finally, we emphasize the urge to develop and use animal models where both pain and depression-like symptoms are considered in conjunction., Competing Interests: 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. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Flores-García, Rizzo, Garçon-Poca, Fernández-Dueñas and Bonaventura.)

Background: Chronic non-cancer pain (CNCP) treatment's primary goal is to maintain physical and mental functioning while improving quality of life. Opioid use in CNCP patients has increased in recent years, and non-pharmacological interventions such as music listening have been proposed to counter it. Unlike other auditive stimuli, music can activate emotional-regulating and reward-regulating circuits, making it a potential tool to modulate attentional processes and regulate mood. This study's primary objective is to provide the first evidence on the distinct (separate) effects of music listening as a coadjuvant maintenance analgesic treatment in CNCP patients undergoing opioid analgesia., Methods and Analysis: This will be a single-centre, phase II, open-label, parallel-group, proof-of-concept randomised clinical trial with CNCP patients under a minimum 4-week regular opioid treatment. We plan to include 70 consecutive patients, which will be randomised (1:1) to either the experimental group (active music listening) or the control group (active audiobooks listening). During 28 days, both groups will listen daily (for at least 30 min and up to 1 hour) to preset playlists tailored to individual preferences.Pain intensity scores at each visit, the changes (differences) from baseline and the proportions of responders according to various definitions based on pain intensity differences will be described and compared between study arms. We will apply longitudinal data assessment methods (mixed generalised linear models) taking the patient as a cluster to assess and compare the endpoints' evolution. We will also use the mediation analysis framework to adjust for the effects of additional therapeutic measures and obtain estimates of effect with a causal interpretation., Ethics and Dissemination: The study protocol has been reviewed, and ethics approval has been obtained from the Bellvitge University Hospital Institutional Review Board, L'Hospitalet de Llobregat, Barcelona, Spain. The results from this study will be actively disseminated through manuscript publications and conference presentations., Trial Registration Number: NCT05726266., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The σ 1 receptor (S1R) is a ligand-regulated non-opioid intracellular receptor involved in several pathological conditions. The development of S1R-based drugs as therapeutic agents is a challenge due to the lack of simple functional assays to identify and classify S1R ligands. We have developed a novel nanoluciferase binary technology (NanoBiT) assay based on the ability of S1R to heteromerize with the binding immunoglobulin protein (BiP) in living cells. The S1R-BiP heterodimerization biosensor allows for rapid and accurate identification of S1R ligands by monitoring the dynamics of association-dissociation of S1R and BiP. Acute treatment of cells with the S1R agonist PRE-084 produced rapid and transient dissociation of the S1R-BiP heterodimer, which was blocked by haloperidol. The effect of PRE-084 was enhanced by calcium depletion, leading to a higher reduction in heterodimerization even in the presence of haloperidol. Prolonged incubation of cells with S1R antagonists (haloperidol, NE-100, BD-1047, and PD-144418) increased the formation of S1R-BiP heteromers, while agonists (PRE-084, 4-IBP, and pentazocine) did not alter heterodimerization under the same experimental conditions. The newly developed S1R-BiP biosensor is a simple and effective tool for exploring S1R pharmacology in an easy cellular setting. This biosensor is suitable for high-throughput applications and a valuable resource in the researcher's toolkit.

G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors and the main drug targets in therapeutics. GPCRs can establish direct receptor-receptor interactions (oligomerization), which can also be considered as targets for drug development (GPCR oligomer-based drugs). However, prior to designing any novel GPCR oligomer-based drug development program, demonstrating the existence of a named GPCR oligomer in native tissues is needed as part of its target engagement definition. Here, we discuss the proximity ligation in situ assay (P-LISA), an experimental approach that reveals GPCR oligomerization in native tissues. We provide a detailed step-by-step protocol to perform P-LISA experiments and visualize GPCR oligomers in brain slices. We also provide instructions for slide observation, data acquisition, and quantification. Finally, we discuss the critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, this protocol may be used to straightforwardly visualize GPCR oligomers in the brain. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Visualization of GPCR oligomers by proximity ligation in situ assay (P-LISA) Support Protocol: Slide observation, image acquisition, and quantification., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Background and Purpose: Opioid-based drugs are the gold standard medicines for pain relief. However, tolerance and several side effects (i.e. constipation and dependence) may occur upon chronic opioid administration. Photopharmacology is a promising approach to improve the benefit/risk profiles of these drugs. Thus, opioids can be locally activated with high spatiotemporal resolution, potentially minimizing systemic-mediated adverse effects. Here, we aimed at developing a morphine photo-derivative (photocaged morphine), which can be activated upon light irradiation both in vitro and in vivo., Experimental Approach: Light-dependent activity of pc-morphine was assessed in cell-based assays (intracellular calcium accumulation and electrophysiology) and in mice (formalin animal model of pain). In addition, tolerance, constipation and dependence were investigated in vivo using experimental paradigms., Key Results: In mice, pc-morphine was able to elicit antinociceptive effects, both using external light-irradiation (hind paw) and spinal cord implanted fibre-optics. In addition, remote morphine photoactivation was devoid of common systemic opioid-related undesired effects, namely, constipation, tolerance to the analgesic effects, rewarding effects and naloxone-induced withdrawal., Conclusion and Implications: Light-dependent opioid-based drugs may allow effective analgesia without the occurrence of tolerance or the associated and severe opioid-related undesired effects., Linked Articles: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The modulation of the A 2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A 2B AR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A 2B AR affinity and pharmacokinetic properties of a collection of A 2B AR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A 2B AR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A 2B AR ligands exhibited remarkable affinity ( K i < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.

Adenosine modulates neurotransmission through inhibitory adenosine A 1 receptors (A 1 Rs) and stimulatory A 2A receptors (A 2A Rs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G279 7.44 S) within the transmembrane helix (TM) 7 of A 1 R (A 1 R G279S ) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A 1 R and the A 1 R-A 2A R heteromer. Our results revealed that the G279 7.44 S mutation does not alter A 1 R expression, ligand binding, constitutive activity or coupling to transducer proteins (G αi , G αq , G α12/13 , G αs , β-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A 1 R G279S weakened the ability of A 1 R to heteromerize with A 2A R, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A 1 R imposes on the constitutive activity and agonist-induced activation of the A 2A R. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G279 7.44 S mutation in TM 7 of A 1 R weakens the TM 5/6 interface of the A 1 R-A 2A R heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A 2A R not forming heteromers with A 1 R could represent a main pathogenetic mechanism of the EOPD associated with the G279 7.44 S ADORA1 mutation., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A 2A receptor (A 2 AR)-dopamine D 2 receptor (D 2 R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Adenosine receptors (ARs) play many important roles in physiology and have been recognized as potential targets for pain relief. Here, we introduce three photoswitchable adenosine derivatives that function as light-dependent agonists for ARs and confer optical control to these G protein-coupled receptors. One of our compounds, AzoAdenosine-3, was evaluated in the classical formalin model of pain. The molecule, active in the dark, was not metabolized by adenosine deaminase and effectively reduced pain perception in a light-dependent manner. These antinociceptive effects suggested a major role for A 1 R and A 3 R in peripheral-mediated pain sensitization, whereas an average adenosine-mediated antinociceptive effect will be facilitated by A 2A R and A 2B R. Our results demonstrate that a photoswitchable adenosine derivative can be used to map the contribution of ARs mediating analgesia in vivo .

Objective: α-Synuclein has been studied as a potential biomarker for Parkinson's disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD., Methods: GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer's disease (AD) patients., Results: GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered., Conclusion: GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A 2A receptors (A 2A Rs) or their degree of functional heteromerization with dopamine D 2 receptors (D 2 R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A 2A R knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A 2A R-D 2 R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A 2A R knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D 2 R without changes in A 2A R expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A 2A R-D 2 R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A 2A R-D 2 R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D 2 R and A 2A R were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A 2A R-D 2 R heteromers. The degree of A 2A R-D 2 R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter' systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu 5 R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu 5 Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu 5 R allosteric modulators., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic-cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D 2 and muscarinic acetylcholine M 1 receptors (D 2 R and M 1 R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D 2 R-M 1 R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET 1 and NanoBiT ® ) assays, performed in transiently transfected HEK293T cells. Subsequently, a D 2 R-M 1 R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA ® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D 2 R-selective agonist (sumanirole) and/or an M 1 R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D 2 R/M 1 R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects., (Copyright © 2020 Crans, Wouters, Valle-León, Taura, Massari, Fernández-Dueñas, Stove and Ciruela.)

Background: It has been hypothesized that heteromers of adenosine A 2A receptors (A2AR) and cannabinoid CB 1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals., Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A 1 receptors or dopamine D 2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level., Conclusions: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine's exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from A 2A R-deficient mice (A 2A R -/- ) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A 2A R antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A 2A R. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A 1 R and A 2A R co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from A 2A R -/- mice. Notably, while guanosine was not able to modify MRS7396 binding to A 2A R-expressing cells, a partial blockade was observed in cells co-expressing A 1 R and A 2A R. The relevance of the A 1 R and A 2A R interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A 2A R agonist-mediated effects in doubly expressing A 1 R and A 2A R cells. Interestingly, while guanosine did not affect A 1 R/A 2A R heteromer formation, it reduced A 2A R agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A 1 R and A 2A R co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ 9 -tetrahydrocannabinol (Δ 9 -THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ 9 -THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ 9 -THC-induced cognitive impairment in an adenosine A 2A receptor (A 2A R)-dependent manner. Furthermore, we reveal the existence of A 2A R and cannabinoid CB 1 receptor (CB 1 R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A 2A R-CB 1 R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A 2A R/CB 1 R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A 2A R-CB 1 R interactions in the brain.

Background : Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods : Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results : Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions : The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT 3 on calcium ( 45 Ca 2+ ) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT 3 . Our results indicate that rT 3 induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT 3 occurred in <5 min in Sertoli cells. In addition, the rT 3 metabolite, T 2 , also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT 3 also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na + /K + ATPase pump. The rapid response of rT 3 is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adenosine A 2A receptors (A 2A R) play a key role in modulating dopamine-dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A 2A R blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A 2A R function in vivo, we aimed to test whether the A 2A R-mediated sensitization of locomotor activity is GPR37-dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A 2A R antagonist, SCH58261 (1 mg/kg, i.p.), daily for 14 days, and the locomotor sensitization, striatum-dependent cued learning, and corticostriatal synaptic plasticity (i.e., long-term depression) were compared in wild-type and GPR37 -/- mice. Notably, GPR37 deletion promoted A 2A R-associated locomotor sensitization but not striatum-dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A 2A R blockade potentiated striatal long-term depression in corticostriatal synapses of GPR37 -/- but not of wild-type mice, thus correlating well with neurochemical alterations of the adenosinergic system. Overall, these results revealed the importance of GPR37 regulating A 2A R-dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

Optopharmacology is a very promising approach based on the use of light-deliverable drugs, which allows manipulating physiological processes with high spatiotemporal resolution. Light-dependent drugs (i.e. caged-compounds) targeting G protein-coupled receptors (GPCRs) have been developed to provide great pharmacological precision on the control of pain. Metabotropic glutamate type 5 (mGlu 5 ) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu 5 receptor negative allosteric modulators (NAMs) have consistently shown analgesic activity in experimental animal models of inflammatory pain. Accordingly, we synthesized a light-deliverable drug (i.e. caged compound) using the chemical structure of raseglurant, a mGlu 5 receptor NAM, as a molecular scaffold. And thereafter, we evaluated the analgesic activity of the caged compound in formalin-injected (hind paw) mice upon light irradiation (405 nm). Of note, light was both delivered at the peripheral (i.e. hind paw) and central level (i.e. thalamus), by means of brain-implanted fiber-optics. The novel light-deliverable drug, JF-NP-26, showed antinociceptive activity upon violet light irradiation either of the hind paw or the thalamus, demonstrating the ability of precisely activating, in time and space, the caged compound. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin nociception test in mice using an optopharmacology approach (i.e. light-deliverable compounds).

Proximity ligation assay (PLA) is an antibody-based method that permits studying protein-protein interactions with high specificity and sensitivity. In brief, when a pair of specific antibodies is in close proximity, the complementary DNA strands they bear engage into a rolling circle amplification and generate, in situ, a single fluorescent signal, which indicates the presence of a protein-protein interaction. Proper image analysis methods are needed to provide accurate quantitative assessment of the obtained fluorescent signals, namely, PLA data. In this chapter, we outline basic aspects of image analysis (including software, data import, image processing functions, and analytical tools) that can be used to extract PLA data from confocal microscopy images using ImageJ. A step-by-step protocol to determine and quantify PLA fluorescence signals is included. Overall, the accurate capture and subsequent analysis of PLA confocal images constitutes a crucial step to properly interpret data obtained with this powerful experimental approach.

The function of guanine-based purines (GBPs) is mostly attributed to the intracellular modulation of heteromeric and monomeric G proteins. However, extracellular effects of guanine derivatives have also been recognized. Thus, in the central nervous system (CNS), a guanine-based purinergic system that exerts neuromodulator effects, has been postulated. The thesis that GBPs are neuromodulators emerged from in vivo and in vitro studies, in which neurotrophic and neuroprotective effects of these kinds of molecules (i.e., guanosine) were demonstrated. GBPs induce several important biological effects in rodent models and have been shown to reduce seizures and pain, stabilize mood disorder behavior and protect against gliomas and diseases related with aging, such as ischemia or Parkinson and Alzheimer diseases. In vitro studies to evaluate the protective and trophic effects of guanosine, and of the nitrogenous base guanine, have been fundamental for understanding the mechanisms of action of GBPs, as well as the signaling pathways involved in their biological roles. Conversely, although selective binding sites for guanosine have been identified in the rat brain, GBP receptors have not been still described. In addition, GBP neuromodulation may depend on the capacity of GBPs to interact with well-known membrane proteins in glutamatergic and adenosinergic systems. Overall, in this review article, we present up-to-date GBP biology, focusing mainly on the mechanisms of action that may lead to the neuromodulator role of GBPs observed in neurological disorders.

Adenosine A 2A receptor (A 2A R) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson's disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan A 2A R antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human A 2A R, since it antagonized A 2A R agonist-mediated cAMP accumulation and impedance responses with K B values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new A 2A R-based label-free assay as a robust and sensitive approach to characterize A 2A R ligands. Next, we evaluated the efficacy of PBF509 reversing motor impairments in several rat models of movement disorders, including catalepsy, tremor, and hemiparkinsonism. Thus, PBF509 (orally) antagonized haloperidol-mediated catalepsy, reduced pilocarpine-induced tremulous jaw movements and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in unilaterally 6-OHDA-lesioned rats. Moreover, PBF509 (3 mg/kg) inhibited L-DOPA-induced dyskinesia (LID), showing not only its efficacy on reversing parkinsonian motor impairments but also acting as antidyskinetic agent. Overall, here we describe a new orally selective A 2A R antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. The main treatment of PD consists of medication with dopamine-based drugs, which palliate the symptoms but may produce adverse effects after chronic administration. Accordingly, there is a need to develop novel neuroprotective therapies. Several studies suggest that omega-3 polyunsaturated fatty acids ( n -3 PUFA) might provide protection against brain damage. Here, we studied several experimental models of PD, using striatal neuronal cultures, striatal slices, and mice, to assess the neuroprotective effects of docosahexaenoic acid (DHA), the main n -3 PUFA in the brain, administered in its triglyceride form (TG-DHA). Hence, we determined the beneficial effects of TG-DHA on neural viability following 6-hydroxydopamine (6-OHDA)-induced neurotoxicity, a well-established PD model. We also implemented a novel mouse behavioral test, the beam walking test, to finely assess mouse motor skills following dopaminergic denervation. This test showed potential as a useful behavioral tool to assess novel PD treatments. Our results indicated that TG-DHA-mediated neuroprotection was independent of the net incorporation of PUFA into the striatum, thus suggesting a tight control of brain lipid homeostasis both in normal and pathological conditions.

The molecular mechanism by which progesterone (P4) modulates the transport of ova and embryos along the oviduct is not fully resolved. We report a rapid response to P4 and agonists of γ-aminobutyric acid receptors A and B (GABA A/B ) in the mouse oviduct that was characterized by oscillatory Ca 2+ signals and increased ciliary beat frequency (CBF). Pharmacological manipulation, genetic ablation, and siRNA-mediated knockdown in oviductal cells, as well as overexpression experiments in HEK 293T cells, confirmed the participation of the cationic channel TRPV4, different subunits of GABA A (α1 to α3, β2, and β3), and GABA B1 in P4-induced responses. TRPV4-mediated Ca 2+ entry in close proximity to the inositol trisphosphate receptor was required to initiate and maintain Ca 2+ oscillations after P4 binding to GABA A and transactivation of G i/o protein-coupled GABA B receptors. Coimmunoprecipitation experiments and imaging of native tissue and HEK 293T cells demonstrated the close association of GABA A and GABA B1 receptors and the activation of G i/o proteins in response to P4 and GABA receptor agonists, confirming a molecular mechanism in which P4 and GABAergic agonists cooperatively stimulate cilial beating., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G protein-coupled adenosine receptors are promising therapeutic targets for a wide range of neuropathological conditions, including Parkinson's disease (PD). However, the ubiquity of adenosine receptors and the ultimate lack of selectivity of certain adenosine-based drugs have frequently diminished their therapeutic use. Photopharmacology is a novel approach that allows the spatiotemporal control of receptor function, thus circumventing some of these limitations. Here, we aimed to develop a light-sensitive caged adenosine A 2A receptor (A 2A R) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective A 2A R antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in A 2A R-expressing cells. Upon photoactivation, MRS7145 precluded A 2A R ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block A 2A R in a light-dependent manner was assessed in vivo. To this end, A 2A R antagonist-mediated locomotor activity potentiation was evaluated in brain (striatum) fiber-optic implanted mice. Upon irradiation (405 nm) of the dorsal striatum, MRS7145 induced significant hyperlocomotion and counteracted haloperidol-induced catalepsy and pilocarpine-induced tremor. Finally, its efficacy in reversing motor impairment was evaluated in a PD animal model, namely the hemiparkinsonian 6-hydroxydopamine (6-OHDA)-lesioned mouse. Photo-activated MRS7145 was able to potentiate the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (l-DOPA). Overall, MRS7145 is a new light-operated A 2A R antagonist with potential utility to manage movement disorders, including PD., (Published by Elsevier B.V.)

Dopamine D 2 receptor (D 2 R) activation triggers both G protein- and β-arrestin-dependent signaling. Biased D 2 R ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of D 2 R to heteromerize with adenosine A 2A receptor (A 2A R) has been associated to D 2 R agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the A 2A R dependence of D 2 R/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of D 2 R/A 2A R homomers and heteromers, we demonstrated that the D 2 R agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when A 2A R/D 2 R heteromers were expressed. Subsequently, the role of A 2A R in the antipsychotic-like activity of UNC9994 was assessed in wild-type and A 2A R -/- mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in A 2A R -/- mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that D 2 R/A 2A R heteromerization facilitates D 2 R β-arrestin recruitment, and furthermore, reveal a pivotal role for A 2A R in the antipsychotic-like activity of the β-arrestin-biased D 2 R ligand, UNC9994.

Pridopidine is in clinical trials for Huntington's disease treatment. Originally developed as a dopamine D 2 receptor (D 2 R) ligand, pridopidine displays about 100-fold higher affinity for the sigma-1 receptor (sigma-1R). Interestingly, pridopidine slows disease progression and improves motor function in Huntington's disease model mice and, in preliminarily reports, Huntington's disease patients. The present study examined the anti-amnesic potential of pridopidine. Thus, memory impairment was produced in mice by administration of phencyclidine (PCP, 10 mg/kg/day) for 10 days, followed by 14 days' treatment with pridopidine (6 mg/kg/day), or saline. Finally, novel object recognition performance was assessed in the animals. Mice receiving PCP and saline exhibited deficits in novel object recognition, as expected, while pridopidine treatment counteracted PCP-induced memory impairment. The effect of pridopidine was attenuated by co-administration of the sigma receptor antagonist, NE-100 (10 mg/kg). Our results suggest that pridopidine exerts anti-amnesic and potentially neuroprotective actions. These data provide new insights into the therapeutic potential of pridopidine as a pro-cognitive drug.

G protein-coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A 2A receptors (A 2A R) and dopamine D 2 receptors (D 2 R) predominantly form A 2A R-D 2 R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A 2A R and D 2 R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain-related differences, a new D 2 R-deficient mouse with the same genetic background (CD-1) than the A 2A R knock-out mouse was generated. Locomotor activity, pre-pulse inhibition (PPI) and drug-induced catalepsy were then evaluated in wild-type, A 2A R and D 2 R knock-out mice, with and without the concomitant administration of either the D 2 R agonist sumanirole or the A 2A R antagonist SCH442416. SCH442416-mediated locomotor effects were demonstrated to be dependent on D 2 R signaling. Similarly, a significant dependence on A 2A R signaling was observed for PPI and for haloperidol-induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A 2A R-D 2 R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders., (© 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)