Introduction

ATP is a multifactorial signaling molecule in the brain, involved in the communication between glia cells as well as in the bidirectional communication between glia and neurons (reviewed in [1]). Extracellular ATP is also produced upon synaptic activity in accordance with the accumulation of ATP in synaptic vesicles and its release with different neurotransmitters (e.g. [2, 3]), namely in the hippocampus [4,5,6], a brain region where synaptic plasticity processes are proposed to encode reference memory traits [7]. Although hippocampal synapses are endowed with different ATP-activated P2 receptors (e.g. [8]), the most evident role of extracellular ATP is to be a substrate for the action of ecto-nucleotidases, regulated by ecto-5’-nucleotidase or CD73 [9], to form extracellular adenosine to selectively activate adenosine A2A receptors (A2AR) [10,11,12]. A2AR are selectively engaged to control synaptic plasticity processes [13,14,15,16,17] and control memory and neurodegeneration (reviewed in [18]).

The adenosine neuromodulation system is a classical neuromodulation system, with a powerful inhibitory effect operated by A1R and a selective recruitment of A2AR to control synaptic plasticity [18], which involve a discrete facilitation of neurotransmitter release [19,20,21], the attenuation of the predominant A1R-mediated inhibition [19, 22, 23] and a post-synaptic facilitation of NMDA receptor-mediated responses [14, 24]. Since ATP release selectively occurs at high frequency simulation [4, 25] and selectively feeds A2AR to control synaptic plasticity processes, we now explored if A2AR control of ATP release from nerve terminals is a putative feedback loop involving CD73-mediated formation of adenosine from released ATP to activate A2AR-mediated facilitation of ATP release, as occurs for astrocytic ATP release [26]. Furthermore, we also aimed at understanding if ATP release is affected by the A1R-mediated inhibitory system, which robustly inhibits the release of classical neurotransmitters.

Methods

Animals

We used 32 male and female mice (20.8±0.2 g, 8-10 weeks old) from our inbred colony of forebrain A2A receptor knockout mice with a C57BL/6 genetic background [11] and wild type C57BL/6 obtained from Charles River (Barcelona, Spain). Mice were housed in collective cages with an enriched environment in HEPA-filtered ventilated racks (n=3-5 per cage) under a controlled environment (12 h light-dark cycle, lights on at 7 AM, and room temperature 22±1°C) with ad libitum access to food and water. The study was approved by the Ethical Committee of the Center for Neuroscience and Cell Biology (ORBEA n° 138-2016/15072016), following European Union guidelines (2010/63).

Preparation of synaptosomes

In spite of their artificial nature, difficulties in experimentally triggering neurotransmitter release with a ‘physiological’ pattern and their heterogeneity, synaptosomes are still the most adequate preparation to unambiguously ascribe mechanisms as occurring presynaptically [27]. Hippocampal synaptosomes (purified synapses) were prepared as previously described [28]. After deep anesthesia under halothane atmosphere, each mouse was decapitated, the two hippocampi were dissected and homogenized in sucrose (0.32 M) solution containing 1 mM EDTA, 10 mM HEPES, 1 mg/mL bovine serum albumin (Sigma), pH 7.4 at 4 °C, supplemented with a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF 0.1 mM), a cocktail of inhibitors of proteases (CLAP 1%, Sigma) and the antioxidant dithiothreitol (1 μM). The homogenate was centrifuged at 3,000 x g for 10 min at 4 °C and the resulting supernatant was further centrifuged at 14,000 x g for 12 min at 4 °C. The resulting pellet (P2 fraction) was resuspended in 1 mL of a 45% (v/v) Percoll solution in Krebs-HEPES buffer (140 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM EDTA, 10 mM glucose; pH 7.4). After centrifugation at 14,000 x g for 2 min at 4 °C, the white top layer was collected (synaptosomal fraction), resuspended in 1 mL Krebs-HEPES buffer and further centrifuged at 14,000 x g for 2 min at 4 °C. The pellet was then resuspended in Krebs-HEPES solution. The purity of this synaptic fraction has been previously quantified as >95% [28].

ATP release

The release of ATP was measured on-line using the luciferin-luciferase assay, as previously described [11]. Briefly, a suspension containing synaptosomes, an ATP assay mix (with luciferin and luciferase; from Sigma) and Krebs-HEPES solution was equilibrated at 25 °C during 10 min to ensure the functional recovery of nerve terminals. The suspension was then transferred to a white 96-well plate and measurements were performed in a luminometer (Victor3). After 60 seconds to measure basal ATP outflow, the evoked release of ATP was triggered with 32 mM of KCl (isomolar substitution of NaCl in the Krebs-HEPES solution), a well-established neurochemical strategy to trigger optimal signal-to-noise calcium-dependent vesicular release from synaptosomes without damage to these artificial synaptic structures [27]. The evoked release of ATP was calculated by integration of the area of the peak upon subtraction of the estimated basal ATP outflow [11].

Pharmacological manipulations

We used the A2AR agonist 2-[4-(2-p-carboxyethyl)phenylamino]-5’-N-ethylcarboxamidoadenosine (CGS21680, Tocris) in a selective concentration range (10-100 nM; [29]), the A2AR antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine (SCH58261; Tocris) at a supra-maximal but selective concentration (50 nM; [14]), the selective A1R agonist N6-cyclopentyladenosine (CPA, Tocris) in a selective concentration range (10-100 nM; [30]), the A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, Tocris) at a supra-maximal but selective concentration (100 nM [30]) and the ecto-5’-nucleotidase or CD73 inhibitor α,β-methylene ADP (AOPCP, Sigma-Aldrich) at a supra-maximal but selective concentration (100 μM [11]). AOPCP was directly prepared in Krebs solution, whereas all adenosine receptor ligands were prepared as 5 mM stock solutions in dimethylsulfoxide.

Statistics

The values are presented as mean±S.E.M. The percentage effect of drugs was calculated in each individual experiment and the S.E.M. is relative to the variance of this percentage effect. To test the significance of the effect of drugs versus control, a paired Student’s t test was used. When making comparisons from a different set of experiments with control, a one-way analysis of variance (ANOVA) was used, followed by a Dunnett’s test. P < 0.05 was considered to represent a significant difference.

Results

A2A receptors increase the evoked release of ATP

The K+-induced release of ATP from nerve terminals likely reflects a vesicular release of ATP [2, 5, 31, 32], as now confirmed by the dependency of this evoked ATP release on the presence of extracellular free calcium. Thus, the elevation of extracellular K+ to 30 mM to depolarize synaptosomes triggered a release of ATP (Fig. 1A, black line); the average K+-evoked ATP release was 21.9±2.3 pmol/mg protein (n=6). This K+-induced release of ATP was reduced by over 90% in a Krebs medium without added calcium (n=4; Fig. 1A, grey line).

Fig. 1
figure 1

Adenosine A2A receptors (A2AR) increase the evoked release of ATP that sustains the activation of A2AR through CD73-mediated formation of extracellular adenosine in hippocampal synaptosomes. (A) Representative recording of luminescence emitted by luciferase as a measure of extracellular ATP in hippocampal synaptosomes depolarized with addition of KCl (30 mM) in the presence (black) and absence of extracellular calcium (grey), showing that the evoked release of ATP is expected to be vesicular in nature. (B) The A2AR agonist CGS21680 (10-100 nM) enhanced the evoked release of ATP, whereas the A2AR antagonist SCH58261 (50 nM) decreased the evoked release of ATP and prevented any action of CGS21680. (C) The ecto-5’-nucleotidase (CD73) inhibitor α,β-methylene ADP (AOPCP, 100 μM) inhibited the evoked release of ATP and neither AOPCP nor A2AR ligands modified the evoked ATP release in hippocampal synaptosomes from A2AR knockout mice. Data are mean SEM of n=4-6 different mice in (B,C). *p<0.05 one-way Student’s t test vs. 0%; #p<0.05 two-tailed Student’s t test between genotypes.

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Since the activation of A2AR facilitates the release of different classical neurotransmitters from hippocampal nerve terminals [19, 21, 33], we now tested if A2AR activation also facilitated the release of ATP. The A2AR agonist CGS21680 increased the evoked release of ATP and this facilitation was larger (p=0.044) at 30 nM CGS21680 (40.1±8.0% facilitation, n=6) than at 10 nM (15.7±2.3%, n=4) and saturated at 100 nM (30.4±4.1%, n=5) (Fig. 1B). The selective A2AR antagonist SCH58261 (50 nM) decreased the evoked release of ATP by 41.7±4.5% (n=6), indicating that endogenous adenosine tonically activates A2AR to bolster ATP release (Fig. 1B). Furthermore, CGS21680 (30 nM) was devoid of effects (p=0.391; n=4) in the presence of 50 nM SCH58261 (Fig. 1B), further confirming the involvement of A2AR in the control of ATP release from hippocampal synaptosomes. Importantly, neither CGS21680 (10-100 nM) nor SCH58261 (50 nM) modified the basal outflow of ATP in the absence of K+-induced depolarization (data not shown).

CD73-mediated ATP-derived adenosine feeds A2A receptors to control ATP release

Since A2AR are selectively activated by CD73-mediated extracellular ATP-derived adenosine [10,11,12, 32, 34], we probed if CD73 was involved in a putative feedback facilitating loop of ATP release from nerve terminals, as previously observed for ATP release from astrocytes [26]. Thus, we tested the impact of the CD73 inhibitor AOPCP on the evoked release of ATP from hippocampal nerve terminals. As shown in Fig. 1C, AOPCP (100 μM) inhibited the K+-evoked release of ATP by 20.3±2.7% (n=6) in hippocampal synaptosomes from wild type mice, where AOPCP was devoid of effects (p=0.351; n=4) in hippocampal synaptosomes from A2AR knockout mice (Fig. 1C) and did not modify the basal outflow of ATP in the absence of K+-induced depolarization in wild type or A2AR knockout mice (data not shown). Moreover, the evoked release of ATP from hippocampal synaptosomes from A2AR knockout mice was not modified by either 30 nM CGS21680 (p=0.947; n=4) or 50 nM SCH58261 (p=0.653; n=4) (Fig. 1C), further reenforcing a putative feedback modulation role of A2AR in bolstering ATP release as a consequence of extracellular ATP-derived adenosine formation.

A2A receptors dampen A1 receptor-mediated inhibition of ATP release

The concluded robust effect of A2AR in the control of ATP release is somewhat surprising in view of the discrete impact of A2AR in the control of different classical neurotransmitters from hippocampal nerve terminals, such as glutamate [19], GABA [33] or acetylcholine [21]. Since A2AR control A1R-mediated effects in nerve terminals [19, 22, 23], we next investigated the impact of A1R on ATP release and the effect of A2AR on this putative A1R-mediated modulation of ATP release.

As shown in Fig. 2A, the selective A1R agonist CPA decreased the evoked release of ATP in a concentration-dependent manner, with inhibitions of 8.5±2.0% at 10 nM (n=4), 17.8±3.6% at 30 nM (n=4) and 26.1±4.4% at 100 nM (n=4). In the presence of the selective A1R antagonist DPCPX (100 nM), CPA (30 nM) was devoid of effects on the evoked release of ATP (p=0.077; n=4), confirming the involvement of A1R in the inhibitory effect of CPA on ATP release (Fig. 2A). Notably, DPCPX (100 nM) did not significantly modify the evoked release of ATP (p=0.622; n=4), indicating that endogenous adenosine does not tonically activate A1R to inhibit ATP release (Fig. 2A). Neither CPA (10-100 nM) nor DPCPX (100 nM) modified the basal outflow of ATP in the absence of K+-induced depolarization (data not shown).

Fig. 2
figure 2

Adenosine A2A receptors dampen the adenosine A1 receptor-mediated inhibition of the evoked release of ATP from hippocampal synaptosomes. (A) The A1R agonist CPA (10-100 nM) decreased the evoked release of ATP, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects as such but prevented any action of CPA. (B) The blockade of A2AR in the presence of SCH58261 (50 nM) amplified the inhibitory effect of CPA and revealed a facilitatory effect of DPCPX. Data are mean SEM of n=4 different mice. *p<0.05 one-way Student’s t test vs. 0%; #p<0.05 two-tailed Student’s t test vs. absence of DPCPX; $p<0.05 vs. absence of SCH58261.

Full size image

To test if A2AR controlled A1R-mediated inhibition of ATP release, we next tested the ability of A1R to modulate ATP release upon blockade of A2AR. In the presence of 50 nM SCH58261, CPA at lower concentrations triggered a more robust inhibition of the evoked release of ATP, which was 20.1±2.2% at 10 nM (n=4; p=0.008 vs. the effect of 10 nM CPA in the absence of SCH58261), 31.3±2.3% at 30 nM (n=4; p=0.020 vs. the effect of 30 nM CPA in the absence of SCH58261) and 36.4±7.7% at 100 nM (n=4; p=0.267 vs. the effect of 100 nM CPA in the absence of SCH58261) (Fig. 2B). This more robust effect of CPA in the presence of SCH58261 only involved A1R activation since CPA (30 nM) was devoid of effects in the presence of 100 nM DPCPX (p=0.622; n=4) (Fig. 2B). Importantly, in the presence of SCH58261, DPCPX (100 nM) increased the evoked release of ATP by 17.7±2.7% (n=4), indicating that A2AR are dampening the ability of A1R to inhibit ATP release from hippocampal synaptosomes (Fig. 2B).

Discussion

The present study shows that the release of ATP from nerve terminals is controlled in a dual and opposite manner by adenosine inhibitory A1 receptors (A1R) and facilitatory A2A receptors (A2AR). The release of ATP from nerve terminals was mainly controlled by A2AR, which activation caused a robust increase of ATP with an efficacy far superior to that controlling other classical neurotransmitters such as glutamate [19, 20, 35], GABA [33, 36] or acetylcholine [21, 27, 37, 38]. Moreover, A2AR blockade revealed a tonic activation of A2AR bolstering the release of ATP, which was not observed when studying the evoked release of classical neurotransmitter [38,39,40]. In contrast, whereas A1R activation triggers a robust inhibition of the release of classical neurotransmitters such as glutamate [19, 20, 41] and acetylcholine [28, 39, 42, 43], A1R agonists caused a comparatively lower inhibition of the evoked release of ATP. Furthermore, whereas there is a constant A1R tonic inhibition by endogenous extracellular adenosine of the evoked release of glutamate [15, 30, 44] or acetylcholine [28, 39, 42, 43], the A1R antagonist DPCPX was devoid of effects on the evoked release of ATP from hippocampal nerve terminals, in contrast to the reported A1R-mediated inhibition of ATP release from superior cervical ganglion [45] or in cultures enriched in cholinergic amacrine-like neurons [46]. This suggests a different relative organization of A1R and A2AR to control the presynaptic release of ATP and of classical neurotransmitters in different neuronal circuits (c.f. [15, 17, 47]).

A striking particularity of the modulation by adenosine of the evoked release of ATP from hippocampal nerve terminals is the control of A1R-mediated inhibition by A2AR. In fact, we observed that the blockade of A2AR augmented the ability of A1R to inhibit ATP release, which indicates that A2AR curtails A1R function. The mechanism underlying this ability of A2AR to control A1R function may either involve the eventual release of an intermediate soluble messenger or a direct interaction between A2AR-A1R heteromers [35]. Indeed, previous neurochemical studies showed that A2AR and A1R are located in the same individual hippocampal nerve terminal [48] and that A2AR activation decreases A1R binding in hippocampal synaptosomes [22, 23]. This translates into an ability of A2AR to shut down inhibitory A1R to allow the implementation of synaptic plasticity, which would otherwise be impeded by the over-activation of A1R upon increased extracellular purine release at higher frequencies of nerve stimulation [4, 25]. This ability of A2AR to control A1R is further illustrated by the dependency of A2AR-mediated facilitation of glutamatergic transmission on an on-going A1R-mediated inhibition, as observed in the hippocampus [19] or in the visual [49] or neocortex [50]. Importantly, although we now observed that A2AR curtailed A1R function, the ability of A2AR to enhance synaptic ATP release is not dependent on A1R since A2AR enhanced ATP release and A1R blockade was devoid of effects. Altogether, these findings indicate that the adenosine modulation of the evoked release of ATP from hippocampal nerve terminals seems to be different from the control of the evoked release of classical neurotransmitters such as glutamate or acetylcholine: thus, the release of ATP from hippocampal nerve terminals is predominantly controlled by A2AR rather than A1R, as also previously reported for the control of neuronal ATP release in the retina [51] and ATP currents in the habenula [52].

The presently reported different modulation by adenosine of the presynaptic release of ATP and of classical neurotransmitters joins previous observations of a different calcium channel dependence [46, 53], different requirements of intensity/frequency of stimulation [4, 25] and a temporal and pharmacological dissociation of the release of ATP from the release of classical neurotransmitters in different preparations [5, 54,55,56,57,58,59]. Given the observed calcium sensitivity of the presynaptic release of ATP and previous reports that this presynaptic ATP release is vesicular in nature [2, 5, 31, 32], it remains to be determined if the presynaptic release of ATP and of classical neurotransmitter occurs from different nerve terminals (see [41]) or from different vesicles within the same nerve terminal (see [5]), as hinted by the peculiar distribution of vesicular nucleoside transporters in different synaptic vesicles [6]. Furthermore, it cannot be excluded that part of the presynaptic release of ATP might be non-vesicular, given that there are several proposed mechanisms for ATP release in different preparations [60,61,62]. Clearly, the mechanism of ATP release from nerve terminals remains to be adequately characterized to better understand the mechanistic basis of the observed different modulation by adenosine of the release of ATP and of classical neurotransmitters.

The presently observed ability of A2AR to bolster ATP release and the conclusion that the activation of A2AR depends on CD73-mediated ATP-derived adenosine indicates the existence of a putative feedback facilitatory loop in synapses linking ATP release/CD73 activity/A2AR activation. This neuronal ATP release/CD73/A2AR activation loop is qualitatively similar to that present in astrocytes [26] but has a different physiological meaning. In fact, the astrocytic ATP release/CD73/A2AR activation loop is expected to sustain a paracrine ATPergic activation of the astrocytic network (reviewed in [63]) in parallel with an adenosinergic inhibition (A1R-mediated) of synaptic transmission [64, 65], which contributes to implement a process of heterosynaptic depression (see [66, 67]). In contrast, the neuronal ATP release/CD73/A2AR activation loop is proposed to be an autocrine adenosinergic potentiation (A2AR-mediated) of glutamate release restricted to the ‘activated’ synapse responsible for the A2AR-mediated control of synaptic plasticity [13,14,15,16,17, 24, 68], which is selectively dependent on CD73-mediated formation of ATP-derived extracellular adenosine [11, 12, 14, 16, 32]. These different conclusions should not be viewed as antagonic but rather complementary, contributing to the implementation of salience of information encoding [69] by bolstering the activity of an ‘activated’ synapse and simultaneously decreasing the activity of surrounding synapses. This illustrates the numerous intertwined roles of the purinergic modulation system in different brain compartments [1], which further stresses the need to study the spatiotemporal gradients of extracellular ATP and adenosine in relation to the different adenosine receptors to better grasp the physiopathological roles of adenosine. This complexity is further increased by the need to recognize that apart being a substrate to ecto-nucleotidases generating adenosine, extracellular ATP also exerts direct roles as a neurotransmitter and neuromodulator [70, 71] through numerous post-synaptic and presynaptic P2X and P2Y receptors [8, 72], as championed by the group of María Teresa Miras-Portugal (e.g. [73, 74]). Importantly, it should be kept in mind that this proposed feedback facilitatory loop linking A2AR activation and an increased ATP release to sustain A2AR activation was so far only documented in purified synaptosomes and it remains to be confirmed if a similar mechanism is present in more integrated brain preparations, namely in an in vivo situation. Furthermore, future studies should investigate the possible contribution of this proposed feedback facilitatory loop to the synaptic dysfunction characteristic of different neuropsychiatric diseases, in view of the previously reported up-regulation of synaptic A2AR in numerous brain diseases (e.g. [11, 12, 18, 24, 68], as well as the established role of released ATP as a danger signal in the brain [71].