Abstract
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.
Highlights
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
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 A2A receptors (A2AR) (Rebola et al, 2008; Augusto et al, 2013; Carmo et al, 2019; Gonçalves et al, 2019), as well as with A2AR 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)
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)
Summary
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). A2AR are mainly located in synapses (Rebola et al, 2005), and 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 A2AR 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 A2AR polymorphisms with anxiety and depression (Hamilton et al, 2004; Hohoff et al, 2010; Oliveira et al, 2019). As a first step to test the existence of such an interplay, we exploited a rat model of repeated restraint stress to test if P2X7R blockade with BBG would impact A2AR up-regulation and, if caffeine blockade of A2AR could interfere with P2X7R up-regulation
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