Luísa V. Lopes

Adenosine A2A receptor facilitation of hippocampal synaptic transmission is dependent on tonic A1 receptor inhibition

Abstract Adenosine tonically inhibits synaptic transmission through actions at A 1 receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A 2A receptor activation or from indirect control of inhibitory GABAergic transmission. The A 2A receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA A and GABA B receptor antagonists, picrotoxin (50 μM) and CGP 55845 (1 μM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A 2A receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A 1 receptors with N 6 -cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K + -evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 μM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 μM) and bisindolylmaleimide (1 μM), but not by the protein kinase A inhibitor, H-89 (1 μM). Isoproterenol (30 μM), but not CGS 21680 (10–300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A 1 receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([ 3 H]DPCPX, 0.5 nM) with an EC 50 of 1 μM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A 1 receptors. Our results suggest that A 2A receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A 1 receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A 2A receptors to allow frequency-dependent plasticity phenomena without compromising the A 1 receptor-mediated neuroprotective role of adenosine.

Decrease of adenosine A1 receptor density and of adenosine neuromodulation in the hippocampus of kindled rats.

Adenosine is a neuromodulator that has been proposed to be a major endogenous anticonvulsant acting via A1 receptors. We tested if implementation of kindling through stimulation of the amygdala affected A1 receptor‐mediated neuromodulation in hippocampal slices taken from rats 4 weeks after the last stage 5 seizure. The A1 receptor agonist, N6‐cyclopentyladenosine (CPA) (6–100 nm), inhibited field excitatory postsynaptic potential (fEPSP) slope with an EC50 of 19.1–19.5 nm in control and sham‐operated rats, but was less potent in kindled rats (EC50 = 42.7 nm). This might result from a decreased number of A1 receptors in hippocampal nerve terminal membranes, because A1 receptor immunoreactivity decreased by 28 ± 3% and the binding density of the A1 receptor agonist [3H]R‐PIA decreased from 1702 ± 64 to 962 ± 78 fmol/mg protein in kindled compared with control rats. The tonic inhibition of hippocampal synaptic transmission by endogenous adenosine was also lower in kindled rats, because A1 receptor blockade with 50 nm 1,3‐dipropyl‐8‐cyclopentyladenosine (DPCPX) enhanced fEPSP slope by 23 ± 3% and θ‐burst‐induced long‐term potentiation by 94 ± 4% in control rats but was virtually devoid of effects in kindled rats. The evoked release of adenosine from hippocampal slices or nerve terminals was 56–71% lower in kindled rats probably due to the combined decrease in the capacity of adenosine transporters and decreased release of adenosine 5′‐triphosphate (ATP), which was partially compensated by a higher extracellular catabolism of ATP into adenosine in kindled rats. These results indicate that, although adenosine might inhibit the onset of epileptogenesis, once kindling is installed, the efficiency of the adenosine inhibitory system is impaired.

Extracellular Alpha-Synuclein Oligomers Modulate Synaptic Transmission and Impair LTP Via NMDA-Receptor Activation

Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

Enhancement of LTP in Aged Rats is Dependent on Endogenous BDNF

Long-term potentiation (LTP), considered the neurophysiological basis for learning and memory, is facilitated by brain-derived neurotrophic factor (BDNF), an action more evident when LTP is evoked by weak θ-burst stimuli and dependent on co-activation of adenosine A2A receptors (A2AR), which are more expressed in aged rats. As θ-burst stimuli also favor LTP in aged animals, we hypothesized that enhanced LTP in aging could be related to changes in neuromodulation by BDNF. The magnitude of CA1 LTP induced by a weak θ-burst stimuli delivered to the Schaffer collaterals was significantly higher in hippocampal slices taken from 36 to 38 and from 70 to 80-week-old rats, when compared with LTP magnitude in slices from 4 or 10 to 15-week-old rats; this enhancement does not impact in cognitive improvement as aged rats revealed an impairment on hippocampal-dependent learning and memory performance, as assessed by the Morris water maze tests. The scavenger for BDNF, TrkB-Fc, and the inhibitor of Trk phosphorylation, K252a, attenuated LTP in slices from 70 to 80-week-old rats, but not from 10 to 15-week-old rats. When exogenously added, BDNF significantly increased LTP in slices from 4 and 10 to 15-week-old rats, but did not further increased LTP in 36 to 38 or 70 to 80-week-old rats. The effects of exogenous BDNF on LTP were prevented by the A2AR antagonist, SCH58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine). These results indicate that the higher LTP magnitude observed upon aging, which does not translate into improved spatial memory performance, is a consequence of an increase in the tonic action of endogenous BDNF.

Long-term Effect of Convulsive Behavior on the Density of Adenosine A1 and A2A Receptors in the Rat Cerebral Cortex

Summary:  Purpose: Adenosine is a neuromodulator that has been proposed to act as an anticonvulsant mainly via inhibitory A1 receptors, but recent data show that genetic deletion of facilitatory A2A receptors might also attenuate convulsions. Since both A1 and A2A receptors are prone to down‐ and upregulation in different stressful situations, we investigated if convulsive behavior leads to a long‐term change in A1 and A2A receptor density in the rat cerebral cortex.

Increase in the Number, G Protein Coupling, and Efficiency of Facilitatory Adenosine A2A Receptors in the Limbic Cortex, but not Striatum, of Aged Rats

Abstract : Adenosines effects result from a balanced activation of inhibitory A1 and facilitatory A2A receptors. Because in aged animals there is an increased number of A2A receptors, we now compared the efficiency of A2A receptors in cortical and striatal preparations of young adult (6‐week‐old) and aged (2‐year‐old) rats. In cortical, in contrast to striatal, membranes from aged rats, A2A receptors were more tightly coupled to G proteins, because 5′‐guanylylimidodiphosphate (100 μM) increased by 321% the Ki of the A2A agonist CGS21680 as a displacer of binding of the A2A antagonist [3H]ZM241385 (1 nM), compared with a 112% increase in young rats. In cortical slices, CGS21680 (30‐1,000 nM) was virtually devoid of effect on cyclic AMP accumulation in young rats but increased cyclic AMP accumulation with an EC50 of 153 nM in aged rats, whereas the efficiency of CGS21680 was similar in striatal slices of young and aged rats. CGS21680 (30 nM) was virtually devoid of effect on acetylcholine release from hippocampal CA1 slices of young rats but caused a 55% facilitation in aged rats. These results show that the number of A2A receptors, their coupling to G proteins, and their efficiency are enhanced in the limbic cortex of aged rats, suggesting a greater involvement of facilitation in adenosine responses.

Binding of the prototypical adenosine A2A receptor agonist CGS 21680 to the cerebral cortex of adenosine A1 and A2A receptor knockout mice

2‐p‐(2‐carboxyethylphenethylamino‐5′‐ethylcarboxamidoadenosine) (CGS 21680) is considered the reference compound to study adenosine A2A receptors. However, CGS 21680 binding in the cerebral cortex, where adenosine A1 receptors are predominant, displays a mixed A2A/A1 receptor pharmacology. We now use adenosine A1 and A2A receptor knockout mice to investigate the characteristics of cortical [3H]CGS 21680 binding. [3H]CGS 21680 binding to the cerebral cortex was strongly reduced in adenosine A1 receptor knockout mice, but only slightly reduced in A2A receptor knockout mice compared with the corresponding wild‐type littermates. Another selective A2A receptor ligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine ([3H]SCH 58261), displayed a saturable binding to mouse cortical membranes, albeit with a binding density 20 times lower than that of striatal membranes, and this [3H]SCH58261 binding was abolished in both striatal and cortical membranes of A2A receptor knockout mice and unchanged in A1 receptor knockout mice. The presence of A2A receptors in cortical neurons was further confirmed by Western blot in mouse cortical nerve terminal membranes. It is concluded that, although A2A receptors are present in the cerebral cortex, the purportedly selective A2A receptor agonist [3H]CGS 21680 binds in the cerebral cortex to an entity that requires the presence of adenosine A1 receptors. Thus, CGS 21680 should be used with care in all preparations where adenosine A1 receptors out‐number A2A receptors.

A2A adenosine receptor deletion is protective in a mouse model of Tauopathy

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer’s disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.

Adenosine A 2A receptor blockade reverts hippocampal stress-induced deficits and restores corticosterone circadian oscillation

Maternal separation (MS) is an early life stress model that induces permanent changes in the central nervous system, impairing hippocampal long-term potentiation (LTP) and spatial working memory. There are compelling evidences for a role of hippocampal adenosine A2A receptors in stress-induced modifications related to cognition, thus opening a potential window for therapeutic intervention. Here, we submitted rats to MS and evaluated the long-lasting molecular, electrophysiological and behavioral impairments in adulthood. We then assessed the therapeutic potential of KW6002, a blocker of A2A receptors, in stress-impaired animals. We report that the blockade of A2A receptors was efficient in reverting the behavior, electrophysiological and morphological impairments induced by MS. In addition, this effect is associated with restoration of the hypothalamic-pituitary-adrenal axis (HPA-axis) activity, as both the plasma corticosterone levels and hippocampal glucocorticoid receptor expression pattern returned to physiological-like status after the treatment. These results reveal the involvement of A2A receptors in the stress-associated impairments and directly in the stress response system by showing that the dysfunction of the HPA-axis as well as the long-lasting synaptic and behavioral effects of MS can be reverted by targeting adenosine A2A receptors. These findings provide a novel evidence for the use of adenosine A2A receptor antagonists as potential therapy against psychopathologies.

Adenosine and Related Drugs in Brain Diseases: Present and Future in Clinical Trials

Adenosine is a naturally occurring nucleoside present ubiquitously throughout the body as a metabolic intermediate. Besides its metabolic role within the cells, adenosine is released into the extracellular space either by neurons or astrocytes acting as a neuromodulator. Extracellular adenosine exerts its action by activating multiple G-protein coupled receptors (subtypes A(1), A(2A), A(2B) and A(3)) having a wide range of physiological effects in the brain. Adenosine levels rise markedly in response to ischemia, hypoxia, excitotoxicity or inflammation being a neuroprotectant under these conditions. However, adenosine may also contribute to neuronal damage and cell death in other circumstances. These actions are firmly established using multiple animal models. Therefore, increasing attention is now given to the role of adenosine in human brain function and its potential benefit for clinical applications. This review covers recent studies undertaken mostly in humans revealing the actions of adenosine and related drugs in cognition and memory as well as in various pathological situations such as psychiatric disorders, drug addiction and neurodegenerative disorders. The actual use of adenosine or adenosine receptor ligands in ongoing clinical trials for the treatment of schizophrenia, panic disorder and anxiety, cocaine dependence and Parkinsons disease is discussed. The evidence herein reviewed highlights the promising potential of adenosine or adenosine receptor ligands as therapeutic agents in several brain disorders.