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The adenosine modulation system is mostly composed by inhibitory A1 receptors (A1R) and the less abundant facilitatory A2A receptors (A2AR), the latter selectively engaged at high frequency stimulation associated with synaptic plasticity processes in the hippocampus. A2AR are activated by adenosine originated from extracellular ATP through ecto-5'-nucleotidase or CD73-mediated catabolism. Using hippocampal synaptosomes, we now investigated how adenosine receptors modulate the synaptic release of ATP. The A2AR agonist CGS21680 (10-100 nM) enhanced the K+-evoked release of ATP, whereas both SCH58261 and the CD73 inhibitor α,β-methylene ADP (100 μM) decreased ATP release; all these effects were abolished in forebrain A2AR knockout mice. The A1R agonist CPA (10-100 nM) inhibited ATP release, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects. The presence of SCH58261 potentiated CPA-mediated ATP release and uncovered a facilitatory effect of DPCPX. Overall, these findings indicate that ATP release is predominantly controlled by A2AR, which are involved in an apparent feedback loop of A2AR-mediated increased ATP release together with dampening of A1R-mediated inhibition. This study is a tribute to María Teresa Miras-Portugal. [ABSTRACT FROM AUTHOR]

Adenosine operates a modulation system fine-tuning the efficiency of synaptic transmission and plasticity through A1 and A2A receptors (A1R, A2AR), respectively. Supramaximal activation of A1R can block hippocampal synaptic transmission, and the tonic engagement of A1R-mediated inhibition is increased with increased frequency of nerve stimulation. This is compatible with an activity-dependent increase in extracellular adenosine in hippocampal excitatory synapses, which can reach levels sufficient to block synaptic transmission. We now report that A2AR activation decreases A1R-medated inhibition of synaptic transmission, with particular relevance during high-frequency-induced long-term potentiation (LTP). Thus, whereas the A1R antagonist DPCPX (50 nM) was devoid of effects on LTP magnitude, the addition of an A2AR antagonist SCH58261 (50 nM) allowed a facilitatory effect of DPCPX on LTP to be revealed. Additionally, the activation of A2AR with CGS21680 (30 nM) decreased the potency of the A1R agonist CPA (6–60 nM) to inhibit hippocampal synaptic transmission in a manner prevented by SCH58261. These observations show that A2AR play a key role in dampening A1R during high-frequency induction of hippocampal LTP. This provides a new framework for understanding how the powerful adenosine A1R-mediated inhibition of excitatory transmission can be controlled to allow the implementation of hippocampal LTP. [ABSTRACT FROM AUTHOR]

Extracellular ATP can be a danger signal, but its role in striatal circuits afflicted in Parkinson's disease (PD) is unclear and was now investigated. ATP was particularly released at high stimulation intensities from purified striatal nerve terminals of mice, which were endowed with different ATP-P2 receptors (P2R), although P2R antagonists did not alter corticostriatal transmission or plasticity. Instead, ATP was extracellularly catabolized into adenosine through CD73 to activate adenosine A2A receptors (A2AR) modulating corticostriatal long-term potentiation (LTP) in mice. In the presymptomatic phase of a 6-hydroxydopamine rat model of PD, ATP release from striatal nerve terminals was increased and was responsible for a greater impact of CD73 and A2AR on corticostriatal LTP. These observations identify increased ATP release and ATP-derived formation of extracellular adenosine bolstering A2AR activation as a key pathway responsible for abnormal synaptic plasticity in circuits involved in the onset of PD motor symptoms. The translation of these findings to humans prompts extending the use of A2AR antagonists from only co-adjuvants of motor control in Parkinsonian patients to neuroprotective drugs delaying the onset of motor symptoms. [ABSTRACT FROM AUTHOR]

Adenosine A2A receptors (A2AR) control fear memory and the underlying processes of synaptic plasticity in the amygdala. In other brain regions, A2AR activation is ensured by ATP-derived extracellular adenosine formed by ecto-5′-nucleotidase or CD73. We now tested whether CD73 is also responsible to provide for the activation of A2AR in controlling fear memory and amygdala long-term potentiation (LTP). The bilateral intracerebroventricular injection of the CD73 inhibitor αβ-methylene ADP (AOPCP, 1 nmol/ventricle/day) phenocopied the effect of the A2AR blockade by decreasing the expression of fear memory, an effect disappearing in CD73-knockout (KO) mice and in forebrain neuronal A2AR-KO mice. In the presence of PPADS (20 μM) to eliminate any modification of ATP/ADP-mediated P2 receptor effects, both AOPCP (100 μM) and the A2AR antagonist, SCH58261 (50 nM), decreased LTP magnitude in synapses of projection from the external capsula into the lateral amygdala, an effect eliminated in slices from both forebrain neuronal A2AR-KO mice and CD73-KO mice. These data indicate a key role of CD73 in the process of A2AR-mediated control of fear memory and underlying synaptic plasticity processes in the amygdala, paving the way to envisage CD73 as a new therapeutic target to interfere with abnormal fear-like emotional processing. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying circuit re-wiring in the mature brain remains ill-defined. An eloquent example of adult circuit remodelling is the hippocampal mossy fiber (MF) sprouting found in diseases such as temporal lobe epilepsy. The molecular determinants underlying this retrograde re-wiring remain unclear. This may involve signaling system(s) controlling axon specification/growth during neurodevelopment reactivated during epileptogenesis. Since adenosine A2A receptors (A2AR) control axon formation/outgrowth and synapse stabilization during development, we now examined the contribution of A2AR to MF sprouting. A2AR blockade significantly attenuated status epilepticus(SE)-induced MF sprouting in a rat pilocarpine model. This involves A2AR located in dentate granule cells since their knockdown selectively in dentate granule cells reduced MF sprouting, most likely through the ability of A2AR to induce the formation/outgrowth of abnormal secondary axons found in rat hippocampal neurons. These A2AR should be activated by extracellular ATP-derived adenosine since a similar prevention/attenuation of SE-induced hippocampal MF sprouting was observed in CD73 knockout mice. These findings demonstrate that A2AR contribute to epilepsy-related MF sprouting, most likely through the reactivation of the ability of A2AR to control axon formation/outgrowth observed during neurodevelopment. These results frame the CD73-A2AR axis as a regulator of circuit remodeling in the mature brain. [ABSTRACT FROM AUTHOR]

Depressive conditions precipitated by repeated stress are a major socio-economical burden in Western countries. Previous studies showed that ATP-P2X7 receptors (P2X7R) and adenosine A2A receptors (A2AR) antagonists attenuate behavioral modifications upon exposure to repeated stress. Since it is unknown if these two purinergic modulation systems work independently, we now investigated a putative interplay between P2X7R and A2AR. Adult rats exposed to restraint stress for 14 days displayed an anxious (thigmotaxis, elevated plus maze), depressive (anhedonia, increased immobility), and amnesic (modified Y maze, object displacement) profile, together with increased expression of Iba-1 (a marker of microglia "activation") and interleukin-1β (IL1β) and tumor necrosis factor α (TNFα; proinflammatory cytokines) and an up-regulation of P2X7R (mRNA) and A2AR (receptor binding) in the hippocampus and prefrontal cortex. All these features were attenuated by the P2X7R-preferring antagonist brilliant blue G (BBG, 45 mg/kg, i.p.) or by caffeine (0.3 g/L, p.o.), which affords neuroprotection through A2AR blockade. Notably, BBG attenuated A2AR upregulation and caffeine attenuated P2X7R upregulation. In microglial N9 cells, the P2X7R agonist BzATP (100 μM) or the A2AR agonist CGS26180 (100 nM) increased calcium levels, which was abrogated by the P2X7R antagonist JNJ47965567 (1 μM) and by the A2AR antagonist SCH58261 (50 nM), respectively; notably JNJ47965567 prevented the effect of CGS21680 and the effect of BzATP was attenuated by SCH58261 and increased by CGS21680. These results provide the first demonstration of a functional interaction between P2X7R and A2AR controlling microglia reactivity likely involved in behavioral adaptive responses to stress and are illustrative of a cooperation between the two arms of the purinergic system in the control of brain function. [ABSTRACT FROM AUTHOR]

Background and Purpose: Parkinson's disease (PD) involves an initial loss of striatal dopamine terminals evolving into degeneration of dopamine neurons in substantia nigra (SN), which can be modelled by 6-hydroxydopamine (6-OHDA) administration. Adenosine A2A receptor blockade attenuates PD features in animal models, but the source of the adenosine causing A2A receptor over-activation is unknown. As ATP is a stress signal, we have tested if extracellular catabolism of adenine nucleotides into adenosine (through ecto-5'-nucleotidase or CD73) leads to A2A receptor over-activation in PD.Experimental Approach: Effects of blocking CD73 with α,β-methylene ADP (AOPCP) were assayed in 6-OHDA-treated rats and dopamine-differentiated neuroblastoma SH-SY5Y cells.Key Results: 6-OHDA increased ATP release and extracellular conversion into adenosine through CD73 up-regulation in SH-SY5Y cells. Removing extracellular adenosine with adenosine deaminase, blocking CD73 with AOPCP, or blocking A2A receptors with SCH58261 were equi-effective in preventing 6-OHDA-induced damage in SH-SY5Y cells. In vivo striatal exposure to 6-OHDA increased ATP release and extracellular formation of adenosine from adenosine nucleotides and up-regulated CD73 and A2A receptors in striatal synaptosomes. Intracerebroventricular administration of AOPCP phenocopied effects of SCH58261, attenuating 6-OHDA-induced (a) increase of contralateral rotations after apomorphine, (b) reduction of dopamine content in striatum and SN, (c) loss of TH staining in striatum and SN, (d) motor dysfunction in the cylinder test, and (e) short-term memory impairment in the object recognition test.Conclusion and Implications: Our data indicate that increased ATP-derived adenosine formation is responsible for A2A receptor over-activation in PD, suggesting CD73 as a new target to manage PD. [ABSTRACT FROM AUTHOR]

Adenosine A2A receptors (A2AR) play a key role in modulating dopamine‐dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A2AR blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A2AR function in vivo, we aimed to test whether the A2AR‐mediated sensitization of locomotor activity is GPR37‐dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A2AR 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 A2AR‐associated locomotor sensitization but not striatum‐dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A2AR 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 A2AR‐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/ Adenosine A2A receptor (A2AR) function in vivo is modulated by a direct protein–protein interaction with the orphan receptor GPR37 upon certain physiological conditions. Therefore, we aimed to determine whether the well‐known A2AR‐mediated sensitization of locomotor activity is GPR37 dependent and if synaptic plasticity adaptations are involved. We show that while under normal physiological conditions, the expression of GPR37 does not modify striatal A2AR functionality, upon chronic A2AR blockade, deletion of GPR37 prompted A2AR‐associated locomotor sensitization, striatal long‐term depression (LTD) in corticostriatal synapses, and reduced adenosine content in the striatum. Overall, GPR37 emerges as an important regulator of A2AR‐dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/ [ABSTRACT FROM AUTHOR]

Background: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS). Methods: Retinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies. Results: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP. Conclusions: This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders. [ABSTRACT FROM AUTHOR]

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A2 BR) are Gs/q-protein-coupled receptors with low expression in the brain. As A2 BR function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A2 BR-knockout (A2 BR- KO) and C57/ BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A2 BR agonist BAY60-6583 (300 n m) decreased the paired-pulse stimulation ratio, an effect prevented by the A2 BR antagonist MRS 1754 (200 nM) and abrogated in A2 BR- KO mice. Accordingly, A2 BR immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A1R control of synaptic transmission, both the A1R inhibition caused by 2-chloroadenosine (0.1-1 μ m) and the disinhibition caused by the A1R antagonist DPCPX (100 n m), both effects prevented by MRS 1754 and abrogated in A2 BR- KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A1R inhibition ( CPA 100 n m). A2 BR- KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A2 BR are present in hippocampal glutamatergic terminals where they counteract the predominant A1R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour. [ABSTRACT FROM AUTHOR]

Depressive conditions precipitated by repeated stress are a major socio-economical burden in Western countries. Previous studies showed that ATP-P 2X7 receptors (P 2X7 R) and adenosine A 2A receptors (A 2A R) antagonists attenuate behavioral modifications upon exposure to repeated stress. Since it is unknown if these two purinergic modulation systems work independently, we now investigated a putative interplay between P 2X7 R and A 2A R. Adult rats exposed to restraint stress for 14 days displayed an anxious (thigmotaxis, elevated plus maze), depressive (anhedonia, increased immobility), and amnesic (modified Y maze, object displacement) profile, together with increased expression of Iba-1 (a marker of microglia "activation") and interleukin-1β (IL1β) and tumor necrosis factor α (TNFα; proinflammatory cytokines) and an up-regulation of P 2X7 R (mRNA) and A 2A R (receptor binding) in the hippocampus and prefrontal cortex. All these features were attenuated by the P 2X7 R-preferring antagonist brilliant blue G (BBG, 45 mg/kg, i.p.) or by caffeine (0.3 g/L, p.o .), which affords neuroprotection through A 2A R blockade. Notably, BBG attenuated A 2A R upregulation and caffeine attenuated P 2X7 R upregulation. In microglial N9 cells, the P 2X7 R agonist BzATP (100 μM) or the A 2A R agonist CGS26180 (100 nM) increased calcium levels, which was abrogated by the P 2X7 R antagonist JNJ47965567 (1 μM) and by the A 2A R antagonist SCH58261 (50 nM), respectively; notably JNJ47965567 prevented the effect of CGS21680 and the effect of BzATP was attenuated by SCH58261 and increased by CGS21680. These results provide the first demonstration of a functional interaction between P 2X7 R and A 2A R controlling microglia reactivity likely involved in behavioral adaptive responses to stress and are illustrative of a cooperation between the two arms of the purinergic system in the control of brain function., Competing Interests: RC is a scientific consultant of the Institute for Scientific Information on Coffee (ISIC). The remaining 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., (Copyright © 2021 Dias, Lopes, Gonçalves, Nunes, Pochmann, Machado, Tomé, Agostinho and Cunha.)