Instituto de Farmacologia e Neurociências (FM-IFN)
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- Activation of adenosine A2A receptor facilitates brain-derived neurotrophic factor modulation of synapticPublication . Diógenes, Maria José; Fernandes, Catarina Cunha; Sebastião, Ana M; Ribeiro, Joaquim A.Both brain-derived neurotrophic factor (BDNF) and adenosine influence neuronal plasticity. We now investigated how adenosine influences the action of BDNF on synaptic transmission in the CA1 area of the rat hippocampal slices. Alone, BDNF (20 –100 ng/ml) did not significantly affect field EPSPs (fEPSPs). However, a 2 min pulse of high-K+ (10 mM) 46 min before the application of BDNF (20 ng/ml)triggered an excitatory action, an effect blocked by the TrkB receptor inhibitor K252a (200 nM), by the adenosine A2A receptor antagonist ZM241385 (50 nM), and by the protein kinaseAinhibitor H-89 (1µM). Presynaptic, rather than postsynaptic depolarization was required to trigger the BDNF action because after K+ depolarization BDNF also increased EPSCs recorded from pyramidal neurons voltageclamped at -60 mV, and transient postsynaptic depolarization was unable to unmask the BDNF action. A weak theta burst stimulation of the afferents could elicit potentiation of synaptic transmission only when applied in the presence of BDNF. Activation of adenosineA2A receptors with CGS 21680 (10 nM), or the increase in extracellular adenosine levels induced by 5-iodotubercidin (100 nM) triggered the excitatory action of BDNF, a process prevented by ZM 241385 and by H-89. In the presence of dibutyryl-cAMP (0.5 mM), BDNF also increased fEPSPs but postsynaptic cAMP (0.5mM) was unable to trigger the BDNF action. It is concluded that presynaptic activity-dependent release of adenosine, through activation of A2A receptors, facilitates BDNF modulation of synaptic transmission at hippocampal synapses.
- Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts : implications for neuromodulationPublication . Assaife-Lopes, Natália; Sousa, Vasco C.; Pereira, Daniela B.; Ribeiro, Joaquim A.; Chao, Moses V.; Sebastião, Ana MBrain-derived neurotrophic factor (BDNF) signaling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been hown to be necessary for the activation of specific signaling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A2A receptor activation, we hypothesized that activation of A2A receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A2A receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansylcadaverine(100µM) did not modify the effects of theA2A receptor agonists but significantly impairedBDNFeffects on TrkB recruitment to lipid rafts. The effect of A2A receptor activation in TrkB localization was mimicked by 5 µM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14 –22), and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts induced by activation of adenosine A2A receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.
- Adenosine : does it have a neuroprotective role after allPublication . De Mendonça, Alexandre; Sebastião, Ana M; Ribeiro, Joaquim A.A neuroprotective role for adenosine is commonly assumed. Recent studies revealed that adenosine may unexpectedly, under certain circumstances, have the opposite effects contributing to neuronal damage and death. The basis for this duality may be the activation of distinct subtypes of adenosine receptors, interactions between these receptors, differential actions on neuronal and glial cells, and various time frames of adenosinergic compounds administration. If these aspects are understood, adenosine should remain an interesting target for therapeutical neuroprotective approaches after all.
- Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomesPublication . Vaz, Sandra H.; Lerias, Sofia; Parreira, Sara; Diógenes, Maria José; Sebastião, Ana MAdenosine, through A(2A) receptor (A(2A)R) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A(2A)R on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K(+)-evoked [(3)H]glutamate release and inhibited the K(+)-evoked [(3)H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A(2A)R since for both neurotransmitters, the BDNF action was blocked by the A(2A)R antagonist SCH 58261 (50 nM). In the presence of the A(2A)R agonist, CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A(2A)R by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A(2A)R.
- Adenosine A2A receptor interactions with receptors for other neurotransmitters and neuromodulatorsPublication . Ribeiro, J. AlexandreAdenosine, by activating adenosine A2A receptors, seems to have a crucial function in regulating the activation of multiple receptors that affect neurotransmitter release and/or synaptic transmission, in particular receptors for neuropeptides (calcitonin gene related peptide (CGRP) and vasoactive intestinal peptide (VIP)), and NMDA receptors, metabotropic glutamate receptors, nicotinic autofacilitatory receptors, dopamine receptors and adenosine A1 receptors. The manner in which these A2A receptors are involved in interactions with the receptors for other neurotransmitters and or neuromodulators opens novel avenues for the action of this `omnipresent' nucleoside. Either by direct receptor–receptor modulation or by post-receptor mechanisms, adenosine, in its `obsession' to protect cells from insults, uses as many receptor systems as possible to synchronize synaptic transmission, in order to exert what seems to be the `destiny' of this nucleoside—protection of the nervous system.
- Adenosine A2A receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving micePublication . Fontinha, Bruno M; Delgado-García, José M; Madroñal, Noelia; Ribeiro, Joaquim A; Sebastião, Ana M; Gruart, AgnèsPrevious in vitro studies have characterized the electrophysiological and molecular signaling pathways of adenosine tonic modulation on long-lasting synaptic plasticity events, particularly for hippocampal long-term potentiation(LTP). However, it remains to be elucidated whether the long-term changes produced by endogenous adenosine in the efficiency of synapses are related to those required for learning and memory formation. Our goal was to understand how endogenous activation of adenosine excitatory A2A receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A2A receptor antagonist, SCH58261, upon a well-known associative learning paradigm - classical eyeblink conditioning. We used a trace paradigm, with a tone as the conditioned stimulus (CS) and an electric shock presented to the supraorbital nerve as the unconditioned stimulus(US). A single electrical pulse was presented to the Schaffer collateral–commissural pathway to evoke field EPSPs (fEPSPs) in the pyramidal CA1 area during the CS–US interval. In vehicle-injected animals, there was a progressive increase in the percentage of conditioning responses (CRs) and in the slope of fEPSPs through conditioning sessions, an effect that was completely prevented (and lost) in SCH58261 (0.5 mg/kg, i.p.)-injected animals. Moreover, experimentally evoked LTP was impaired in SCH58261- injected mice. In conclusion, the endogenous activation of adenosine A2A receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.
- Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampusPublication . Duarte, António Pinto; Coelho, Joana E.; Cunha, Rodrigo A.; Ribeiro, Joaquim Alexandre; Sebastião, Ana MAdenosine, a neuromodulator of the CNS, activates inhibitory- A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nM), facilitated adenosine uptake through a PKCdependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nM) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nM), facilitated the evoked release of [3H]acetylcholine under lowfrequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2)either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nM) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.
- Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1Publication . Cristóvão-Ferreira, Sofia; Vaz, Sandra H.; Ribeiro, Joaquim A.; Sebastião, Ana MNeurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)- 9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of KM. This was mimicked by adenylate cyclase activation (forskolin, 10 lM) and prevented by protein kinase A (PKA) inhibition (N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 lM), which per se did not influenceGABAtransport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol- 3-yl]-3-(indol-3-yl) maleimide, 1 lM) facilitated GABA transport whereas PKC activation (4-b-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.
- Adenosine A2A receptors facilitate synaptic NMDA currents in CA1 pyramidal neuronsPublication . Mouro, Francisco; Rombo, Diogo M.; Dias, Raquel Baptista; Ribeiro, Joaquim A.; Sebastião, Ana MBackground and purpose: NMDA receptors play a key role in both synaptic plasticity and neurodegeneration. Adenosine is an endogenous neuromodulator and through membrane receptors of the A2A subtype can influence both synaptic plasticity and neuronal death. The present work was designed to evaluate the influence of adenosine A2A receptors upon NMDA receptor activity in CA1 hippocampal neurons. We discriminated between modulation of synaptic versus extrasynaptic receptors, since extrasynaptic NMDA receptors are mostly associated with neurodegeneration while synaptic NMDA receptors are linked to plasticity phenomena. Experimental approach: Whole-cell patch-clamp recordings were obtained to evaluate NMDA receptor actions on CA1 pyramidal neurons of young adult (5-10 weeks) male Wistar rat hippocampus. Key results: Activation of A2A receptors with CGS 21680 (30 nM) consistently facilitated chemically-evoked NMDA receptor-currents (NMDA-PSCs) and afferent-evoked NMDA-currents (NMDA-EPSCs), an action prevented by an A2A receptor antagonist (SCH58261, 100 nM) and a PKA inhibitor, H-89 (1 μM). These actions did not reflect facilitation in glutamate release since there was no change in NMDA-EPSCs paired pulse ratio. A2A receptor actions were lost in the presence of an open-channel NMDA receptor blocker, MK-801 (10 μM), but persisted in the presence of memantine, at a concentration (10 μM) known to preferentially block extrasynaptic NMDA receptors. Conclusion and implications: These results show that A2A receptors exert a positive postsynaptic modulatory effect over synaptic, but not extrasynaptic, NMDA receptors in CA1 neurons and, therefore, under non-pathological conditions may contribute to shift the dual role of NMDA receptors towards enhancement of synaptic plasticity.
- Adenosine A2A receptors in neuronal outgrowth: a target for nerve regeneration?Publication . Sebastião, Ana M; Ribeiro, FilipaAxonal and dendritic outgrowth are fundamental processes in the development of the nervous system. During this period, neurons change their morphology from a simple bipolar shape into a mature complex shape. Neurons develop dendrites and extend long or short axons that travel through a complex path until reaching target cells and form functional and accurate neuronal circuits.
