European Journal of Neuroscience, pp. 1–11, 2015

doi:10.1111/ejn.12851

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus Francisco Q. Goncßalves,1,* Johny Pires,1,* Anna Pliassova,1,* Rui Beleza,1,* Cristina Lemos,1 Joana M. Marques,1 €falvi,1 Rodrigo A. Cunha1,2 and Daniel Rial1 Ricardo J. Rodrigues,1 Paula M. Canas,1 Attila Ko 1 2

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Keywords: A1 receptors, A2B receptor, Adenosine, hippocampus, nerve terminal, receptor interaction

Abstract Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A2BR) are Gs/q-protein-coupled receptors with low expression in the brain. As A2BR function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A2BR-knockout (A2BR-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A2BR agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A2BR antagonist MRS 1754 (200 nM) and abrogated in A2BR-KO mice. Accordingly, A2BR immunoreactivity was present in 73  5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A1R control of synaptic transmission, both the A1R inhibition caused by 2-chloroadenosine (0.1–1 lM) and the disinhibition caused by the A1R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A2BR-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A1R inhibition (CPA 100 nM). A2BR-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A2BR are present in hippocampal glutamatergic terminals where they counteract the predominant A1R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Introduction Adenosine is a neuromodulator acting through four types of membrane-bound G-protein-coupled adenosine receptors, named A1, A2A, A2B and A3 receptors (Fredholm et al., 2005). The relevance of adenosine in brain synapses is testified by the impact of the adenosine system in responses such as mood, motor control or learning and memory (Gomes et al., 2011); these central effects of adenosine mostly involve A1 and A2A receptors (Fredholm et al., 2005), where A1 receptors play a prominent role in the control of emotional stability (Gimenez-Llort et al., 2002; Lang et al., 2003) and A2A receptors control locomotion and cognition (Cunha & Agostinho, 2010; Shen et al., 2013). The other adenosine receptors, namely adenosine A2B receptors (A2BR), have low expression levels in the brain (Dixon et al., 1996). A2BR are recognised as Gs/q-protein-coupled receptors

Correspondence: Daniel Rial, as above. E-mail: [email protected] *These authors contributed equally to this work. Received 13 October 2014, revised 29 December 2014, accepted 14 January 2015

(Feoktistov & Biaggioni, 1997), although they can interact with different G-protein-coupled receptors to recruit other G-proteins (Ryzhov et al., 2006; Cohen et al., 2010; Liu et al., 2014). Our knowledge about A2BR mostly stems from their peripheral roles controlling cardiac myocite contractility, intestinal tone, asthma, inflammation, cancer and diabetes (Feoktistov & Biaggioni, 1997; Headrick et al., 2013). Most studies on A2BR in the brain have focused on pathological conditions (Trincavelli et al., 2004; Moidunny et al., 2012) and on glial cells (Fredholm & Altiok, 1994; Fiebich et al., 1996; Jimenez et al., 1999; Allaman et al., 2011). This is fuelled by the observation that A2BR display a lower affinity for adenosine than the other adenosine receptors (Fredholm et al., 2001), suggesting that A2BR may be preferentially activated under noxious conditions associated with higher extracellular levels of adenosine (Dunwiddie & Masino, 2001; Popoli & Pepponi, 2012). However, some studies suggest that A2BR might also control neurotransmitter release through interaction with other receptors (Moriyama & Sitkovsky, 2010; Garcß~ao et al., 2013). The parallel advent of new pharmacological ligands with high selectivity and potency for A2BR, together with the availability of A2BRknockout (KO) mice, provides ideal conditions for re-evaluating

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

2 F. Q. Goncßalves et al. whether A2BR control information processing in neuronal networks and impact on behavioural performance. Thus, we now used electrophysiological, neurochemical and behavioural measurements to probe the role of A2BR, focusing on the hippocampus where the presence of A2BR has previously been documented in rodents (Dixon et al., 1996; Zhou et al., 2004) and humans (Perez-Buira et al., 2007).

Material and methods Animals Male C57/BL6 mice (10–12 weeks old) were obtained from Charles River (Barcelona, Spain) and A2BR-KO mice (Belikoff et al., 2011) were kindly donated by Drs Akio Ohta and Michael Sitkovsky (New England Inflammation and Tissue Protection Institute, Northeastern University, Boston, MA, USA). Mice were housed under controlled temperature (23  2 °C), subject to a fixed 12-h light/ dark cycle, with free access to food and water. All studies were approved by the Ethics Committee of the Center for Neuroscience and Cell Biology of Coimbra (ORBEA-78/2013) and conducted according to the European Union guidelines (86/609/EEC). In particular, the mice were deeply anaesthetised with 2-bromo-2-chloro1,1,1-trifluoroethane (halothane; no reaction to handling or tail pinch, while still breathing) before decapitation. Drugs 2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide (BAY 60-6583), N-(4-cyanophenyl)-2-[4-(2,3,6, 7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-ylphenoxy]-acetamide (MRS 1754) and (2R,3R,4S,5R)-2-[6-(cyclopentylamino)purin-9-yl]5-(hydroxymethyl)oxolane-3,4-diol (CPA) were from Tocris Biosciences (Bristol, UK) and 2-chloroadenosine and 8-cyclopentyl-1,3dipropylxanthine (DPCPX) were from Sigma (St Louis, MO, USA). MRS 1754, BAY 60-6583 and DPCPX were made up into 5-mM stocks in dimethylsulfoxide and dissolved in Krebs solution to the desired concentration, with adequate controls for the possible influence of dimethylsulfoxide. All drugs at electrophysiology experiments were applied for a period of at least 20 min before subsequent addition of a new drug. Electrophysiological recordings The experiments were carried out as previously described (Lopes et al., 2002). Mice were decapitated after halothane anesthesia, and the hippocampus was dissected in an ice-cold aCSF solution (in mM: NaCl, 124; KCl, 3; NaH2PO4, 1.25; glucose, 10; NaHCO3 26; MgSO4, 1; and CaCl2, 2) gassed with 95% O2 and 5% CO2. Slices (400 lm) were prepared with a McIlwain chopper and allowed to recover for 30 min at 35 °C and for 30 min at room temperature in a Harvard Apparatus resting chamber with gassed aCSF. Individual slices were transferred to a submersion recording chamber (1 mL capacity) and continuously superfused at a rate of 3 mL/min with gassed aCSF kept at 30.5 °C. A bipolar concentric electrode was placed on the Schaffer collateral–commissural pathway and stimulated every 20 s with rectangular pulses of 0.1 ms. The orthodromically-evoked field excitatory postsynaptic potentials (fEPSPs) were recorded through an extracellular microelectrode pipette filled with 4 M NaCl (2–4 MΩ resistance) and placed in the stratum radiatum of the CA1 area. We first constructed an input– output curve to select the intensity of the stimulus to evoke a fEPSP of ~ 40% of maximal amplitude. Recordings were obtained

with an ISO-80 amplifier (World Precision Instruments, Hertfordshire, UK) and digitised using a ADC-42 board (Pico Technologies, Pelham, NY, USA). Averages of three consecutive responses were continuously monitored on a personal computer with the LTP 1.01 software (Anderson & Collingridge, 2001). Responses were quantified as the initial slope of the averaged fEPSP and the effect of drugs, added to the superfusion solution, was estimated by changes of the fEPSP slope compared with baseline. Short-term plasticity was accessed using paired-pulse stimulation (PPS) with inter-pulse interval of 25 ms. Western blot analysis in total membranes and synaptosomes In order to probe the presence of A2BR in synapses, purified synaptic contacts (synaptosomes) and total membranes from the hippocampus were prepared using sucrose–Percoll differential centrifugations, as previously described (Canas et al., 2009; Cognato et al., 2010). Briefly, one hippocampus was homogenised in a medium containing 0.25 M sucrose and 10 mM HEPES (pH 7.4). The homogenate was spun at 2000 g at 4 °C for 3 min and the supernatant spun again at 9500 g for 13 min. The pellet was re-suspended in 2 mL of 0.25 M sucrose and 10 mM HEPES (pH 7.4) and 2 mL was placed onto 3 mL of Percoll discontinuous gradient containing 0.32 M sucrose, 1 mM EDTA, 0.25 mM dithiothreitol and 3, 10 or 23% Percoll, pH 7.4. The gradients were centrifuged at 25 000 g for 11 min at 4 °C, and the nerve terminals were collected between the 10% and 23% Percoll bands, diluted in 15 mL of HEPES buffered medium (in mM: NaCl, 140; KCl, 5; NaHCO3, 5; NaH2PO4, 1.2; MgCl2, 1; glucose, 10; and HEPES, pH 7.4, 10) and washed by centrifugation at 22 000 g for 11 min at 4 °C. This procedure for preparation of the synaptosomes is crucial to reduce the amount of postsynaptic density material. In fact, immunocytochemical analysis of the synaptosomes obtained with this discontinuous Percoll gradient showed that < 1% of the synaptophysin-positive elements were labelled by an anti-PSD95 antibody (Rodrigues et al., 2005, 2008). Western blot analysis was carried out as previously described (e.g. Canas et al., 2009; Cognato et al., 2010). Briefly, after determining the amount of protein, each synaptosomal sample was diluted with five volumes of SDS-PAGE buffer composed of 30% (v/v) glycerol, 0.6 M dithiothreitol, 10% (w/v) SDS and 375 mM Tris–HCl pH 6.8, boiled at 95 °C for 5 min. These diluted samples and the pre-stained molecular weight markers (GE Healthcare, Amadora, Portugal) were separated by SDS-PAGE (10% with a 4% concentrating gel) under reducing conditions and electro-transferred to polyvinylidene difluoride membranes (0.45 lm; GE Healthcare). After blocking for 2 h at room temperature with 5% milk in Trisbuffered saline, pH 7.6, containing 0.1% Tween 20 (TBS-T), the membranes were incubated overnight at 4 °C with a rabbit antiA2BR antibody (1:250; sc-28996 from Santa Cruz Biotechnology, Santa Cruz, CA, USA). This particular antibody was selected as its selectivity has been previously been validated by eliminating its immunoreactivity upon neutralising A2BR with a siRNA (Eckle et al., 2008). After four washing periods for 10 min with TBS-T containing 0.5% milk, the membranes were incubated with the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2000; GE Healthcare) in TBS-T containing 1% milk for 90 min at room temperature. After five 10-min washes in TBS-T with 0.5% milk, the membranes were incubated with enhanced chemifluorescence for 5 min and then analysed with a VersaDoc 3000 (Bio-Rad, Amadora, Portugal). The membranes were then re-probed and tested for atubulin immunoreactivity (1:10 000; Sigma) to confirm that similar

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

A2B receptors control A1-mediated responses in the hippocampus 3 amounts of protein were applied to the gels, as previously described (e.g. Canas et al., 2009; Cognato et al., 2010). Immunocytochemical analysis of purified nerve terminals To probe the localisation of A2BR in glutamatergic terminals, we used an immunocytochemical analysis of single nerve terminals to determine the presence of A2BR in glutamatergic nerve terminals assessed as immunopositive for the glutamatergic terminal marker vesicular glutamate transporter type 1 (vGluT1), as previously performed (Rodrigues et al., 2005). Hippocampal synaptosomes (prepared as describe above) were plated in coverslips previously coated with poly-D-lysine (0.1 mg/ mL; Sigma Aldrich), fixed with 4% paraformaldehyde (in 4% sucrose and 0.9% NaCl) for 15 min and washed twice with 0.1 M phosphate-buffered saline (PBS) medium. The nerve terminals were permeabilised in PBS with 0.2% Triton X-100 for 10 min, blocked for 1 h in PBS with 3% BSA and 5% normal bovine serum to prevent non-specific binding, washed twice with PBS and then incubated with the primary antibodies, rabbit anti-A2BR (1:5000) and goat anti-vGluT1 (1:1000, Invitrogen), in PBS with 3% BSA for 1 h at room temperature. After washing three times with PBS with 3% BSA, the nerve terminals were immunolabelled with Alexa Fluor 488-conjugated donkey anti-mouse and Alexa Fluor 594-conjugated goat anti-guinea pig (1:200; Invitrogen) for 1 h at room temperature. It was confirmed that none of the secondary antibodies produced any signal in preparations to which the addition of the corresponding primary antibody was omitted. After washing and mounting onto slides with Prolong Gold Antifading (Invitrogen), the preparations were visualised under a Zeiss Imager Z2 fluorescence microscope equipped with a AxioCam HRm and 63 9 Plan-ApoChromat oil objective (1.4 numerical aperture). Each coverslip was analysed by counting six different fields and in each field a total amount of 500 individualised elements were counted, as previously described (Canas et al., 2014). The images, acquired in each colour channel using identical masks, were quantified using ImageJ 1.37v software (NIH, Bethesda, MD, USA). To determine the co-localisation of the different fluorophores in the plated nerve terminals, the colocalisation coefficients were calculated from the red and green two-colour-channel scatter plots (Costes et al., 2004) using a macro routine developed by our group to automatically evaluate Pearson’s correlation between first and the second colour channel with a significance level > 95% (Canas et al., 2014). The values are presented as the percentage of total vGluT1-immunopositive nerve terminals that were labelled with A2BR, as mean  SEM of n = 5 corresponding to preparations obtained from different mice. Hippocampal [14C] glutamate release experiments Experimental procedures were carried out essentially as previously described for release studies in hippocampal slices and synaptosomes (Kofalvi et al., 2000, 2003). For synaptosomal experiments, the brains of C57BL/6 male mice were collected into ice-cold 0.32 M sucrose solution containing 10 mM HEPES, 1 mM EDTA, and 1/500 v/v protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA), pH 7.4. The hippocampi were rapidly dissected and homogenised with a glass potter and teflon homogeniser, and centrifuged at 3000 g for 5 min. The supernatant was collected and centrifuged at 13 000 g for 10 min to obtain the P2 synaptosomal fraction. For slice experiments, the hippocampi were cut into 400lm-thick transverse slices using a McIlwain tissue chopper and the slices were left to recover in Krebs–HEPES buffer (in mM: NaCl,

113; KCl, 3; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 10; HEPES, 15; pH 7.4) under continuous gassing with 95% O2 and 5% CO2 for 1 h at room temperature. Synaptosomes were used instantly after preparation and slices were prepared while the synaptosomes were resuspended in 500 lL of Krebs-HEPES containing [14C]-U-glutamate (20 lM; American Radiolabeled Chemicals, St Louis, MO, USA; specific activity: 200 mCi/mmol; 0.1 mCi/mL) and the glutamate decarboxylase inhibitor aminooxyacetic acid (Aaa, 100 lM) to prevent [14C] glutamate metabolism into [14C]-labelled derivatives (Krebs-HEPES-Aaa solution). After the 10-min loading with the tracer at 37 °C, synaptosomes were diluted in 4 mL Krebs-HEPES-Aaa solution and this suspension were divided among 8 microvolume chambers, where the synaptosomes were trapped over GF/B filters (Whatman, Sigma-Aldrich, Sintra, Portugal). Slices were also loaded with the tracer for 10 min at 37 °C, and, after washing with Krebs-HEPES-Aaa solution, they were gently placed with a pipette in microvolume (100 lL) chambers of a heated homemade multi-channel release system (2 slices/ chamber). The slices and the synaptosomes were washed for 10 min at a rate of 0.8 mL/min with the Krebs-HEPES-Aaa solution at 37 °C till the end of the experiment. After the 10-min washout, 2min samples were collected from the effluents of each chamber. Synaptosomes were stimulated twice with a 10-min interval with two identical pulses of high K+ (25 mM for 60 s; isomolar substitution of Na+) as denoted with S1 and S2. The evoked release of glutamate was previously shown to be largely Ca2+-dependent (Kofalvi et al., 2003). BAY 60-6583, CPA or their vehicle DMSO were bath applied 4 min before S2 when used alone. In some cases, BAY 606583 was given before S1 till the end of the experiment, and CPA was added 4 min before S2 as above. The superfused slices were also challenged twice with high K+ (60 mM for 90 s), with an 18min interval. Drug/vehicle (DMSO) treatments were bath applied 12-min before S2. The radioactivity content of each sample and of the slices as well as of the filters with the trapped synaptosomes was counted by a Tricarb b-counter (Perkin Elmer, Lisbon Portugal). Disintegrations per minute (DPM) values were expressed as fractional release (FR%), i.e. the percent of actual content in the effluent as a function of the total synaptosomal content of radioactivity. Treatment effects were determined based on their effect on the second stimulus (S2), as compared to the first stimulus (S1). Treatmentinduced changes in the S2/S1 ratio were expressed to the S2/S1 ratio of the appropriate DMSO control taken as 100%. Behavioral analyses All behavioural tests were performed between 9:00 and 12:00 PM in a sound-attenuated room under low-intensity light (12 lx). The behavioural analysis was recorded and all the parameters were automatically quantified using the Any-mazeâ video-tracking system (Stoelting Inc., Kiel, WI, USA). Open field The open field apparatus was a 38 cm 9 38 cm 9 38 cm chamber, where mice were placed in the centre and their activity recorded during 10 min, as previously described (Rial et al., 2014). Elevated plus-maze The elevated plus-maze was made of wood, covered with impermeable Formica and placed 60 cm above the floor. The four arms were 18-cm long and 6-cm wide. Two opposite arms were surrounded by

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

4 F. Q. Goncßalves et al. walls (6-cm high, closed arms), while the other two were devoid of enclosing walls (open arms). The four arms were connected by a central platform (6 9 6 cm). Each animal was placed in the centre of the maze facing a closed arm and analysed for 5 min, as previously described (Rial et al., 2009). Y-maze The apparatus consists of three equal closed arms (each arm was 20 cm long, 15 cm high and 6 cm wide) converging to an equal angle. Each mouse was placed at the end of one arm and allowed to freely move through the maze during 5 min. An alternation was defined as consecutive entries in all three arms. The percentage of alternation was calculated as previously described (Dall’Igna et al., 2007).

a

Occupation plot analysis For the analysis of exploratory behaviour, all the arms of the apparatuses described above were divided in two areas, namely centre and outside areas, as shown in Figs 4 and 5. Pixel density was calculated using the individual mouse occupation plot images obtained with the Any-mazeâ video-tracking system and submitted to the following programing code: I = imread(‘nn.png’); J = rgb2gray(I); figure, imshow (I), figure, imshow(J); sum = 0; count = 0; for i = 1:38; for j = 1:50; if (J(i,j) ≥ 0); sum = sum + J(i,j); count = count + 1; end; end; end; disp(‘Average’); Avrg = sum/count;. This code allowed calculating the number of pixels, proportional to the occupancy of each mouse at that spatial point, in the centre and outside areas of the apparatus, which was then converted into a colour code, being red the highest pixel density, and blue the lowest pixel density.

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Fig. 1. Impact of A2BR on synaptic transmission and short-term plasticity in Schaffer collaterals-CA1 pyramid synapses of mouse hippocampal slices. The A2BR antagonist MRS 1754, tested in three different concentrations (20 nM, 200 nM and 2 lM), was devoid of effects on both basal synaptic transmission (a) and paired pulse stimulation (PPS, 25 ms interpulse interval) (b). The data are mean  SEM of n = 3–4. The inserts in (a) are representative fEPSP recordings in the absence (left) and in the presence (right) of 200 nM MRS 1754. The A2BR agonist BAY 60-6583, tested at three different concentrations (30 nM, 300 nM and 3 lM), was devoid of effects on basal synaptic transmission (c) but decreased PPS (25 ms interpulse interval) at the two higher concentrations (d). The data are mean  SEM of n = 3–4. *P < 0.05 compared to control using a Newman–Keuls post hoc test. The effect of BAY 60-6583 (300 nM) on PPS was prevented by 200 nM MRS 1754 (e) and is abrogated in slices from A2BR-KO mice (f); The data are mean  SEM of n = 3–4. *P < 0.05 compared to the absence of BAY 60-6583 (open bars) using a Newman–Keuls post hoc test. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

A2B receptors control A1-mediated responses in the hippocampus 5 Data analysis

a

Data are expressed as means  SEM of the indicated number of independent observations in different animals (n). The statistical analysis was carried out using a Student’s t-test for one variable evaluation and one-way analysis of variance (ANOVA) for multifactorial comparison, followed by post hoc comparisons using the Newman–Keuls test. The significance level was 95% and all tests were performed using the GraphPad Prism software (StatSoft Inc., La Jolla, CA, USA). b

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Results Activation of A2BR decreases short-term plasticity (pairedpulse stimulation) without interfering with basal synaptic transmission To probe the impact of A2BR on the functioning of neuronal circuits, we tested the effect of selective A2BR ligands on synaptic transmission in mouse hippocampal slices. The blockade of A2BR with the A2BR antagonist MRS 1754, tested in 3 different concentrations (20 nM, 200 nM and 2 lM), did not modify (F3,60 = 0.15 P > 0.05) basal synaptic transmission (Fig. 1a). Short-term plasticity, measured as the paired-pulse stimulation (PPS) ratio (with an interpulse interval of 25 ms) was also not modified (F3,60 = 0.3 P > 0.05) by the tested concentrations of MRS 1754 (Fig. 3b). The activation of A2BR with the A2BR agonist BAY 60-6583 also failed to modifiy (F3,60 = 0.2 P > 0.05) basal synaptic transmission at the 3 different concentrations tested (30 nM, 300 nM and 3 lM; Fig. 1c). However, BAY 60-6583 (300 nM and 3 lM) decreased the PPS ratio (F3,60 = 4.94, P < 0.01; Fig. 3d); this effect of BAY 60-6583 (300 nM) was prevented by the A2BR antagonist MRS 1754 (200 nM; Fig. 1e) and was absent in hippocampal slices from A2BRKO mice (F3,60 = 0.8 P > 0.05; Fig. 1f). A2BR are enriched in synaptosomes and located in glutamatergic terminals of the hippocampus As modifications of paired-pulse facilitation in excitatory synapses most often result from a presynaptic effect (Kamiya & Zucker, 1994), we sought to directly demonstrate the presence of A2BR in glutamatergic nerve terminals. Western blot analysis showed the presence of A2BR immunoreactivity in total membranes and in synaptosomes (Fig. 2a). The quantitive comparison between these two preparations showed an increased A2BR immunoreactivity in synaptosomes compared to total membranes (t2,0.05 < 0.05). Furthermore, the immunocytochemical analysis of individual nerve terminals revealed that 73  5% (n = 5) of glutamatergic terminals (i.e. immunopositive for the glutamatergic nerve terminal marker, vGluT1) were also endowed with A2BR immunoreactivity (Fig. 2b–d). This confirmed the presence of A2BR in hippocampal nerve terminals, namely in glutamatergic nerve terminals.

A2BR modulate A1R-mediated inhibition of synaptic transmission and short-term plasticity As adenosine A1 receptors (A1R) play a predominant role in the control of basal synaptic transmission in hippocampal glutamatergic synapses (Dunwiddie et al., 1981; Costenla et al., 2011), we tested whether A2BR would control this predominant A1R action. In accordance with an on-going tonic inhibition of synaptic transmission by endogenous adenosine acting on A1R, a supra-maximal and selective

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Fig. 2. A2BR are present in the hippocampus, in particular in glutamatergic nerve terminals. (a) Representative Western blot (n = 4) illustrating the presence of A2BR immunoreactivity in total membranes and synaptosomes from the mouse hippocampus, and comparing the relative density of A2BR immunoreactivity in total membranes (open bar) and synaptosomal membranes (grey bar), expressed as mean  SEM of the ratio between A2BR and atubulin immunoreactivity (n = 4). *P < 0.05 vs. total membranes. (b-e) Immunocytochemical analysis of platted purified nerve terminals from the mouse hippocampus revealed that nerve terminals immuno-positive for vesicular glutamate transporter type 1 (vGluT1, i.e. glutamatergic terminals, green in b) are endowed with A2BR (red in c), as confirmed by the superimposition of the images (yellow in d), allowing to estimate an the average percentage (expressed as mean  SEM of n = 5) of glutamatergic nerve terminals endowed with A2BR.

concentration of the A1R antagonist DPCPX (100 nM) enhanced synaptic transmission by 47  4% (n = 4). Notably, the addition of BAY 60-6583 (300 nM) prevented this effect, bringing the amplitude of fEPSPs almost to the baseline (5  2%, n = 4; F3,58 = 7.08 P < 0.001 vs. the effect of DPCPX in the absence of BAY 60-6583; Fig. 3a). This effect of BAY 60-6583 was abbrogated by 200 nM MRS 1754 (F3,58 = 0.5 P > 0.05 45  3%, n = 4; Fig. 3b) and was absent in hippocampal slices from A2BR-KO mice (50  7%, (F3,58 = 0.2 P > 0.05, n = 4; Fig. 3c), prompting the conclusion that A2BR activation dampens the tonic activation of A1R by endogenous adenosine. To probe whether A2BR directly controlled A1R function, we compared the inhibitory action of the non-selective A1R agonist 2chloroadenosine in the absence and in the presence of BAY 606583. 2-Chloroadenosine concentration-dependently inhibited synaptic transmission ( 35  5% at 0.1 lM, 50  5% at 0.3 lM and 70  4% at 1 lM; n = 4). As shown in Fig. 3d, the inhibitory effects by 2-chloroadenosine on fEPSP amplitude were attenuated in the presence of 300 nM BAY 60-6583 ( 20  3% at 0.1 lM, 35  1% at 0.3 lM and 55  2% at 1 lM; (F3,58 = 4.2

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

6 F. Q. Goncßalves et al. a

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Fig. 3. The activation of A2BR causes a functional desensitisation of A1R-mediated inhibition of hippocampal synaptic transmission. (a) The A1R antagonist DPCPX (100 nM) caused a disinhibition of synaptic transmission, in accordance with the predominant role of endogenous adenosine tonically inhibiting synaptic transmission through A1R activation; notably, the A2BR agonist BAY 60-6583 (300 nM) decreased this DPCPX-induced increase in basal synaptic transmission. The data are mean  SEM of n = 3–5. This effect of BAY 60-6583 on the DPCPX-induced increase in basal synaptic transmission was prevented in the presence of the A2BR antagonist MRS 1754 (200 nM) (b) and abrogated in slices from A2BR-KO mice (c). The data are mean  SEM of n = 3–5. (d) Average concentration-response curves of the A1R-mediated inhibition of synaptic transmission by 2-chloroadenosine, showing that BAY 60-6583 (300 nM) decreased the inhibition of synaptic transmission caused by the lower concentrations of 2-chloroadenosine (●), an effect abrogated in slices from A2BR-KO mice (▲). The data are mean  SEM of n = 4–5. *P < 0.05 compared to the absence of BAY 60-6583 (□).

P < 0.05 n = 4). Importantly, BAY 60-6583 (300 nM) did not modify the concentration-response curve of 2-chloroadenosine in hippocampal slices from A2BR-KO mice (n = 4; Fig. 3d). A1 and A2B receptors interact to control high K+-evoked release of [14C] glutamate in slices but not in synaptosomes Similarly to our previous reports (Kofalvi et al., 2003), repetitive (S1 and S2) stimulation with high K+ (25 mM for 60 s) evoked the release of similar amounts of [14C] glutamate from superfused synaptosomes (S1, 3.69  0.13 FR%; S2/S1, 0.85  0.06; n = 21; Fig. 4a). The A2BR agonist, BAY 60-6583 (300 nM) did not presynaptically affect the evoked release of [14C] glutamate from synaptosomes (n = 9 animals in duplicate, P > 0.05; Fig. 4a,b). As previously observed (Ambrosio et al., 1997), the A1R agonist, CPA (100 nM) inhibited the evoked release of [14C] glutamate by 35.5  3.3% (n = 10, P < 0.0001), which inhibition persisted in the presence of 300 nM BAY 60-6583 (by 29.3  5.9%; n = 10; P < 0.001; Fig. 4b). As this suggests a lack of presynaptic interaction between the two adenosine receptor subtypes, we moved from isolated presynapses to a more complex system, i.e. superfused hippocampal slices. Similarly to synaptosomes, repetitive (S1 and S2)

stimulation with high K+ (60 mM for 90 s) evoked the release of similar amounts of [14C] glutamate from superfused slices (S1, 3.32  0.17 FR%; S2/S1, 1.11  0.04; n = 5; Fig. 4c). By contrast to synaptosomes, BAY 60-6583 (300 nM) now inhibited the evoked release of [14C] glutamate from slices by 20.6  5.0% (n = 5, P < 0.05; Fig. 4c,d). CPA (100 nM) also inhibited the evoked release of [14C] glutamate from slices by 19.8  5.6% (n = 5, P < 0.05; Fig. 4d); however, in the presence of BAY 60-6583 (300 nM), CPA (100 nM) now failed to significantly affect the evoked release as compared to DMSO control (9.7  8.6% inhibition; n = 5, P > 0.05). The genetic deletion of A2BR modifies the exploratory behavior without altering locomotion or anxiety To test the behavioural relevance of this A2BR-mediated control of A1R function, we probed for possible alterations of behavioural performance focusing on emotional responses, which are known to be controlled by A1R (Gimenez-Llort et al., 2002; Lang et al., 2003) and thus likely to be affected in A2BR-KO mice compared to wild type (WT) mice. In the open field test, A2BR-KO and WT mice travelled the same distance (t2,0.05 > 0.05; data not shown); however,

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

A2B receptors control A1-mediated responses in the hippocampus 7 a

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Fig. 4. A2B and A1 receptors interact in the control of [14C] glutamate release in the hippocampus. (a,c) Fractional release percent (FR%; see Materials and Methods) diagram representing the averaged time-course of [14C] glutamate release from superfused mouse hippocampal (a) synaptosomes or (c) slices under treatment with the A2BR agonist, BAY 60-6583 as indicated by the horizontal bar (300 nM) and the respective control. Stimuli with high-K+ (in the synaptosomes, 25 mM; 2 9 60 s; in the slices, 60 mM; 2 9 90 s) are marked as S1 and S2. (b,d) Bar graph representing the effect of A2BR activation by BAY 606583 and the effect of A1R activation by CPA (100 nM) alone and combined on the high-K+-evoked release of [14C] glutamate from synaptosomes (b) or slices (d). CPA was added 4 min before S2 in synaptosomes and 12 min before S2 in slices. The Y axis represents the effect of the treatment on the S2/S1 ratio, normalised to the vehicle control. All data points and bars represent the mean  SEM derived from 9 to 10 animals (synaptosomes) or 5 animals (slices). *P < 0.05; ***P < 0.001; n.s., not significant vs. Control group.

A2BR-KO mice displayed a tendency for an increase number of entries in the center of the open field arena when compared to WT mice (t2,0.05 = 0.06; Fig. 5b). In the elevated plus maze, none of the parameters classically used to gauge anxiety (% of time in open arms; % of entries in the open arms and closed arms entries; see Dawson & Tricklebank, 1995) were modified between A2BR-KO and WT mice (data not shown). However, as occurred in the open field test, we also observed a different exploratory behavior between A2BR-KO and WT mice in the elevated plus maze: thus, whereas WT mice equally explored the center area and the outside area of the apparatus (t2,0.05 > 0.05; Fig. 5d), A2BR-KO mice explored more the center compared to the outside area (t2,0.05 < 0.0001; Fig. 5e). The direct comparison between genotypes confirmed that A2BR-KO mice explored statistically more the center area than control mice (t2,0.05 < 0.0001; Fig. 5f). Genetic deletion of A2BR modifies the exploratory behavior without cognitive modifications As the pattern of exploratory behavior is critically affected by alterations of working memory, we tested whether A2BR-KO mice displayed a modified working memory. In the Y-maze task, A2BR-KO and WT mice displayed a similar percentage of spontaneous alternations (t2,0.05 > 0.05; Fig. 6b). However, although the distance traveled in the Y-maze was similar in A2BR-KO and WT mice (data not shown), the total number of entries into the arms was larger (t2,0.05 < 0.001) in A2BR-KO mice (Fig. 6c), suggesting a modified exploratory behavior in A2BR-KO mice. This was confirmed by the analysis of the different time spent in the center and in the periphery of the maze (Fig. 6a): thus, control mice spent more time in the outside area (t2,0.05 < 0.001; Fig. 6d) whereas A2BR-KO mice spent the same time in the center and in the outside area (t2,0.05 > 0.05;

Fig. 6e). The direct comparison between genotypes confirmed that A2BR-KO mice explored statistically more the center area than control mice (t2,0.05 < 0.001; Fig. 6f).

Discussion The present study provides the first direct demonstration of a synaptic localisation of A2BR, in particular in glutamatergic terminals. Furthermore, the activation of these A2BR attenuated the predominant A1R-mediated inhibition of excitatory synaptic transmission and of glutamate release and the genetic inactivation of A2BR modified the pattern of exploratory behaviour. The presence of A2BR in the brain has been essentially regarded as residual on the basis of their low expression in the brain (Dixon et al., 1996) and on the ability to account for the effects of adenosine as resulting from A1R to control synaptic transmission and short-term plasticity (Dunwiddie & Masino, 2001) and A2AR to selectively control long-term plasticity (Rebola et al., 2008; Costenla et al., 2011). Our present immunological and functional convergent evidence supporting the presence of A2BR in hippocampal synapses actually shows that all adenosine receptors can be identified in synapses (Rebola et al., 2003, 2005), even the A3R, the function of which is still unclear (Dunwiddie et al., 1997; Lopes et al., 2003). The most prominent effect associated with A2BR function in the hippocampus was the control of A1R function. In fact, both electrophysiological and neurochemical approaches converged to conclude that a selective A2BR agonist blunted the ability of A1R to inhibit both the release of glutamate and glutamatergic transmission. Notably, this effect is unlikely to be presynaptic as it was only observed in slices but not in purified nerve terminals. Furthermore, the present conclusion that A2BR control A1R function, illustrates a new concept of fine-tuning of neuromodulation systems (Sebasti~ao

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

8 F. Q. Goncßalves et al. a

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Fig. 5. Effects of A2BR genetic deletion on locomotor activity, anxiety-like and exploratory behaviour. (a) Comparison of the average occupation plot representing the pattern of exploration of wild type (control) and A2BR-KO mice in the open field test. (b) Average number of entries in the centre area of the open field arena by control and A2BR-KO mice. (c) Average occupation plot representing the pattern of exploration in the elevated plus-maze test by control and A2BR-KO mice, revealing that control mice similarly explored the centre and outside area of the elevated plus-maze (d), whereas A2BR-KO mice spent more time in the centre than in the outside area (e). (f) The direct comparison of the occupation plot analysis (pixel quantification in arbitrary units) in the central area between genotypes, confirmed that A2BR-KO mice spent more time than control mice in the central area of the elevated plus-maze. The data are mean  SEM of n = 8–10. *P < 0.05 compared to control group using a Newman–Keuls post hoc test.

& Ribeiro, 2009), i.e. the ability to adapt the efficiency of neuromodulation systems according to the needs of neuronal networks. The main functional consequence of activating A2BR was the control of A1R function, which play the predominant role in the control of basal synaptic transmission, as best heralded by the lack of effect of adenosine on hippocampal synaptic transmission in A1R knockout mice (Johansson et al., 2001). Thus, this novel A2BR-mediated functional down-regulation of A1R prompts the idea that the adenosine neuromodulation system might display an organisation more complex than that initially anticipated. In fact, we have to include A2BR to refine the initial proposal that A1R control basal transmission and A2AR are selectively engaged to modulate synaptic plasticity processes (Cunha, 2008), which is mechanistically justified by the disproportionally higher release of ATP at higher frequencies of stimulation (Cunha et al., 1996) and the selective activation of A2AR by ATP-derived adenosine (Augusto et al., 2013) together with the ability of A2AR to desensitise A1R (Lopes et al., 1999). One notable property of A2BR is their lower affinity for adenosine compared to the other subtypes of adenosine receptors (Fredholm et al., 2001), which has prompted the idea that A2BR are selectively engaged in pathological conditions (Popoli & Pepponi, 2012)

associated with a substantial increase in the extracellular levels of adenosine (Cunha, 2001; Latini & Pedata, 2001; Dale & Frenguelli, 2009). We now post that this A2BR-mediated functional desensitisation of A1R might be of particular importance in such noxious brain conditions when the total block of excitatory transmission that can be achieved by the supra-maximal activation of A1R (Sebasti~ao et al., 2000) would be attenuated by the engagement of A2BR to allow maintaining some level of functioning of excitatory transmission in neuronal networks. Notably, there are some reports suggesting that A1R are important to control the onset of brain damage, but actually lose their efficiency with the augmentation of brain damage (Sweeney, 1997; Rebola et al., 2003; Olsson et al., 2004; Fedele et al., 2006). This has mostly been interpreted as an autodesensitisation of A1R upon prolonged activation (Abbracchio et al., 1992; Ruiz et al., 1996; Hettinger et al., 1998; Coelho et al., 2006), but the present data now prompt the possibility that the A2BRinduced functional downregulation of A1R may also play a role. The mechanism underlying this ability of A2BR to functionally downregulate A1R still remains to be unravelled. One possibility would be a direct heteromerisation between A2BR and A1R, given that A2BR can heteromerise with different membrane proteins such

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

A2B receptors control A1-mediated responses in the hippocampus 9 a

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Fig. 6. Effects of A2BR genetic deletion on working memory and exploratory behaviour. (a) Comparison of the average occupation plot representing the pattern of exploration of wild type (control) and A2BR-KO mice in the Y-maze. The percentage spontaneous alternation (an index of working memory) was not altered in A2BR-KO compared to control mice (b) whereas the total number of entries into the Y-maze arms was larger in A2BR-KO compared to control mice (c). The occupation plot analysis (pixel quantification in arbitrary units) revealed that control mice spent more time in the outside than central area (d), whereas A2BRKO mice spent similar time in central and outside area (e). (f) The direct comparison of the occupation plot analysis (pixel quantification in arbitrary units) in the central area between genotypes, confirmed that A2BR-KO mice spent more time than control mice in the central area of the Y-maze. The data are mean  SEM of n = 8–10. *P < 0.05 compared to control group using a Newman–Keuls post hoc test.

as receptors (Corset et al., 2000; Moriyama & Sitkovsky, 2010) and enzymes (Antonioli et al., 2014) and A1R can also form dimers with different G-protein-coupled receptors (Gines et al., 2000; Ciruela et al., 2001; Kamikubo et al., 2013). An alternative would be an ability of A2BR to trigger an intracellular cross talk through its diverse G-protein coupling (Ryzhov et al., 2006; Cohen et al., 2010; Liu et al., 2014) to functionally desensitise A1R (Ciruela et al., 1997; Nie et al., 1997; Hashimi et al., 1998; Lopes et al., 1999), in a manner analogous to the ability of A2BR to control ion channels (Garcß~ao et al., 2013), G protein coupled receptors (Feoktistov & Biaggioni, 1997; Conde et al., 2008) or growth factor receptors (Corset et al., 2000). Further studies will be required to clarify the mechanism underlying the ability of A2BR to functionally desensitise A1R in nerve terminals. In spite of this proposal that the A2BR-mediated functional desensitisation of A1R might be particularly relevant in noxious brain conditions, it may also play a role under physiological conditions, given that A2BR display different agonist potency to recruit different intracellular signalling cascades (Gao et al., 2014). This is of particular interest to interpret the observed modification of the pattern of exploratory behaviour of mice with a genetic deletion of A2BR. It is interesting to note that behavioural changes observed in A2BR-KO mice were restricted to exploratory behaviour. The hippocampus, precisely where we identified A2BR-mediated responses, is one of the brain regions involved in exploratory behaviour as the spatial recognition is immediately activated when in a new environment, allowing the subject to cope with spatial-inherent treats and to escape in case of a possible fight of flight conflict (Monaco et al., 2014). These changes of exploratory behaviour found in A2BR-KO mice resemble the different pattern of exploration and different selection of strategy in the Morris water maze of A1R-KO mice (Lang et al., 2003). Furthermore, the influences of A1R in emotional

responses and in arousal are expressed as changes in the swimming pattern of zebrafish (Maximino et al., 2011). These observations prompt the speculation that the modified exploratory behaviour observed in A2BR-KO mice may be related to the observed A2BRmediated desensitisation of A1R responses in the hippocampus. However, this working hypothesis should not rule out the involvement of other central mechanisms that are modulated by A2BR, namely astrocytic responses such as glycogen availability (Allaman et al., 2003) or the release of cytokines (Trincavelli et al., 2004), which are known to impact on behaviour.

Conclusions The present study provides the first direct demonstration for the presence of A2BR in glutamatergic nerve terminals of the mouse hippocampus. The activation of these synaptic A2BR decreased the functioning of the predominant A1R-mediated inhibition of synaptic transmission, suggesting a primary fine-tuning role for synaptic A2BR. Finally, we also report alterations of the pattern of exploratory behaviour in A2BR-KO mice although it remains to be demonstrated whether they are due to this A2BR-mediated functional desensitisation of hippocampal A1R.

Acknowledgements We are in debt to Drs Akio Ohta and Michael Sitkovsky (New England Inflammation and Tissue Protection Institute, Northeastern University, Boston, MA, USA) for their generous gift of A2BR KO mice. Supported by FCT (PTDC/SAU-NSC/122254/2010), QREN (CENTRO-07-ST24-FEDER002006) and the U.S. Army Research Office and the Defense Advanced Research Projects Agency (grant W911NF-10-1-0059) and Programa Ci^encia sem Fronteiras (CNPq, Brazil). J.M.M. acknowledges the support of Projeto Mais Centro - “Aging, Stress And Chronic Diseases: From Mechanisms to

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

10 F. Q. Goncßalves et al. Therapeutics” (CENTRO-07-ST24-FEDER-002006). A.K. acknowledges the support of the FCT grant PTDC/SAU-NEU/100729/2008. The authors declare no conflict of interest.

Abbreviations A1R, adenosine A1 receptor; A2AR, adenosine A2A receptor; A2BR, adenosine A2B receptor(s); aCSF, artificial cerebral spinal fluid; BAY 60-65 83, 2-[[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl] thio]-acetamide; BSA, bovine serum albumin; CPA, (2R,3R,4S,5R)-2-[6-(cyclopentylamino)purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; fEPSP, field excitatory postsynaptic potential; KO, knockout; MRS 1754, N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-ylphenoxy]-acetamide; PBS, phosphate-buffered saline; pH 7.6, containing 0.1% Tween 20; PPS, paired-pulse stimulation; TBS-T, Tris-buffered saline; vGluT1, vesicular glutamate transporter type 1.

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