The amygdala NT3-TrkC pathway underlies inter-individual differences in fear extinction and related synaptic plasticity.

Fear-related pathologies are among the most prevalent psychiatric conditions, having inappropriate learned fear and resistance to extinction as cardinal features. Exposure therapy represents a promising therapeutic approach, the efficiency of which depends on inter-individual variation in fear extinction learning, which neurobiological basis is unknown. We characterized a model of extinction learning, whereby fear-conditioned mice were categorized as extinction (EXT)-success or EXT-failure, according to their inherent ability to extinguish fear. In the lateral amygdala, GluN2A-containing NMDAR are required for LTP and stabilization of fear memories, while GluN2B-containing NMDAR are required for LTD and fear extinction. EXT-success mice showed attenuated LTP, strong LTD and higher levels of synaptic GluN2B, while EXT-failure mice showed strong LTP, no LTD and higher levels of synaptic GluN2A. Neurotrophin 3 (NT3) infusion in the lateral amygdala was sufficient to rescue extinction deficits in EXT-failure mice. Mechanistically, activation of tropomyosin receptor kinase C (TrkC) with NT3 in EXT-failure slices attenuated lateral amygdala LTP, in a GluN2B-dependent manner. Conversely, blocking endogenous NT3-TrkC signaling with TrkC-Fc chimera in EXT-success slices strengthened lateral amygdala LTP. Our data support a key role for the NT3-TrkC system in inter-individual differences in fear extinction in rodents, through modulation of amygdalar NMDAR composition and synaptic plasticity.

International Union of Basic and Clinical Pharmacology. CXII: Adenosine Receptors: A Further Update.

Our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors (2011) contained a number of emerging developments with respect to this G protein-coupled receptor subfamily, including protein structure, protein oligomerization, protein diversity, and allosteric modulation by small molecules. Since then, a wealth of new data and results has been added, allowing us to explore novel concepts such as target binding kinetics and biased signaling of adenosine receptors, to examine a multitude of receptor structures and novel ligands, to gauge new pharmacology, and to evaluate clinical trials with adenosine receptor ligands. This review should therefore be considered a further update of our previous reports from 2001 and 2011. SIGNIFICANCE STATEMENT: Adenosine receptors (ARs) are of continuing interest for future treatment of chronic and acute disease conditions, including inflammatory diseases, neurodegenerative afflictions, and cancer. The design of AR agonists ("biased" or not) and antagonists is largely structure based now, thanks to the tremendous progress in AR structural biology. The A2A- and A2BAR appear to modulate the immune response in tumor biology. Many clinical trials for this indication are ongoing, whereas an A2AAR antagonist (istradefylline) has been approved as an anti-Parkinson agent.

Modification of astrocytic Cx43 hemichannel activity in animal models of AD: modulation by adenosine A2A receptors.

Increasing evidence implicates astrocytic dysfunction in Alzheimer's disease (AD), a neurodegenerative disorder characterised by progressive cognitive loss. The accumulation of amyloid-ß (Aß) plaques is a histopathological hallmark of AD and associated with increased astrocyte reactivity. In APP/PS1 mice modelling established AD (9 months), we now show an altered astrocytic morphology and enhanced activity of astrocytic hemichannels, mainly composed by connexin 43 (Cx43). Hemichannel activity in hippocampal astrocytes is also increased in two models of early AD: (1) mice with intracerebroventricular (icv) administration of Aß1-42, and (2) hippocampal slices superfused with Aß1-42 peptides. In hippocampal gliosomes of APP/PS1 mice, Cx43 levels were increased, whereas mice administered icv with Aß1-42 only displayed increased Cx43 phosphorylation levels. This suggests that hemichannel activity might be differentially modulated throughout AD progression. Additionally, we tested if adenosine A2A receptor (A2AR) blockade reversed alterations of astrocytic hemichannel activity and found that the pharmacological blockade or genetic silencing (global and astrocytic) of A2AR prevented Aß-induced hemichannel dysregulation in hippocampal slices, although A2AR genetic silencing increased the activity of astroglial hemichannels in control conditions. In primary cultures of astrocytes, A2AR-related protective effect was shown to occur through a protein kinase C (PKC) pathway. Our results indicate that the dysfunction of hemichannel activity in hippocampal astrocytes is an early event in AD, which is modulated by A2AR.

Adenosine A2A Receptor Blockade Provides More Effective Benefits at the Onset Rather than after Overt Neurodegeneration in a Rat Model of Parkinson's Disease.

Adenosine A2A receptor (A2AR) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and neuroprotection. PD begins with a synaptic dysfunction and damage in the striatum evolving to an overt neuronal damage of dopaminergic neurons in the substantia nigra. We tested if A2AR antagonists are equally effective in controlling these two degenerative processes. We used a slow intracerebroventricular infusion of the toxin MPP+ in male rats for 15 days, which caused an initial loss of synaptic markers in the striatum within 10 days, followed by a neuronal loss in the substantia nigra within 30 days. Interestingly, the initial loss of striatal nerve terminals involved a loss of both dopaminergic and glutamatergic synaptic markers, while GABAergic markers were preserved. The daily administration of the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) in the first 10 days after MPP+ infusion markedly attenuated both the initial loss of striatal synaptic markers and the subsequent loss of nigra dopaminergic neurons. Strikingly, the administration of SCH58261 (0.1 mg/kg, i.p. for 10 days) starting 20 days after MPP+ infusion was less efficacious to attenuate the loss of nigra dopaminergic neurons. This prominent A2AR-mediated control of synaptotoxicity was directly confirmed by showing that the MPTP-induced dysfunction (MTT assay) and damage (lactate dehydrogenase release assay) of striatal synaptosomes were prevented by 50 nM SCH58261. This suggests that A2AR antagonists may be more effective to counteract the onset rather than the evolution of PD pathology.

Genetic Polymorphisms of P2RX7 but Not of ADORA2A Are Associated with the Severity of SARS-CoV-2 Infection.

SARS-CoV-2 infection ranges from mild to severe presentations, according to the intensity of the aberrant inflammatory response. Purinergic receptors dually control the inflammatory response: while adenosine A2A receptors (A2ARs) are anti-inflammatory, ATP P2X7 receptors (P2X7Rs) exert pro-inflammatory effects. The aim of this study was to assess if there were differences in allelic and genotypic frequencies of a loss-of-function SNP of ADORA2A (rs2298383) and a gain-of-function single nucleotide polymorphism (SNP) of P2RX7 (rs208294) in the severity of SARS-CoV-2-associated infection. Fifty-five individuals were enrolled and categorized according to the severity of the infection. Endpoint genotyping was performed in blood cells to screen for both SNPs. The TT genotype (vs. CT + CC) and the T allele (vs. C allele) of P2RX7 SNP were found to be associated with more severe forms of COVID-19, whereas the association between ADORA2A SNP and the severity of infection was not significantly different. The T allele of P2RX7 SNP was more frequent in people with more than one comorbidity and with cardiovascular conditions and was associated with colorectal cancer. Our findings suggest a more prominent role of P2X7R rather than of A2AR polymorphisms in SARS-CoV-2 infection, although larger population-based studies should be performed to validate our conclusions.

Astrocytic A2A receptors silencing negatively impacts hippocampal synaptic plasticity and memory of adult mice.

Astrocytes are wired to bidirectionally communicate with neurons namely with synapses, thus shaping synaptic plasticity, which in the hippocampus is considered to underlie learning and memory. Adenosine A2A receptors (A2A R) are a potential candidate to modulate this bidirectional communication, since A2A R regulate synaptic plasticity and memory and also control key astrocytic functions. Nonetheless, little is known about the role of astrocytic A2A R in synaptic plasticity and hippocampal-dependent memory. Here, we investigated the impact of genetic silencing astrocytic A2A R on hippocampal synaptic plasticity and memory of adult mice. The genetic A2A R silencing in astrocytes was accomplished by a bilateral injection into the CA1 hippocampal area of a viral construct (AAV5-GFAP-GFP-Cre) that inactivate A2A R expression in astrocytes of male adult mice carrying "floxed" A2A R gene, as confirmed by A2A R binding assays. Astrocytic A2A R silencing alters astrocytic morphology, typified by an increment of astrocytic arbor complexity, and led to deficits in spatial reference memory and compromised hippocampal synaptic plasticity, typified by a reduction of LTP magnitude and a shift of synaptic long-term depression (LTD) toward LTP. These data indicate that astrocytic A2A R control astrocytic morphology and influence hippocampal synaptic plasticity and memory of adult mice in a manner different from neuronal A2A R.

Feedback facilitation by adenosine A2A receptors of ATP release from mouse hippocampal nerve terminals.

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,ß-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.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

The striatum drives the ergogenic effects of caffeine.

Caffeine is one of the main ergogenic resources used in exercise and sports. Previously, we reported the ergogenic mechanism of caffeine through neuronal A2AR antagonism in the central nervous system [1]. We now demonstrate that the striatum rules the ergogenic effects of caffeine through neuroplasticity changes. Thirty-four Swiss (8-10 weeks, 47 ± 1.5 g) and twenty-four C57BL/6J (8-10 weeks, 23.9 ± 0.4 g) adult male mice were studied behaviorly and electrophysiologically using caffeine and energy metabolism was studied in SH-SY5Y cells. Systemic (15 mg/kg, i.p.) or striatal (bilateral, 15 µg) caffeine was psychostimulant in the open field (p < 0.05) and increased grip efficiency (p < 0.05). Caffeine also shifted long-term depression (LTD) to potentiation (LTP) in striatal slices and increased the mitochondrial mass (p < 0.05) and membrane potential (p < 0.05) in SH-SY5Y dopaminergic cells. Our results demonstrate the role of the striatum in the ergogenic effects of caffeine, with changes in neuroplasticity and mitochondrial metabolism.

Aß1-42 peptides blunt the adenosine A2A receptor-mediated control of the interplay between P2X7 and P2Y1 receptors mediated calcium responses in astrocytes.

The contribution of astrocytes to Alzheimer's disease (AD) is still ill defined. AD involves an abnormal accumulation of amyloid-ß peptides (Aß) and increased production of danger signals such as ATP. ATP can direct or indirectly, through its metabolism into adenosine, trigger adaptive astrocytic responses resulting from intracellular Ca2+ oscillations. AD also triggers an upregulation of astrocytic adenosine A2A receptors (A2AR), which blockade prevents memory dysfunction in AD. We now investigated how Aß peptides affect ATP-mediated Ca2+ responses in astrocytes measured by fluorescence live-cell imaging and whether A2AR control astrocytic Ca2+ responses mediated by ATP receptors, mainly P2X7R and P2Y1R. In primary cultures of rat astrocytes exposed to Aß1-42, ATP-evoked Ca2+ responses had a lower amplitude but a longer duration than in control astrocytes and involved P2X7R and P2Y1R, the former potentiating the later. Moreover, Aß1-42 exposure increased protein levels of P2Y1R in astrocytes. A2AR antagonism with SCH58261 controlled in a protein kinase A-dependent manner both P2X7R- and P2Y1R-mediated Ca2+ responses in astrocytes. The interplay between these purinoceptors in astrocytes was blunted upon exposure to Aß1-42. These findings uncover the ability of A2AR to regulate the inter-twinned P2X7R- and P2Y1R-mediated Ca2+ dynamics in astrocytes, which is disrupted in conditions of early AD.

Downregulation of Sirtuin 1 Does Not Account for the Impaired Long-Term Potentiation in the Prefrontal Cortex of Female APPswe/PS1dE9 Mice Modelling Alzheimer's Disease.

Alzheimer's disease (AD), which predominantly affects women, involves at its onset a metabolic deregulation associated with a synaptic failure. Here, we performed a behavioral, neurophysiological and neurochemical characterization of 9-month-old female APPswe/PS1dE9 (APP/PS1) mice as a model of early AD. These animals showed learning and memory deficits in the Morris water maze, increased thigmotaxis and anxiety-like behavior and showed signs of fear generalization. Long-term potentiation (LTP) was decreased in the prefrontal cortex (PFC), but not in the CA1 hippocampus or amygdala. This was associated with a decreased density of sirtuin-1 in cerebrocortical synaptosomes and a decreased density of sirtuin-1 and sestrin-2 in total cerebrocortical extracts, without alterations of sirtuin-3 levels or of synaptic markers (syntaxin, synaptophysin, SNAP25, PSD95). However, activation of sirtuin-1 did not affect or recover PFC-LTP deficit in APP/PS1 female mice; instead, inhibition of sirtuin-1 increased PFC-LTP magnitude. It is concluded that mood and memory dysfunction in 9-month-old female APP/PS1 mice is associated with a parallel decrease in synaptic plasticity and in synaptic sirtuin-1 levels in the prefrontal cortex, although sirtiun1 activation failed to restore abnormal cortical plasticity.

Impact of blunting astrocyte activity on hippocampal synaptic plasticity in a mouse model of early Alzheimer's disease based on amyloid-ß peptide exposure.

Amyloid-ß peptides (Aß) accumulate in the brain since early Alzheimer's disease (AD) and dysregulate hippocampal synaptic plasticity, the neurophysiological basis of memory. Although the relationship between long-term potentiation (LTP) and memory processes is well established, there is also evidence that long-term depression (LTD) may be crucial for learning and memory. Alterations in synaptic plasticity, namely in LTP, can be due to communication failures between astrocytes and neurons; however, little is known about astrocytes' ability to control hippocampal LTD, particularly in AD-like conditions. We now aimed to test the involvement of astrocytes in changes of hippocampal LTP and LTD triggered by Aß1-42 , taking advantage of L-α-aminoadipate (L-AA), a gliotoxin that blunts astrocytic function. The effects of Aß1-42 exposure were tested in two different experimental paradigms: ex vivo (hippocampal slices superfusion) and in vivo (intracerebroventricular injection), which were previously validated to impair memory and hippocampal synaptic plasticity, two features of early AD. Blunting astrocytic function with L-AA reduced LTP and LTD amplitude in hippocampal slices from control mice, but the effect on LTD was less evident, suggesting that astrocytes have a greater influence on LTP than on LTD under non-pathological conditions. However, under AD conditions, blunting astrocytes did not consistently alter the reduction of LTP magnitude, but reverted the LTD-to-LTP shift caused by both ex vivo and in vivo Aß1-42 exposure. This shows that astrocytes were responsible for the hippocampal LTD-to-LTP shift observed in early AD conditions, reinforcing the interest of strategies targeting astrocytes to restore memory and synaptic plasticity deficits present in early AD.

An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer's disease.

The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2-arachidonoyl glycerol (2-AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2-AG, also accepts the surrogate chromogenic substrate 4-nitrophenyl butyrate (4-NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2-AG. The initial velocity of 4-NPB hydrolysis was dependent on protein, substrate, and Ca2+ concentrations, and was sensitive to the non-selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2-AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB1 R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4-NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin - an established model of sporadic AD -, both CB1 R levels and 4-NPB hydrolysis and its DAGL- and MAGL-dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples.

l-α-aminoadipate causes astrocyte pathology with negative impact on mouse hippocampal synaptic plasticity and memory.

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.

Signaling by adenosine receptors-Homeostatic or allostatic control?

Adenosine modulation is considered both a paracrine signal coordinating different cells in a tissue and a stress signal. Both functions are ensured by 4 types of adenosine receptors (ARs), which have been studied individually. Mice with knockout of all ARs (quad-AR-KO) now allow enquiring the overall function of the adenosine modulation system. The observed "normal" physiology of quad-AR-KO mice indicates that ARs do not regulate homeostasis and are likely recruited to selectively control allostasis.

Adenosine A2A receptors blockade attenuates dexamethasone-induced alterations in cultured astrocytes.

Anxiety involves abnormal glucocorticoid signalling and altered glia-neuron communication in brain regions processing emotional responses. Adenosine A2A receptor (A2AR) blockade ameliorates mood and memory impairments by preventing synaptic dysfunction and astrogliosis. Since the glucocorticoid dexamethasone (DEX) can mimic early life-stress conditions, leading to anxiety-like behaviours, we now tested if A2AR blockade prevents alterations in the morphology and function of astrocytes exposed to DEX. Cultured astrocytes exposed to DEX exhibited an up-regulation of astrocytic markers (GFAP, connexin-43 and glutamine synthetase), as well as of A2AR. Moreover, DEX enhanced ATP and glutamate release and increased basal astrocytic Ca2+ levels. The selective A2AR antagonist SCH58261 prevented DEX-induced alterations in ATP release and basal Ca2+ levels but did not affect DEX-induced alteration of glutamate release and astrocytic markers. These findings suggest that alterations in astrocytes function, which might contribute to abnormal glucocorticoid brain signalling, are controlled by A2AR, and therefore, reinforce the relevance of A2AR as a potential therapeutic target to manage mood disorders.

Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

Thebromine Targets Adenosine Receptors to Control Hippocampal Neuronal Function and Damage.

Theobromine is a caffeine metabolite most abundant in dark chocolate, of which consumption is linked with a lower risk of cognitive decline. However, the mechanisms through which theobromine affects neuronal function remain ill-defined. Using electrophysiological recordings in mouse hippocampal synapses, we now characterized the impact of a realistic concentration of theobromine on synaptic transmission and plasticity. Theobromine (30 µM) facilitated synaptic transmission while decreasing the magnitude of long-term potentiation (LTP), with both effects being blunted by adenosine deaminase (2 U/mL). The pharmacological blockade of A1R with DPCPX (100 nM) eliminated the theobromine-dependent facilitation of synaptic transmission, whereas the A2AR antagonist SCH58261 (50 nM), as well as the genetic deletion of A2AR, abrogated the theobromine-induced impairment of LTP. Furthermore, theobromine prevented LTP deficits and neuronal loss, respectively, in mouse hippocampal slices and neuronal cultures exposed to Aß1-42 peptides, considered a culprit of Alzheimer's disease. Overall, these results indicate that theobromine affects information flow via the antagonism of adenosine receptors, normalizing synaptic plasticity and affording neuroprotection in dementia-related conditions in a manner similar to caffeine.

CD73-Mediated Formation of Extracellular Adenosine Is Responsible for Adenosine A2A Receptor-Mediated Control of Fear Memory and Amygdala Plasticity.

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 αß-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.

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.