Crosstalk Between ATP-P2X7 and Adenosine A2A Receptors Controlling Neuroinflammation in Rats Subject to Repeated Restraint Stress

Depressive conditions precipitated by repeated stress are a major socio-economical burden in Western countries. Previous studies showed that ATP-P2X7 receptors (P2X7R) and adenosine A2A receptors (A2AR) antagonists attenuate behavioral modifications upon exposure to repeated stress. Since it is unknown if these two purinergic modulation systems work independently, we now investigated a putative interplay between P2X7R and A2AR. Adult rats exposed to restraint stress for 14 days displayed an anxious (thigmotaxis, elevated plus maze), depressive (anhedonia, increased immobility), and amnesic (modified Y maze, object displacement) profile, together with increased expression of Iba-1 (a marker of microglia “activation”) and interleukin-1β (IL1β) and tumor necrosis factor α (TNFα; proinflammatory cytokines) and an up-regulation of P2X7R (mRNA) and A2AR (receptor binding) in the hippocampus and prefrontal cortex. All these features were attenuated by the P2X7R-preferring antagonist brilliant blue G (BBG, 45 mg/kg, i.p.) or by caffeine (0.3 g/L, p.o.), which affords neuroprotection through A2AR blockade. Notably, BBG attenuated A2AR upregulation and caffeine attenuated P2X7R upregulation. In microglial N9 cells, the P2X7R agonist BzATP (100 μM) or the A2AR agonist CGS26180 (100 nM) increased calcium levels, which was abrogated by the P2X7R antagonist JNJ47965567 (1 μM) and by the A2AR antagonist SCH58261 (50 nM), respectively; notably JNJ47965567 prevented the effect of CGS21680 and the effect of BzATP was attenuated by SCH58261 and increased by CGS21680. These results provide the first demonstration of a functional interaction between P2X7R and A2AR controlling microglia reactivity likely involved in behavioral adaptive responses to stress and are illustrative of a cooperation between the two arms of the purinergic system in the control of brain function.


INTRODUCTION
Depression represents the major burden of disease in Europe (Andlin-Sobocki et al., 2005) and the constellation of mood alterations associated with depression can be recapitulated in animal models repeatedly exposed to different stressors (de Kloet et al., 2005;Berton et al., 2012). The use of animal models converges with imaging studies to identify modifications of different brain regions, such as the hippocampus, prefrontal, and limbic cortices, that are associated with mood dysfunction (de Kloet et al., 2005) and provide compelling evidence for the involvement of neuroinflammation Deng et al., 2020;Troubat et al., 2021) and of synaptic dysfunction (Duman and Aghajanian, 2012;Vose and Stanton, 2017) as key processes in the etiology of major depression. However, the identification of molecular systems that may be targeted to correct depressive symptoms has still failed to yield novel and effective anti-depressants (Ménard et al., 2016).
One candidate system is operated by purines, which fulfill numerous roles controlling neuronal communication, neuron-glia communication, and neuroinflammation (Agostinho et al., 2020). ATP is a danger signal in the brain (Rodrigues et al., 2015) and one of its receptors, P 2X7 receptors (P 2X7 R), has been associated with mood dysfunction (reviewed in Ribeiro et al., 2019;Illes et al., 2020), based on the association of particular P 2X7 R haplotypes with depression (Czamara et al., 2018) and with the ability of genetic deletion or pharmacological antagonism of P 2X7 R to control mood dysfunction in different animal models of repeated stress (Iwata et al., 2016;Yue et al., 2017;Farooq et al., 2018;Aricioglu et al., 2019). The mechanism underlying the impact of P 2X7 R on mood is still undefined, but the control of glia, mainly microglia, which contributes to the build-up of neuroinflammation, stems as a promising candidate mechanism (Yue et al., 2017;Bhattacharya and Jones, 2018). Together with possible neuronal effects of P 2X7 R, the control of neuroinflammation can account for the general neuroprotective properties of P 2X7 R antagonists, such as the blood-brain barrierpermeant drug, brilliant blue G (BBG; Díaz-Hernández et al., 2009Arbeloa et al., 2012;Carmo et al., 2014;Wang et al., 2015;Yue et al., 2017;Farooq et al., 2018;Aricioglu et al., 2019).
The purinergic system is particularly enticing since it encompasses two parallel signaling systems: one involving ATP and P 2 R and the other involving the dephosphorylation product of ATP, adenosine, which acts on P 1 or adenosine receptors, mainly inhibitory A 1 receptors and facilitatory A 2A receptors (A 2A R) in the brain (Fredholm et al., 2005). The extracellular conversion of ATP into adenosine is mediated by ectonucleotidases (Cunha, 2001;Zimmermann et al., 2012) and we have shown that the extracellular formation of ATP-derived adenosine is selectively associated with the activation of neuronal A 2A R (Rebola et al., 2008;Augusto et al., 2013;Carmo et al., 2019;Gonçalves et al., 2019), as well as with A 2A R located in other cell types (e.g., Deaglio et al., 2007;Flögel et al., 2012;Flores-Santibáñez et al., 2015;Mahmut et al., 2015;Meng et al., 2019). A 2A R are mainly located in synapses (Rebola et al., 2005), but also control microglia and neuroinflammation (Orr et al., 2009;Rebola et al., 2011;Madeira et al., 2016;Duarte et al., 2019) to robustly impact neurodegeneration (reviewed in Cunha, 2016). Both selective A 2A R antagonists and the non-selective adenosine receptor antagonist caffeine (Fredholm et al., 1999), can control mood and memory alterations in rodents exposed to repeated stress (Yamada et al., 2013;Kaster et al., 2015), as per the mood normalizing properties afforded by the intake of caffeine in humans (reviewed in Grosso et al., 2016) and the association of A 2A R polymorphisms with anxiety and depression (Hamilton et al., 2004;Hohoff et al., 2010;Oliveira et al., 2019).
Thus, the available evidence indicates P 2X7 R as well as A 2A R as major players in the control of mood dysfunction, with both receptors systems undergoing an up-regulation in animal models exposed to repeated stress (Cunha et al., 2006;Kongsui et al., 2014;Kaster et al., 2015;Aricioglu et al., 2019). However, it has never been explored if there is any interplay between both receptors systems in the control of mood dysfunction. As a first step to test the existence of such an interplay, we now exploited a rat model of repeated restraint stress to test if P 2X7 R blockade with BBG would impact A 2A R up-regulation and, conversely, if caffeine blockade of A 2A R could interfere with P 2X7 R up-regulation.

Animals
Male Wistar rats (adults, 220-250 g, n = 78: 18 controls treated with vehicle, nine controls treated with BBG, nine controls treated with caffeine; 18 stressed treated with vehicle, nine stressed treated with BBG, nine stressed treated with caffeine, six for electrophysiology) were obtained from Charles River (Barcelona, Spain) and were maintained at 23-25 • C, with 12 h light / 12 h dark cycle and standard chow and tap water ad libitum. All procedures in this study were conducted following the principles and procedures outlined as ''3Rs'' in the guidelines of the European Union (2010/63/EU), FELASA, and ARRIVE, and were approved by the Portuguese Ethical Committee (DGAV) and by the Institution's Ethics' Committee (ORBEA 238-2019/14102019). Since the behavioral alterations caused by this protocol of restraint stress were so far only validated in male rats, the ''3Rs'' guidelines imposed the use of only male rats to obtain the first proof-of-concept supporting the existence of any interaction between P 2X7 R and A 2A R.

In vivo Drug Treatments
As done previously (Carmo et al., 2014), the blood-brain barrierpermeant and efficacious P 2X7 R antagonist brilliant blue G (BBG, 45 mg/kg dissolved in saline; from Sigma-Aldrich, Portugal) or saline were administered intraperitoneally every 48 h at 7 PM, starting 3 days before the protocol of restraint stress, until the sacrifice of the animals. The tested dose of BBG has previously been shown to yield a brain concentration of 200-220 nM (Díaz-Hernández et al., 2012), which is within the effective and selective range of BBG towards central P 2X7 R and is without evident side-effects in control rodents (Donnelly-Roberts and Jarvis, 2007).
Caffeine (Sigma, Portugal) was administered through the drinking water as previously reported (Duarte et al., 2009;Cognato et al., 2010) at a dose (0.3 g/L) estimated to correspond to a daily intake of 3-4 cups of coffee by humans (Fredholm et al., 1999), which rodents consume without modification of their water intake (Duarte et al., 2009Silva et al., 2013). This yields a concentration of circa 30 µM in the brain parenchyma (Costenla et al., 2010;Silva et al., 2013), which selectively targets adenosine receptors  and mimics the neuroprotective impact of A 2A R antagonists, rather than of A 1 R (Cunha et al., 2006;Dall'Igna et al., 2007), namely in animal models of stress and depression (Kaster et al., 2015;Machado et al., 2017). Caffeine intake was allowed only overnight (7 PM-7 AM), starting 3 days before the protocol of restraint stress, until the sacrifice of the animals and this repeated exposure to caffeine is expected to afford neuroprotection without major modification of behavioral or physiological parameters in control rodents (Duarte et al., 2009;Yang et al., 2009;Cognato et al., 2010).

Restraint Stress
The stress model used consisted of a repeated physical restraint of rats, as done previously (Cunha et al., 2006). The rats were individually placed in a room adjacent to their colony in an independent plastic compartment and immobilized in a 25 × 7 cm plastic bottle, with a plastic taper on the outside and a 1 cm hole at one end for breathing. After the termination of each daily restraint stress session, the rats were returned to their home cages. The schedule of sub-chronic restraint stress consisted of a daily 4 h immobilization period (between 10 AM and 4 PM) during 14 consecutive days, the time previously defined to be required to cause stable behavioral modifications for at least 1 week in adult male rats (Cunha et al., 2006). Control age-matched rats were handled as their tested littermates except that they were not isolated or immobilized.

Behavioral Evaluation
Behavioral tests were carried out from 9 AM until 4 PM on the 15 th until the 18 th day after beginning the restraint stress protocol (Figure 1). As shown in Figure 1, the animals were subject to a tight schedule of behavioral characterization, with a minimal time interval between each test, which could lead to crosstesting interferences. However, the analysis of the performance of control animals in the successive tests did not show evident differences from historic controls where rats of the same age and strain were tested in each different test with wider time gaps between the different tests (Cunha et al., 2006;Cognato et al., 2010;Carmo et al., 2014;Coelho et al., 2014;Matheus et al., 2016). All behavior tests were carried out by two experimenters who were unaware of the phenotypes or drug treatments, in a soundattenuated room with an eight lux illumination and visual cues on the walls, to which the animals were previously habituated. The apparatuses were cleaned with 20% ethyl alcohol to remove any odors after testing each animal.
Locomotion and exploratory behavior were monitored using an open-field arena made of dark gray PVC measuring 100 × 100 cm 2 (divided by white lines into 25 squares of 20 × 20 cm 2 ) and was surrounded by 40-cm high walls. Each rat was placed in the center of the open field and the following variables were recorded for 10 min: number of peripheral squares (adjacent to the walls) crossed (peripheral locomotion), number of central squares (away from the walls) crossed (central locomotion) and total locomotion (peripheral locomotion plus central locomotion).
Anxiety was further assessed using the elevated plus-maze, which consisted of four arms of the same size (40 cm × 5 cm) arranged in the form of a cross and raised 50 cm above the floor. Two opposed arms were surrounded by 30 cm high opaque black Plexiglas walls, except for the entrance (closed arms) while the other two had no walls (open arms). Each animal was placed on the central square of the maze facing an enclosed arm and was allowed to explore the maze for 5 min. The number of entries and the time spent in both open and closed arms were recorded, considering an entry only when the whole body and four paws were inside an arm.
The depressive-like behavior was evaluated in the forced swimming test, where rats were placed in individual glass cylinders (40 cm in height and 17 cm in diameter) containing water (water depth was 30 cm, kept at 25 ± 1 • C) to measure the total duration of immobility, climbing, and swimming during a 10-min session. A rat was regarded as immobile when floating motionless or making only those movements necessary to keep its head above the water. The climbing behavior was defined as upward-directed movements of the forepaws usually along the side of the swimming chamber and the swimming behavior is defined as movement (usually horizontal) throughout the swimming chamber; diving and face shaking behaviors were not considered.
Anhedonic-like behavior was evaluated with the sucrose preference test, where rats were first single-housed in a cage with two bottles and free access to food. After 4 h of habituation, one bottle was randomly switched to contain 1.2% sucrose solution and the total consumption of water and sucrose solution was measured at the end of a 16 h test period (12 h dark phase plus 4 h light phase). Sucrose preference was calculated as the ratio of sucrose vs. total intake. Spatial memory was evaluated using a 2-trials Y-maze paradigm (Dellu et al., 1997). The test was carried out in a Plexiglas apparatus with equal three arms (10 cm wide, 35 cm long, and walls of 25 cm height) in a Y-shape, separated by equal angles. The test consists of two sessions of 5 min duration separated by a 2-h inter-trial interval. During the first session, the rat was placed at the end of one arm and allowed to explore the two available arms since the third arm (the novel arm) was blocked by a guillotine door. During the second session, the '''novel''' arm was opened and the rat was again placed in the start arm and allowed to explore the three arms. Memory performance was evaluated by measuring the time spent exploring the ''novel'' arm compared to the exploration of the other two arms. An entry into an arm was defined as the placement of all four paws into the arm.
Hippocampal-dependent memory was also evaluated using the object displacement test, where rats were exposed to two identical objects in the same open field apparatus in which they were habituated and were allowed to explore for 5 min the objects fixed in opposite corners 10 cm away from walls and 70 cm apart from each other. In the test trial, carried out 2-h after, rats were again placed for 5 min in the open field arena, except that one of the objects was moved to a novel position. Memory performance was quantified with an object displacement index defined as the ratio between the time exploring the object in the novel location over the total time exploring both objects. Exploration of an object is defined as directing the nose to the object at a distance equal to or less than 2 cm from the object and/or touching it with the nose; rearing on to the object was not considered exploratory behavior.
The sequence of the tests is indicated in Figure 1.

mRNA Expression
After completion of the battery of behavior analysis, rats were sacrificed by decapitation under deep anesthesia upon exposure to a halothane-saturated atmosphere. One hippocampus or part of the prefrontal cortex of each rat was used to extract total RNA with a MagNA Lyser Instrument and a MagNA Pure Compact RNA Isolation kit (Roche, Portugal), according to the manufacturer's instructions. The integrity, quantity, and purity of the RNA yields were checked by electrophoresis and spectrophotometry. Reverse transcription for first-strand cDNA synthesis from each sample was performed using a random hexamer primer with the Transcriptor First Strand cDNA Synthesis kit (Roche), according to the manufacturer's instructions. The resulting cDNAs were used as templates for real-time PCR, which was carried out on the LightCycler instrument (Roche) using the FastStart DNA Master SYBR Green I kit (Roche). The mRNA expression of the marker of microglia ''activation'' Iba1 (ionized calcium-binding adaptor molecule 1), of the pro-inflammatory cytokines interleukin-1β (IL1β) and tumor necrosis factor α (TNFα) and of P 2X7 R, was calculated relative to GADPH (glyceraldehyde 3-phosphate dehydrogenase) mRNA expression, using the following primers . Quantification was carried out based on standard curves run simultaneously with the test samples generated by conventional PCR amplification, as previously described (Costenla et al., 2011;Rebola et al., 2011). The purity and specificity of the resulting PCR products were assessed by melting curve analysis and electrophoresis. Control reactions were performed to verify that no amplification occurred without cDNA.
The binding assays were performed as previously described (Cunha et al., 2006), using the second hippocampus and the rest of the prefrontal cortex from each rat. After purifying whole membranes by centrifugation-based fractionation (Rebola et al., 2005), the membranes were resuspended in Tris-Mg solution (containing 50 mM Tris and 10 mM MgCl 2 , pH 7.4) with 4 U/ml of adenosine deaminase (to remove endogenous adenosine). Binding with 2 nM of 3 H-SCH58261 (specific activity of 77 Ci/mmol; prepared by GE Healthcare and offered by E.Ongini, Schering-Plough, Italy), a supramaximal concentration of this selective A 2A R ligand (Lopes et al., 2004), was performed for 1 h at room temperature with 286-343 (hippocampus) or 54-71 µg of protein (prefrontal cortex), with constant swirling. The binding reactions were stopped by the addition of 4 ml of ice-cold Tris-Mg solution and filtration through Frontiers in Cellular Neuroscience | www.frontiersin.org Whatman GF/C filters (GE Healthcare). The radioactivity was measured with 2 ml of scintillation liquid (AquaSafe 500 Plus, Zinsser Analytic). The specific binding was expressed as fmol/mg protein and was estimated by subtraction of the non-specific binding, which was measured in the presence of 12 µM of xanthine amine congener (XAC; Sigma), an antagonist of adenosine receptors. All binding assays were performed in duplicate.
The fluorescence data were background-corrected by subtracting the mean fluorescence value of N9 cells that were not incubated with Fluo-4-AM. Intracellular calcium concentration was estimated for each time point using the formula: , F is the fluorescence recorded at each time point, F max is the maximal fluorescence, obtained upon ionomycin application, and F min is the minimal fluorescence. The magnitude of [Ca 2+ ] i transients evoked by each stimulus (∆[Ca 2+ ] i ) was obtained subtracting the mean of basal levels from the maximum value after stimulus application.

Electrophysiological Recordings
Rats were decapitated after anesthesia and the brain was quickly removed and placed in ice-cold, oxygenated (95% O 2 , 5% CO 2 ) artificial cerebrospinal fluid (ACSF; in mM: 124.0 NaCl, 4.4 KCl, 1.0 Na 2 HPO 4 , 25.0 NaHCO 3 , 2.0 CaCl 2 , 1.2 MgCl 2 , 10.0 glucose). Using a McIlwain tissue chopper (Brinkmann Instruments, NY, USA), slices (400 µm-thick) from the dorsal hippocampus were cut transverse to its long axis and placed in a holding chamber with oxygenated ACSF. Slices were allowed to recover for at least 1 h before being transferred to a submerged recording chamber and superfused at 3 mL/min with oxygenated ACSF kept at 30.5 • C.
Extracellular field excitatory post-synaptic potential (fEPSP) were recorded as previously described (Costenla et al., 2011) with the stimulating bipolar concentric electrode placed in the proximal CA1 stratum radiatum for stimulation of the Schaffer collaterals and the recording electrode, filled with 4 M NaCl (2-5 M resistance), placed in the CA1 stratum radiatum targeting the distal dendrites of pyramidal neurons. Stimulation was delivered every 20 s with rectangular pulses of 0.1 ms duration using either a Grass S44 or a Grass S48 square pulse stimulator (Grass Technologies, RI, USA). After amplification (ISO-80, World Precision Instruments, Hertfordshire, UK), the recordings were digitized (BNC-2110, National Instruments, Newbury, UK), averaged in groups of three, and analyzed using the WinLTP version 2.10 software (WinLTP Limited, Bristol, UK; Anderson and Collingridge, 2007). The intensity of stimulation was chosen between 30-50% of maximal fEPSP response, determined based on input/output curves in which the fEPSP slope was plotted vs. stimulus intensity. Alterations of basal synaptic transmission were quantified as the percentage change of the average value of the fEPSP slope taken from 15-20 min after beginning exposure to the tested drug applied through the superfusion medium, relative to the average value of the fEPSP slope during the 5 min that preceded the application of each modifying drug. Long-term potentiation (LTP) was induced by a high-frequency stimulation (HFS) train (100 Hz for 1 s). LTP was quantified as the percentage change of the average fEPSP slope taken between 55 and 60 min after LTP induction relative to the average slope of the fEPSP measured during the 10 min that preceded LTP induction. The effect of drugs on LTP was assessed by comparing LTP magnitude in the absence and presence of the Frontiers in Cellular Neuroscience | www.frontiersin.org drug in experiments carried out in different slices from the same animal.

Statistics
Data are presented as the mean ± SEM of n experiments (i.e., n independent rats or cell cultures). The comparison of control and stressed rats and the effect of drugs was analyzed using a two-tailed unpaired Student's t-test. When testing the impact of a drug on the effects of stress, the data were first analyzed with a two-way ANOVA followed by a Newman-Keuls post hoc test. The comparison between the effect of multiple drugs was carried out using a Dunnett's test. All tests were performed using Prism 6.0 software (GraphPad, San Diego, CA, USA) considering significance at a 95% confidence interval.

RESULTS
The model of repeated restraint stress triggers robust and reproducible behavioral alterations of mood and memory in adult rats (see Figure 2). Thus, whereas there was no significant change of spontaneous locomotion (n = 18; t = 0.991, p = 0.328, unpaired Student's t-test; Figure 2A), stressed rats displayed a thigmotaxic behavior indicative of an increased anxiety-like profile, as indicated by the decreased number of crossings in the center of the open field (n = 18; t = 7.229, p < 0.001, unpaired Student's t-test; Figure 2B). This was confirmed in the elevated plus-maze where stressed rats displayed a decreased number of entries in the open arms (n = 18; t = 9.002, p < 0.001, unpaired Student's t-test; Figure 2C) and decreased time in the open arms (n = 18; t = 8.628, p < 0.001, unpaired Student's t-test). Stressed rats also displayed anhedonic behavior in a sucrose preference test (n = 18; t = 5.673, p < 0.001, unpaired Student's t-test; Figure 2D) and an increased immobility time in the forced swimming test (n = 18; t = 9.959, p < 0.001, unpaired Student's t-test; Figure 2E), as well as a decreased time spent climbing the walls of the swimming container (n = 18; t = 7.069, p < 0.001, unpaired Student's t-test; Figure 2F), indicative of depressive-like behavior. Short-term memory was also deteriorated in stressed compared to control rats, as observed by a decreased time searching the novel (previously hidden) arm of a Y-maze (n = 18; t = 6.033, p < 0.001, unpaired Student's t-test; Figure 2G) and a decreased preference to explore the displaced object (t = 8.009, p < 0.001 between displaced and non-displaced object in control rats and t = 1.885, p = 0.069 between displaced and non-displaced object in stressed rats, unpaired Student's t-test; Figure 2H).

Impact of the P 2X7 R Antagonist BBG
The P 2X7 R-prefering antagonist Brillant Blue G (BBG, 45 mg/kg) was devoid of effects in control rats but attenuated or prevented the behavioral and neurochemical alterations caused by repeated stress (Figures 4, 5) Figure 4E), the stress-induced decrease of the time climbing the wall in the forced swimming test (effect of stress F (1,32) = 16.35, p < 0.001; effect of BBG F (1,32) = 36.11, p < 0.001; interaction F (1,32) = 12.87, p = 0.001; two-way ANOVA; Figure 4F), the stress-induced decrease of the time spent in the novel arm of the Y-maze (effect of stress F (1,32) = 9.243, p = 0.005; effect of BBG F (1,32) = 6.434, p = 0.016; interaction F (1,32) = 3.596, p = 0.067; two-way ANOVA; Figure 4G), and the stress-induced decrease of the relative time exploring the displaced object (t = 1.928, p = 0.072 between displaced and non-displaced object in stressed rats treated with vehicle and t = 6.246, p < 0.001 between displaced and non-displaced object , and in the elevated plus-maze (C) tests, anhedonia as evaluated in the sucrose preference test (D), helpless-like behavior as evaluated by the forced-swimming test (E,F) and impaired memory performance as evaluated by a modified Y maze test (G) and an object-displacement test (H). Data are shown as mean ± SEM; n = 16-18 rats per group. *P < 0.001 using a Student's t-test.
FIGURE 3 | Male adult Wistar rats (8-10 weeks old) subject to a protocol of restraint stress (4 h/day) during 14 days display an increased expression of inflammatory markers and an up-regulation of P 2X7 and A 2A receptors in the hippocampus (black) and prefrontal cortex (gray). Compared with non-stressed control rats (open bars), stressed rats (red checkered bars) displayed an increased expression of the microglia marker Iba1 (A), of interleukin 1β (IL1β; B), of tumor necrosis factor α (TNFα; C), and P 2X7 receptors (P 2X7 R; D) as well as an increased density of A 2A receptors (A 2A R; E) as assessed by the binding density of a supramaximal concentration of the selective A 2A R antagonist 3 H-SCH58261 (2 nM). Data are shown as mean ± SEM; n = 11-12 rats per group. *P < 0.001 vs. control using a Student's t-test.
in stressed rats treated with BBG, unpaired Student's t-test; Figure 4H).  , helpless-like behavior as evaluated by the forced-swimming test (E,F) and impaired memory performance as evaluated by a modified Y maze test (G) and an object-displacement test (H). Data are shown as mean ± SEM; n = 8-9 rats per group. *P < 0.05 vs. control-water, **P < 0.05 vs. stress-water using a Tukey's multiple comparisons post hoc test after a two-way ANOVA.
FIGURE 5 | Male adult Wistar rats (8-10 weeks old) subject to a protocol of restraint stress (4 h/day) during 14 days display an increased expression of inflammatory markers and an up-regulation of P 2X7 and A 2A receptors in the hippocampus (black, dark green) and prefrontal cortex (gray, light green) which were prevented by the P 2X7 receptor antagonist Brillant Blue G (BBG). Whereas BBG treatment (45 mg/kg, ip, daily, beginning 3 days before the stress protocol and until the sacrifice of the animals; green) was devoid of effects in non-stressed control rats (open bars), BBG prevented all alterations of stressed rats (red checkered bars), namely the increased expression of the microglia marker Iba1 (A), of interleukin 1β (IL1β; B), of tumor necrosis factor α (TNFα; C), and P 2X7 receptors (P 2X7 R; D) as well as an increased density of A 2A receptors (A 2A R; E) as assessed by the binding density of a supramaximal concentration of the selective A 2A R antagonist 3 H-SCH58261 (2 nM). Data are shown as mean ± SEM; n = 5-7 rats per group. *P < 0.05 vs. control-water, **P < 0.05 vs. stress-water using a Tukey's multiple comparisons post hoc test after a two-way ANOVA.
The treatment with BBG also attenuated the stress-induced up-regulation of P 2X7 R in the hippocampus (effect of stress
Caffeine also attenuated the stress-induced increase in the expression of the marker of ''activated'' microglia Iba1 in the hippocampus (

P 2X7 R -A 2A R Interaction in Microglial N9 Cells
Since we observed crosstalk between BBG and caffeine upon restraint stress, whereby BBG controlled the up-regulation of A 2A R and caffeine controlled the upregulation of P 2X7 R expression, and the stress-induced behavioral modifications were accompanied by a parallel control of markers of microglia ''activation'' and neuroinflammation, we next used a microglial N9 cell line to directly investigate a putative crosstalk between P 2X7 R and A 2A R, since both receptors are present and functional in this microglia cell model (e.g., Ferrari et al., 1996;Gomes et al., 2013).
The P 2X7 R-preferring agonist BzATP (100 µM) evoked an elevation of intracellular free Ca 2+ levels (∆[Ca 2+ ] i ) of 94.8 ± 14.5 nM (n = 16), which was inhibited (−76.51 ± 20.02%, n = 10-16, F (3, 44) = 13.21, p = 0.029) in the presence of the FIGURE 6 | Male adult Wistar rats (8-10 weeks old) subject to a protocol of restraint stress (4 h/day) during 14 days display the expected features of depressed rats, which were prevented by the adenosine receptor antagonist caffeine (caff). Whereas caffeine consumption (0.3 g/L, po, beginning 3 days before the stress protocol and until the sacrifice of the animals; blue) was devoid of effects in non-stressed control rats (open bars), caffeine prevented all behavioral modifications of stressed rats (red checkered bars): without modification of locomotor activity as evaluated in the open field (A), caffeine prevented anxiety-like behavior as evaluated in the open field (B) and in the elevated plus-maze (C) tests, anhedonia as evaluated in the sucrose preference test (D), helpless-like behavior as evaluated by the forced-swimming test (E,F) and impaired memory performance as evaluated by a modified Y maze test (G) and an object-displacement test (H). Data are shown as mean ± SEM; n = 8-9 rats per group. *P < 0.05 vs. control-water, **P < 0.05 vs. stress-water using a Tukey's multiple comparisons post hoc test after a two-way ANOVA.
FIGURE 7 | Male adult Wistar rats (8-10 weeks old) subject to a protocol of restraint stress (4 h/day) during 14 days display an increased expression of inflammatory markers and an up-regulation of P 2X7 and of A 2A receptors in the hippocampus (black, dark blue) and prefrontal cortex (gray, light blue) which were prevented by the adenosine antagonist caffeine (caff). Whereas caffeine consumption (0.3 g/L, po, beginning 3 days before the stress protocol and until the sacrifice of the animals; blue) was devoid of effects in non-stressed control rats (open bars), caffeine prevented all alterations of stressed rats (red checkered bars), namely the increased expression of the microglia marker Iba1 (A), of interleukin 1β (IL1β; B), of tumor necrosis factor α (TNFα; C) and P 2X7 receptors (P 2X7 R; D) as well as an increased density of A 2A receptors (A 2A R; E) as assessed by the binding density of a supramaximal concentration of the selective A 2A R antagonist 3 H-SCH58261 (2 nM). Data are shown as mean ± SEM; n = 5-7 rats per group. *P < 0.05 vs. control-water, **P < 0.05 vs. stress-water using a Tukey's multiple comparisons post hoc test after a two-way ANOVA.

P 2X7 R -A 2A R Interaction in the Control of Hippocampal Synaptic Plasticity
Since we and others have collected evidence for a role of synaptic dysfunction underlying stress-associated behavioral alterations (Duman and Aghajanian, 2012;Kaster et al., 2015) and suggestions of P 2X7 R-mediated synaptic dysfunction add-up to the well-established ability of A 2A R to control synaptic function (reviewed in Cunha, 2016), we next investigated if P 2X7 R and A 2A R might interact in the control of synaptic plasticity in excitatory synapses of the dorsal hippocampus.
We first tested the effect of P 2X7 R agonist BzATP on basal synaptic transmission. BzATP (30 µM) decreased hippocampal synaptic transmission by 54.75 ± 3.96% (n = 4); this effect recovered fully upon washout of BzATP and repeated administrations of 30 µM BzATP caused a similar depression of synaptic transmission (p > 0.05). This allowed exploring the pharmacology of BzATP (30 µM)-induced decreased The time course recordings are from representative experiments, whereas the bar graphs correspond to n = 6-18 independent cultures of N9 microglial cells. *p < 0.05 one-way ANOVA followed by a Dunnett's post hoc test compared to the first bar from the left (stimulus only, without modifiers, which were added 5 min before the stimulus).
hippocampal synaptic transmission: this effect was unaffected in the presence of 1 µM BBG (−48.98 ± 4.96%, n = 4, t = 1.245, p = 0.260 vs. the effect of BzATP alone) and was fully prevented in the presence of the adenosine A 1 receptor antagonist, DPCPX (50 nM; 2.97 ± 17.91% alteration of fEPSP slope, n = 4; t = 3.319, p = 0.016 vs. the effect of BzATP alone; Figure 9A). This shows the inexistence of a P 2X7 Rmediated effect (lack of effect of BBG) and indicates that BzATP is rapidly converted by ectonucleotidases (Cunha et al., 1998) into an adenosine analog to indirectly alter hippocampal synaptic transmission through inhibitory A 1 adenosine receptors (prevention by DPCPX), as previously proposed (Kukley et al., 2004). This precludes the use of BzATP to search for P 2X7 Rmediated effects in hippocampal slices. Instead, we tested the impact of P 2X7 R antagonists on high-frequency induced LTP in Schaffer collaterals-CA1 pyramidal cell synapses. LTP magnitude was not significantly altered by either 1 µM BBG (n = 8, t = 0.493, p = 0.630 vs. LTP magnitude in control conditions, i.e., in the FIGURE 9 | Lack of direct effects of P 2X7 receptors on hippocampal synaptic plasticity or its modulation by A 2A receptors. (A) The P 2X7 R agonist BzATP (30 µM) decreased synaptic transmission in Schaffer collaterals-CA1 pyramid synapses of hippocampal slices from adult rats (10-12 weeks old), but this effect was likely mediated through A 1 R since it was prevented by the A 1 R antagonist DPCPX (50 nM) but not by the P 2X7 R antagonist BBG (1 µM). Data are shown as mean ± SEM of n = 4; *p < 0.05 vs. control (100%, dashed line). (B-D) The P 2X7 R antagonists BBG (1 µM) or JNJ47965567 (JNJ, 1 µM) did not significantly modify the magnitude of Long-term potentiation (LTP; change in field excitatory post-synaptic potential (fEPSP) slope at 50-60 min) induced by a high-frequency stimulation (HFS) train concerning pre-HFS values (B,C) and also failed to alter the inhibition of LTP magnitude caused by the A 2A R antagonist SCH58261 (SCH, 50 nM; D). The inserts show recordings obtained in representative experiments of fEPSP responses obtained before (filled line) and 50-60 min after (dotted line) LTP induction in the presence or in the absence (control) of BBG; each trace comprises the stimulus artifact, followed by the presynaptic volley and the fEPSP. All values are shown as mean ± SEM of 5-8 experiments; *p < 0.05 vs. LTP magnitude in the absence of drugs (control). ns: non-significant.
absence of tested drugs) or 1 µM JNJ47965567 (n = 6; t = 0.754, p = 0.468 vs. control LTP magnitude; Figures 9B,C). This does not support a role of P 2X7 R in the control of synaptic plasticity.

DISCUSSION
The present study provides compelling novel evidence for a hitherto unrecognized interaction between P 2X7 R and A 2A R in the control of brain dysfunction. This conclusion is based on the parallel effects of BBG, a P 2X7 R preferring antagonist, and of caffeine, which antagonizes A 2A R, to prevent neuroinflammation and behavioral alterations upon repeated restraint stress and on the ability of caffeine to prevent P 2X7 R upregulation and of BBG to prevent A 2A R up-regulation; although these in vivo evidence are only suggestive of a P 2X7 R-A 2A R interaction, this contention is further supported by the independent in vitro experiments showing that P 2X7 R and A 2A R closely interact in the control of calcium responses in N9 microglial cells. This indicates that these two, so far considered independent, arms of the purinergic system (Agostinho et al., 2020), operated by ATP-P 2 R and by adenosine-P 1 R might actually cooperate to control adaptative brain function. Importantly, this proofof-concept, so far only confirmed to occur in male rats (selected to cope with the ''3R'' guidelines), still needs to be extended to female rats, an issue of particular importance since there are gender differences in the A 2A R modulation of microglia and neuroinflammatory-like responses in rodents (Caetano et al., 2017;Simões-Henriques et al., 2020).
The present study extends to a model of repeated restraint stress the ability of P 2X7 R blockade to attenuate behavioral modifications upon chronic stress (Iwata et al., 2016;Yue et al., 2017;Farooq et al., 2018;Aricioglu et al., 2019;reviewed in Illes et al., 2020). This is in agreement with the association of P 2X7 R polymorphisms with depressive symptoms (see meta-analysis in Czamara et al., 2018) and reinforces the concept of ATP as a danger signal in brain dysfunction (reviewed in Rodrigues et al., 2015). As observed by others in different animal models of brain dysfunction (Jimenez-Pacheco et al., 2013;Wang et al., 2017;Martínez-Frailes et al., 2019;Song et al., 2019), namely upon chronic stress (Yue et al., 2017;Dang et al., 2018; but see Kongsui et al., 2014), we identified an up-regulation of P 2X7 R and an ability of P 2X7 R to control different markers of neuroinflammation, as also reported in other animal models of depression (Yue et al., 2017;Bhattacharya and Jones, 2018), to mediate stress-induced behavioral modifications Deng et al., 2020;Troubat et al., 2021).
The present study also provides the first demonstration that a prolonged (days) intake of caffeine prevents behavioral modifications caused by repeated restraint stress, as has been observed in other animal models of stress (Pechlivanova et al., 2012;Kaster et al., 2015;Yin et al., 2015;Kasimay Cakir et al., 2017) and in individuals with mood dysfunction, namely depression (reviewed in Grosso et al., 2016;Wang et al., 2016) and suicide ideation (e.g., Lucas et al., 2014;Park et al., 2019).
The protective effects of caffeine in animal stress models are mimicked by selective A 2A R blockade (Kaster et al., 2015) and A 2A R polymorphisms are associated with the incidence of major depression (Oliveira et al., 2019). We also observed an up-regulation of A 2A R, as occurs in different conditions of brain dysfunction (reviewed in Cunha, 2016), namely upon repeated stress (Cunha et al., 2006;Kaster et al., 2015). A 2A R, as well as caffeine, can control abnormal synaptic plasticity and synaptic dysfunction (e.g., Kaster et al., 2015;Temido-Ferreira et al., 2020) and also control microglia reactivity and neuro-inflammation (e.g., Brothers et al., 2010;Rebola et al., 2011;Mao et al., 2020), but the exact mechanism underlying the ability of A 2A R to control mood dysfunction upon chronic stress remains to be defined.
Apart from establishing the ability of BBG and caffeine to attenuate behavioral alterations in this particular model of repeated restraint stress, the major finding of the present study is the existence of putative crosstalk between the two purinergic signaling systems operated by each of these antagonists. The inhibition of the stress-induced up-regulation of A 2A R by BBG and, conversely, the inhibition of the stress-induced up-regulation of P 2X7 R by caffeine is suggestive of crosstalk between the two types of purinergic receptors in vivo. This was reinforced by parallel experiments studying calcium transients in microglial N9 cells. In fact, in microglial N9 cells, A 2A R activation increased and A 2A R blockade decreased BzATPinduced calcium transients, which was mediated by P 2X7 R, and conversely, a selective P 2X7 R antagonist attenuated CGS26180induced calcium transients, which was largely mediated by A 2A R. Since synaptic alterations have also been proposed to underlie stress-induced alterations of brain function (Duman and Aghajanian, 2012;Vose and Stanton, 2017), we also investigated if there was crosstalk between P 2X7 R and A 2A R in synaptic alterations, namely in the process of LTP in the hippocampus. While we have previously established a selective role of A 2A R controlling synaptic plasticity without an effect on basal synaptic transmission (Costenla et al., 2011;Gonçalves et al., 2019;Temido-Ferreira et al., 2020), a putative role of P 2X7 R on the control of hippocampal synaptic transmission has been controversial (Armstrong et al., 2002;Kukley et al., 2004;Klaft et al., 2012;Khan et al., 2019) and an eventual role of P 2X7 R on the control of synaptic plasticity had not yet been tested. We now show that BzATP decreases synaptic transmission, but this effect is blocked by the selective A 1 R antagonist DPCPX (see Kukley et al., 2004), following the remarkable efficiency of ectonucleotidases to metabolize ATP derivates into their adenosine derivative counterparts (Cunha et al., 1998) to activate the abundant and efficient presynaptic A 1 R that decrease excitatory transmission in the hippocampus (reviewed in Dunwiddie and Masino, 2001). Thus, we resorted to testing the impact of P 2X7 R antagonists (BBG and JNJ47965567) on hippocampal LTP and concluded that P 2X7 R does not seem to control hippocampal LTP under physiological conditions. Furthermore, we did not observe the ability of P 2X7 R antagonists to modify the decrease of LTP caused by the blockade of A 2A R.
In contrast to the inconclusive effects on a putative P 2X7 R-A 2A R interaction in the control of synaptic plasticity, the crosstalk between P 2X7 R and A 2A R in the control of microglial responses suggests that the interplay between P 2X7 R and A 2A R to control brain maladaptive function upon repeated stress might mostly be due to crosstalk in the control of neuroinflammation rather than of synaptic plasticity. Interestingly, crosstalk between P 2 and P 1 receptors in the control of microglia was first documented by Kettenmann's group (Färber et al., 2008) and further developed by Koizumi's group (reviewed in Koizumi et al., 2013); however, these P 2 R-P 1 R interactions in microglia were not characterized to involve P 2X7 R and A 2A R, although parallel effects of P 2X7 R and A 2A R have previously been described to control inflammatory processes (Savio et al., 2017) and brain injury (Ye et al., 2018). We now demonstrate direct crosstalk between both receptors in the control of microglial N9 cell responses, which is paralleled by the ability of antagonists of each receptor to control the other's up-regulation upon repeated stress. This is highly suggestive of direct cooperation between the two arms of the purinergic modulation system to control neuro-inflammation and the adaptive central responses to repeated stress. However, future studies still need to detail if the P 2X7 R-A 2A R interaction only occurs in microglia or might also take place in astrocytes. In fact, P 2X7 R (reviewed in Franke et al., 2012) and A 2A R (reviewed in Cunha, 2016) also have profound effects on the pathophysiological roles of astrocytes and the involvement of astrocytes in the control neuroinflammation and neuronal function as well as adaptation to repeated stress (reviewed in Rial et al., 2016) cannot exclude them as a possible major locus of P 2X7 R-A 2A R interactions to control the observed behavioral modifications upon repeated restraint stress.
The detailed mechanisms of this P 2X7 R-A 2A R interactions also remain to be unraveled and they can involve different possibilities: one possibility is the formation of heteromers, which has been documented for P 2X7 R (Antonio et al., 2011) and for A 2A R (reviewed in Ferré and Ciruela, 2019) and between different P 2 R and P 1 R (Namba et al., 2010); another possibility is the use of transducing systems of each receptor to control the other receptor function, as has been shown for P 2X7 R controlling metabotropic receptors (reviewed in Miras-Portugal et al., 2019), A 2A R controlling ionotropic receptors (e.g., Garção et al., 2013;Temido-Ferreira et al., 2020) and between different P 2 R and P 1 R (George et al., 2016); a third possibility is a key role of ecto-nucleotidases metabolizing ATP into adenosine in a rapid (Dunwiddie et al., 1997;Cunha et al., 1998) and highly controlled manner (James and Richardson, 1993;Cunha, 2001) to format the balanced activation of both receptors (Kukley et al., 2004;Liston et al., 2020). After this first step establishing an interaction between A 2A R and P 2X7 R, future work will be required to detail the mechanistic basis of this A 2A R-P 2X7 R interaction.
In conclusion, the present study provides evidence for crosstalk between P 2X7 R and A 2A R in the control of neuroinflammation and adaptive responses to restraint stress. The importance of these findings is best heralded by the new prospects to simultaneously target P 2X7 R and A 2A R to maximize the neuroprotective potential of the purinergic system. The present findings place at the center-stage the need to study the purinergic system as a whole and understand the relative contribution of its different constituents to provide the required integrative views (see Agostinho et al., 2020) to justify robust protective strategies to control maladaptation of brain function characteristic of neuropsychiatric disorders.

DATA AVAILABILITY STATEMENT
Data will be made available upon reasonable and justified request. Requests to access the datasets should be directed to cunharod@gmail.com.

ETHICS STATEMENT
The animal study was reviewed and approved by the Portuguese Ethical Committee (DGAV) and by the Institution's Ethics Committee (ORBEA 238-2019/14102019).