Nelson Rebola

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2A Receptor Heteromers

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R–A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R–A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R–A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R–A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses.

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Co‐localization and functional interaction between adenosine A2A and metabotropic group 5 receptors in glutamatergic nerve terminals of the rat striatum

The anti‐Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A2A receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinsons disease. mGluR5 and A2A antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the anti‐Parkinsonian and neuroprotective effects of A2A and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A2A agonist, CGS21680 (1–30 nm) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A2A antagonist, SCH58261 (50 nm). The mGluR5 agonist, CHPG (300–600 μm) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 μm). Both mGluR5 and A2A receptors were located in the active zone and 57 ± 6% of striatal glutamatergic nerve terminals possessed both A2A and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nm) and CHPG (100 μm) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nm CGS21680 was prevented by 10 μm MPEP, whereas facilitation by 300 μm CHPG was prevented by 10 nm SCH58261. These results provide the first direct evidence that A2A and mGluR5 receptors are co‐located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinsons disease.

Different synaptic and subsynaptic localization of adenosine A2A receptors in the hippocampus and striatum of the rat

Adenosine A(2A) receptors are most abundant in the striatum where they control the striatopallidal pathway thus controlling locomotion. Extra-striatal A(2A) receptors are considerably less abundant but their blockade confers robust neuroprotection. We now have investigated if striatal and extra-striatal A(2A) receptors have a different neuronal location to understand their different functions. The binding density of the A(2A) antagonist, [(3)H]-7-(2-phenylethyl)-5-amino-2-(2-furyl)pyrazolo[4,3e][1,2,4]triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), was enriched in nerve terminals membranes (B(max)=103+/-12 fmol/mg protein) compared with total membranes (B(max)=29+/-4 fmol/mg protein) from the hippocampus, the same occurring with A(2A) receptor immunoreactivity. In contrast, there was no enrichment of [(3)H]SCH 58261 binding or A(2A) receptor immunoreactivity in synaptosomal compared with total membranes from the striatum. Further subcellular fractionation of hippocampal nerve terminals revealed that A(2A) receptor immunoreactivity was enriched in the active zone of presynaptic nerve terminals, whereas it was predominantly located in the postsynaptic density in the striatum, although a minority of striatal A(2A) receptors were located in the presynaptic active zone. These results indicate that A(2A) receptors in the striatum are not enriched in synapses in agreement with the preponderant role of A(2A) receptors in signal processing in striatopallidal neurons. In contrast, hippocampal A(2A) receptors are enriched in synapses, mainly in the active zone, in accordance with their role in controlling neurotransmitter release. This regional variation in the neuronal distribution of A(2A) receptors reinforces the care required to extrapolate our knowledge from striatal A(2A) receptors to other brain preparations.

Adenosine A2A receptor antagonists exert motor and neuroprotective effects by distinct cellular mechanisms

To investigate whether the motor and neuroprotective effects of adenosine A2A receptor (A2AR) antagonists are mediated by distinct cell types in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of Parkinsons disease.

Activity-dependent synaptic plasticity of NMDA receptors

Activity‐dependent, bidirectional control of synaptic efficacy is thought to contribute to many forms of experience‐dependent plasticity, including learning and memory. Although most excitatory synapses contain both AMPA and N‐methyl‐d‐aspartate receptors (AMPARs and NMDARs), most studies have focused on the plasticity of synaptic AMPARs, and on the pivotal role of NMDA receptors for its induction. Here we review evidence that synaptic NMDARs themselves are subject to long‐term activity‐dependent changes by mechanisms that may differ from that of synaptic AMPARs. The bidirectional modulation of NMDAR‐mediated synaptic responses is likely to have important functional implications for NMDAR‐dependent forms of synaptic plasticity.

Subcellular localization of adenosine A1 receptors in nerve terminals and synapses of the rat hippocampus

Adenosine is a neuromodulator in the CNS that mainly acts through pre- and postsynaptic A(1) receptors to inhibit the release of excitatory neurotransmitters and NMDA receptor function. This might result from a highly localized distribution of A(1) receptors in the active zone and postsynaptic density of CNS synapses that we now investigated in the rat hippocampus. The binding density of the selective A(1) receptor antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX), was enriched in membranes from Percoll-purified nerve terminals (B(max)=1839+/-52 fM/mg protein) compared to total membranes from the hippocampus (B(max)=984+/-31 fM/mg protein), the same occurring with A(1) receptor immunoreactivity. [3H]DPCPX binding occurred mainly to the plasma membrane rather than to intracellular sites, since the binding of the membrane permeable A(1) receptor ligand [3H]DPCPX to intact hippocampal nerve terminals (B(max)=1901+/-192 fM/mg protein) was markedly reduced (B(max)=321+/-30 fM/mg protein) by the membrane impermeable adenosine receptor antagonist, 8-sulfophenyltheophilline (25 microM). Further subcellular fractionation of hippocampal nerve terminals revealed that A(1) receptor immunoreactivity was strategically located in the active zone of presynaptic nerve terminals, as expected to understand the efficiency of A(1) receptors to depress neurotransmitter release. A(1) Receptors were also present in nerve terminals outside the active zone in accordance with the existence of a presynaptic A(1) receptor reserve. Finally, A(1) receptor immunoreactivity was evident in the postsynaptic density together with NMDA receptor subunits 1, 2A and 2B and with N-and P/Q-type calcium channel immunoreactivity, emphasizing the importance of A(1) receptors in the control of dendritic integration.

A Critical Role of the Adenosine A2A Receptor in Extrastriatal Neurons in Modulating Psychomotor Activity as Revealed by Opposite Phenotypes of Striatum and Forebrain A2A Receptor Knock-Outs

The function of striatal adenosine A2A receptors (A2ARs) is well recognized because of their high expression levels and the documented antagonistic interaction between A2ARs and dopamine D2 receptors in the striatum. However, the role of extrastriatal A2ARs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A2ARs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A2ARs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A2AR knock-out (st-A2AR KO) mice in comparison with forebrain-specific A2AR KO (fb-A2AR KO) mice. In contrast to fb-A2AR KO (deleting A2ARs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A2AR KO mice exhibited Cre-mediated selective deletion of the A2AR gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A2AR KO but attenuated in fb-A2AR KO mice. Furthermore, selective inactivation of the A2ARs in extrastriatal cells by administering the A2AR antagonist KW6002 into st-A2AR KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A2ARs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A2ARs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A2ARs.

Adenosine A1 and A2A receptors are co-expressed in pyramidal neurons and co-localized in glutamatergic nerve terminals of the rat hippocampus

Adenosine is a neuromodulator that controls neurotransmitter release through inhibitory A1 and facilitatory A2A receptors. Although both adenosine receptor-mediated inhibition and facilitation of glutamate release have been observed, it is not clear whether both A1 and A2A receptors are located in the same glutamatergic nerve terminal or whether they are located on different populations of these terminals. Thus, we have tested if single pyramidal glutamatergic neurons from the hippocampus simultaneously expressed A1 and A2A receptor mRNA and if A1 and A2A receptors were co-localized in hippocampal glutamatergic nerve terminals. Single cell PCR analysis of visually identified pyramidal neurons revealed the simultaneous presence of A1 and A2A receptor mRNA in four out 16 pyramidal cells possessing glutamatergic markers but not GABAergic or astrocytic markers. Also, A1 and A2A receptor immunoreactivities were co-localized in 26 +/- 4% of nerve terminals labeled with antibodies against vesicular glutamate transporters type 1 or 2, i.e. glutamatergic nerve terminals. This indicates that glutamatergic neurons in the hippocampus co-express A1 and A2A receptors and that these two receptors are co-localized in a subset of glutamatergic nerve terminals.

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.