Maria Teresa Tebano

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus : a possible key mechanism in the modulation of N-methyl-D-aspartate effects

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA‐mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A2ARs) modulated mGlu5R‐dependent effects in the hippocampus, as they do in the striatum. Co‐application of the A2AR agonist CGS 21680 with the mGlu5R agonist (RS)‐2‐chloro‐s‐hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A2ARs seemed to be required to enable mGlu5R‐mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A2AR antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A2AR knockout mice. Evidence for the interaction between A2ARs and mGlu5Rs was further strengthened by demonstrating their co‐localization in hippocampal synapses. This is the first evidence showing that hippocampal A2ARs and mGlu5Rs are co‐located and act synergistically, and that A2ARs play a permissive role in mGlu5R receptor‐mediated potentiation of NMDA effects in the hippocampus.

Adenosine A2A receptors are required for normal BDNF levels and BDNF-induced potentiation of synaptic transmission in the mouse hippocampus

Brain‐derived neurotrophic factor (BDNF), a member of neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Both BDNF and its tyrosine kinase receptors (TrkB) are highly expressed in the hippocampus, where an interaction with adenosine A2A receptors (A2ARs) has been recently reported. In the present paper, we evaluated the role of A2ARs in mediating functional effects of BDNF in hippocampus using A2AR knock‐out (KO) mice. In hippocampal slices from WT mice, application of BDNF (10 ng/mL) increased the slope of excitatory post‐synaptic field potentials (fEPSPs), an index of synaptic facilitation. This increase of fEPSP slope was abolished by the selective A2A antagonist ZM 241385. Similarly, genetic deletion of the A2ARs abolished BDNF‐induced increase of the fEPSP slope in slices from A2AR KO mice The reduced functional ability of BDNF in A2AR KO mice was correlated with the reduction in hippocampal BDNF levels. In agreement, the pharmacological blockade of A2Rs by systemic ZM 241385 significantly reduced BDNF levels in the hippocampus of normal mice. These results indicate that the tonic activation of A2ARs is required for BDNF‐induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus.

The cannabinoid receptor agonist WIN 55,212-2 attenuates the effects induced by quinolinic acid in the rat striatum

The ability of CB(1) receptors to regulate the release of glutamate in the striatum, together with the finding that, in experimental models of Huntington disease (HD), both endocannabinoid levels and CB(1) receptor densities are reduced, has prompted the investigation on the neuroprotective role of the cannabinoids in HD. Quinolinic acid (QA) is an excitotoxin that, when injected in the rat striatum reproduces many features of HD and that acts by stimulating glutamate outflow. The aim of the present study was to test the ability of the cannabinoid receptor agonist WIN 55,212-2 to prevent the effects induced by QA in the rat striatum. In microdialysis experiments, probe perfusion with WIN 55,212-2 significantly and dose-dependently prevented the increase in extracellular glutamate induced by QA. In electrophysiological recordings in corticostriatal slices, the application of WIN 55,212-2 prevented QA-induced reduction of the field potential amplitude. Both effects of WIN 55,212-2 were prevented by the CB(1) receptor antagonist AM 251. In in vivo experiments, intrastriatal WIN 55,212-2 significantly attenuated the striatal damage induced by QA, although no significant effects were observed on a behavioural ground. These data demonstrate that the stimulation of CB(1) receptors might lead to neuroprotective effects against excitotoxic striatal toxicity.

Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre‐ and postsynaptic sites in the rat striatum

Adenosine A2A receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A2A receptor antagonists in models of pre‐ and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A2A receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose‐dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30–100 nM) significantly reduced 4‐aminopyridine (4‐AP)‐induced paired‐pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N‐methyl‐D‐aspartate (NMDA; 12.5 and 50 μM). The A2A antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A2A agonist CGS 21680 (100 nM) showed a protective influence toward 50 μM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca2+]i or the release of lactate dehydrogenase (LDH) induced by 100 and 300 μM NMDA, respectively. The ability of ZM 241385 to prevent QA‐induced glutamate outflow and 4‐AP‐induced effects confirms that A2A receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA‐induced effects suggest that A2A receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A2A antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission

J. Neurochem. (2011) 116, 273–280.

Permissive role of adenosine A2A receptors on metabotropic glutamate receptor 5 (mGluR5)-mediated effects in the striatum.

The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) have been reported to functionally interact in the striatum. The aim of the present work was to verify the hypothesis that the state of activation of A2A Rs could influence mGlu5R‐mediated effects in the striatum. In electrophysiological experiments (extracellular recording in rat corticostriatal slices), the ability of the selective mGlu5R agonist CHPG to potentiate the reduction of the field potential amplitude induced by NMDA was prevented not only by the selective mGlu5R antagonist MPEP, but also by the selective A2AR antagonist ZM 241385. Analogously, the application of CHPG potentiated NMDA‐induced toxicity (measured by LDH release) in cultured striatal neurons, an effect that was abolished by both MPEP and ZM 241385. Finally, the A2AR agonist CGS 21680 potentiated CHGP effects, an action that was reproduced and abolished, respectively, by forskolin (an activator of the cAMP/protein kinase A, PKA, pathway) and KT 5720 (a PKA inhibitor). The results indicate that A2ARs exert a permissive role on mGlu5R‐induced effects in the striatum. Such an interaction may represent an additional target for the development of therapeutic strategies towards striatal disorders.

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A2A, cannabinoid CB1 and metabotropic glutamate 5 (mGlu5) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB1R) agonist WIN55,212‐2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A2ARs. Such a permissive effect of A2ARs towards CB1Rs does not seem to occur pre‐synaptically as the ability of WIN55,212‐2 to increase the R2/R1 ratio under a protocol of paired‐pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212‐2 were reduced in slices from mice lacking post‐synaptic striatal A2ARs. The selective mGlu5R agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212‐2, and such a potentiation was abolished by A2AR blockade. Unlike the synaptic effects, the ability of WIN55,212‐2 to prevent NMDA‐induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A2ARs regulates the synaptic effects of CB1Rs and that A2ARs may control CB1 effects also indirectly, namely through mGlu5Rs.

Neuroprotective effects of the mGlu5R antagonist MPEP towards quinolinic acid-induced striatal toxicity: involvement of pre- and post-synaptic mechanisms and lack of direct NMDA blocking activity.

The aim of this work was to investigate the potential neuroprotective effects of the metabotropic glutamate receptor 5 (mGlu5R) antagonist 2‐Methyl‐6‐(phenylethynyl)‐pyridine (MPEP) towards quinolinic acid (QA)–induced striatal excitoxicity. Intrastriatal MPEP (5 nmol/0.5 µL) significantly attenuated the body weight loss, the electroencephalographic alterations, the impairment in spatial memory and the striatal damage induced by bilateral striatal injection of QA (210 nmol/0.7 µL). In a second set of experiments, we aimed to elucidate the mechanisms underlying the neuroprotective effects of MPEP. In microdialysis studies in naive rats MPEP (80–250 µm through the dialysis probe) significantly reduced the increase in glutamate levels induced by 5 mm QA. In primary cultures of striatal neurons MPEP (50 µm) reduced the toxicity induced by direct application of glutamate [measured as release of lactate dehydrogenase [LDH]). Finally, we found that 50 µm MPEP was unable to directly block NMDA‐induced effects (namely field potential reduction in corticostriatal slices, as well as LDH release and intracellular calcium increase in striatal neurons). We conclude that: (i) MPEP has neuroprotective effects towards QA‐induced striatal excitotoxicity; (ii) both pre‐ and post‐synaptic mechanisms are involved; (iii) the neuroprotective effects of MPEP do not appear to involve a direct blockade of NMDA receptors.

Opposite effects of the A2A receptor agonist CGS21680 in the striatum of Huntington's disease versus wild-type mice

Huntingtons disease (HD) is an inherited neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) are involved in excitotoxic/neurodegenerative processes, and A(2A)R ligands may be neuroprotective in models of HD. However, changes in the transcription, expression and function of A(2A)Rs have been reported to occur in HD models. The aim of the present work was to verify whether A(2A)R-mediated effects are altered in the striatum of transgenic HD (R6/2) versus wild-type (WT) mice. Extracellular field potentials (FPs) were recorded in corticostriatal slices from R6/2 mice in early (7-8 weeks) or frankly (12-13 weeks) symptomatic phases, and age-matched WT. In 12-13 weeks aged WT animals, the application of 75 microM NMDA induced a transient disappearance of the FP followed by an almost complete recovery at washout. In slices from HD mice, the mean FP recovery was significantly reduced (P<0.01 versus WT). A(2A)R activation oppositely modulated NMDA-induced toxicity in the striatum of HD versus WT mice. Indeed, the A(2A)R agonist CGS21680 reduced the FP recovery in slices from WT mice, while it significantly increased it in slices from R6/2 mice. In early symptomatic (7-8 weeks) mice, no differences were observed between WT and HD animals in terms of basal synaptic transmission and response to NMDA. At the same age, the behavioural effects elicited by CGS21680 were qualitatively identical in WT and HD mice. These findings may have very important implications for the neuroprotective potential of A(2A)R ligands in HD.

Role of Adenosine A2A Receptors in Modulating Synaptic Functions and Brain Levels of BDNF: a Possible Key Mechanism in the Pathophysiology of Huntington's Disease

In the last few years, accumulating evidence has shown the existence of an important cross-talk between adenosine A2A receptors (A2ARs) and brain-derived neurotrophic factor (BDNF). Not only are A2ARs involved in the mechanism of transactivation of BDNF receptor TrkB, they also modulate the effect of BDNF on synaptic transmission, playing a facilitatory and permissive role. The cAMP-PKA pathway, the main transduction system operated by A2ARs, is involved in such effects. Furthermore, a basal tonus of A2ARs is required to allow the regulation of BDNF physiological levels in the brain, as demonstrated by the reduced protein levels measured in A2ARs KO mice. The crucial role of adenosine A2ARs in the maintenance of synaptic functions and BDNF levels will be reviewed here and discussed in the light of possible implications for Huntingtons disease therapy, in which a joint impairment of BDNF and A2ARs seems to play a pathogenetic role.