Of adenosine and the blues: the adenosinergic system in the pathophysiology and treatment of major depressive disorder

Pharmacological Research 163 (2021) 105363 Available online 4 December 2020 1043-6618/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Of adenosine and the blues: The adenosinergic system in the pathophysiology and treatment of major depressive disorder Joana I. Gomes a , b , 1 , Miguel Farinha-Ferreira a , b , 1 , N´ adia Rei a , b , Joana Gonçalves-Ribeiro a , b , Joaquim A. Ribeiro a , b , Ana M. Sebasti˜ ao a , b , Sandra H. Vaz a , b , * a Instituto de Farmacologia e Neurociˆ encias, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal b Instituto de Medicina Molecular Jo˜ ao Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal ARTICLE INFO Keywords: Adenosinergic system Adenosine Major Depressive Disorder Anxiety Antidepressants ABSTRACT Major depressive disorder (MDD) is the foremost cause of global disability, being responsible for enormous personal, societal, and economical costs. Importantly, existing pharmacological treatments for MDD are partially or totally ineffective in a large segment of patients. As such, the search for novel antidepressant drug targets, anchored on a clear understanding of the etiological and pathophysiological mechanisms underpinning MDD, becomes of the utmost importance. The adenosinergic system, a highly conserved neuromodulatory system, appears as a promising novel target, given both its regulatory actions over many MDD-affected systems and processes. With this goal in mind, we herein review the evidence concerning the role of adenosine as a potential player in pathophysiology and treatment of MDD, combining data from both human and animal studies. Altogether, evidence supports the assertions that the adenosinergic system is altered in both MDD patients and animal models, and that drugs targeting this system have considerable potential as putative antidepressants. Furthermore, evidence also suggests that modifications in adenosine signaling may have a key role in the effects of several pharmacological and non-pharmacological antidepressant treatments with demonstrated efficacy, such as electroconvulsive shock, sleep deprivation, and deep brain stimulation. Lastly, it becomes clear from the available literature that there is yet much to study regarding the role of the adenosinergic system in the pathophysiology and treatment of MDD, and we suggest several avenues of research that are likely to prove fruitful. 1. Introduction Major Depressive Disorder (MDD) is a debilitating condition, affecting 246–286 million people worldwide [1], characterized by persistently depressed mood, inability to feel pleasure (anhedonia), motivational deficits, increased anxiety, somatic symptoms, cognitive impairments, sleep dysfunction, as well as suicidal thoughts and/or attempts [2,3]. The personal, societal, and economic burden of MDD is difficult to overstate. Indeed, MDD is the single greatest cause of global disability [4], representing a major burden upon public healthcare systems, and on the global economy at large. This impact is even greater when taking into account the fact that MDD is widely recognized major risk factor for other disabling conditions, such as substance abuse disorders [5], and cardiovascular disease [6]. The impact of MDD is largely amplified by the fact that the existing treatments, which include psychotherapy [7], pharmacological treatment, and other non-pharmacological biological interventions, such as transcranial magnetic stimulation [8] or electroconvulsive therapy (ECT) [9], are often partially or totally ineffective. In fact, a 2006 large-scale research effort found that approximately 30 % of patients do not respond to any drug treatment [10]. Furthermore, even when effective, the existing antidepressants are often not well tolerated, causing a myriad of undesirable and often serious side-effects leading to treatment discontinuation [11], and seem to primarily target emotional symptoms. MDD-induced cognitive dysfunction has been much less targeted, despite its role in patient quality of life [12,13]. These problems underline the need of developing novel, effective, and safe antidepressant compounds. Unfortunately, research efforts have largely failed in this regard, likely due to a combination of factors. For one, MDD is characterized by an extremely diverse and complex * Corresponding author at: Instituto de Medicina Molecular Jo˜ ao Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. E-mail address: [email protected] (S.H. Vaz). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs https://doi.org/10.1016/j.phrs.2020.105363 Received 15 September 2020; Received in revised form 30 November 2020; Accepted 1 December 2020 Pharmacological Research 163 (2021) 105363 2 pathophysiology [3,4,14]. In fact, at the neurobiological level, in addition to monoaminergic dysfunctions [15], MDD is characterized by disruptions in glutamate [16,17], γ-amino-butyric-acid (GABA) [17,18], and endocannabinoid signaling [19], altered hypothalamic-pituitary-adrenal (HPA) axis activity [20,21], increased neuroinflammation [22,23], decreased neuroand synaptogenesis [24–26], diminished brain-derived neurotrophic factor (BDNF) signaling [27,28], and impaired synaptic plasticity [29,30], among other numerous alterations. Furthermore, the etiology of MDD is not yet fully understood, with no individual, or group of, causative factors being capable of reliably predicting MDD diagnosis. Nonetheless, it is known that multiple factors play a role in the development of this disorder, including biological (e.g., genetic predisposition, hormonal imbalance, inflammatory or metabolic disease), environmental (e.g., early childhood adversity, acute trauma, chronic stress), and personal factors (e.g., personality traits, cognitive and coping styles, poor social relationships), often interacting with one another [31–36]. On the other hand, investment in neuropsychopharmaceutical development has continually decreased in the past decades [37], in part due to the poor investment-to-reward ratio that has characterized this field of drug development. Importantly, this has also likely influenced the type of drugs that have been developed, with most newly developed antidepressants being based upon the widely popular monoaminergic hypothesis of MDD, in essence being small improvements upon the commercially successful monoamine reuptake inhibitors. Indeed, in the last 10–20 years, less than a handful of compounds with partially or entirely monoamine-independent mechanisms of action [38–40] have been brought to market. One as of yet largely unexplored target for antidepressant drug development is the adenosinergic system [41–43], here defined as the system that encompasses the enzymes responsible for the synthesis and catabolism of adenosine, the nucleoside transporters responsible for adenosine transport, and the P1 family of purinergic receptors. The inattention to the adenosinergic system as a target for MDD is somewhat surprising, considering that this ubiquitous neuromodulatory system has a modulatory role over most MDD-implicated dysfunctions. Indeed, the adenosinergic system has been demonstrated to have functional interactions with the monoaminergic [44,45], glutamatergic [46], GABAergic [47], and endocannabinoid systems [48,49], to modulate HPA axis [50] and neuroinflammatory activity [51], to be involved in the regulation of neuroand synaptogenesis [52,53], as well as of BDNF signaling [54], and to impact synaptic plasticity [55,56]. Furthermore, adenosinergic drugs are already commercially available, and are known to be mostly safe. In fact, the most widely used psychoactive substance in the world – caffeine – exerts its effects through adenosine receptor antagonism [42]. Thus, the adenosinergic system appears as a prime target for the development of novel antidepressant drugs. Moreover, given its widespread interactions with multiple neurobiological systems and processes affected in MDD pathophysiology, understanding how changes in the adenosinergic system may cause, and/or be caused by depressive states would not only inform additional drug development, but also further increase our understanding of the pathophysiology of MDD. As such, the aim of the present review is to provide a broad overview of adenosinergic system in relation to both the pathophysiology and treatment of MDD. Specifically, after an initial outline of the key elements in adenosinergic system, as well as of their distribution and functioning under physiological conditions, we will sequentially review the evidence regarding how each of those elements is affected in MDD conditions, including studies in MDD patients and in rodent models of MDD. Furthermore, we will review how manipulations of each of those elements of the adenosinergic system impact depressive symptoms and associated neurobiological dysfunctions. Lastly, we will provide a critical appraisal of the overall literature allowing suggestions for future research on this subject. 2. Overview of the adenosinergic system Adenosine is one of the most ubiquitous and conserved neuromodulators in the central nervous system (CNS). In this section we will provide a brief overview of the receptors, transporters, and the enzymatic pathways responsible for the synthesis and breakdown of adenosine (see Fig. 1), so as to better contextualize the findings relating each of those constituents to the pathophysiology and treatment of MDD. 2.1. Adenosine synthesis, transport and catabolism Extracellular adenosine levels in CNS are regulated by a complex machinery comprised of synthetic and degradative enzymes, as well as transporters. The relative relevance of each element depends on the cell metabolic state and, in the case of neuronal cells, their excitability status. Under basal conditions extracellular adenosine concentration is in the range of 25–250 nM [57], being sufficient for tonic activation of a substantial fraction of adenosine receptors. However, pathological situations involving abnormally high neuronal and astrocytic activity – such as hypoxia, ischemia, and seizures – result in markedly elevated extracellular concentrations of adenosine, largely due to an increase in the extracellular metabolism of adenine nucleotides to adenosine [57–59]. Congruently, many adenosine-mediated effects that are observed to a lesser extent under normal conditions (e.g. presynaptic inhibition of glutamate release), are greatly augmented during such pathological events, representing a neuroprotective mechanism [59–61]. Adenosine is synthetized both intraand extracellularly. Intracellular adenosine synthesis occurs through the dephosphorylation of 5′-adenosine monophosphate (AMP) by the cytosolic enzyme 5′-nucleotidase, or through the hydrolysis of S-adenosil-homocysteine (SAH) by the enzyme SAH hydrolase. Two soluble 5′-nucleotidases have been identified: an inosine monophosphate (IMP)-selective cytosolic 5′-nucleotidase, and an AMP-selective cytosolic 5′-nucleotidase. The intracellular concentration of AMP under physiological conditions (0.1−0.5 mM) is much lower than the K m values for AMP cytosolic-5′-nucleotidases (1−14 mM). As such, these enzymes only respond to abnormally high concentrations of AMP, which are primarily associated with increased metabolic activity. A small variation in adenosine triphosphate (ATP) catabolism can induce a large increase in AMP concentration, as the intracellular concentration of ATP is about 50 times higher than that of AMP [58]. This fact contributes to the proposal that the intracellular formation of adenosine from catabolism of cytosolic ATP is a highly sensitive signal of increased metabolic rate or metabolic stress [58,62]. Another source of adenosine is the transmethylation pathway, where adenosine results from SAH hydrolysis by SAH hydrolase (SAHH), which simultaneously produces l-homocysteine [63]. SAHH is involved in transmethylation mechanisms, since SAH results from transmethylase activity upon S-adenosylmethionine (SAM). Furthermore, SAM is a methyl donor in cells, and through SAM-dependent methyltransferases these methyl groups can be transferred to several types of substrates, such as nucleic acid, protein, phospholipids, and monoamine neurotransmitters, being deeply involved in epigenetic modifications [63,64]. SAH is able to inhibit SAM-dependent transmethylation reactions, with this inhibition being limited by the metabolic conversion of SAH to adenosine and l-homocysteine [63,64]. Brain SAHH expression is highest in the cortex and cerebellum, but under non-pathological conditions SAHH has low impact upon neuronal excitability [65]. This suggests a minor role for SAHH in the control of neuronal cytoplasmic adenosine levels, in contrast to what appears to occur in cardiac muscle cells [66]. At the extracellular level, adenosine is produced by the conversion of released adenine nucleotides (especially ATP) via the ectonucleotidase pathway, and of cyclic adenosine monophosphate (cAMP) via the ectophosphodiesterase pathway. Specifically, ATP released as neurotransmitter, neuromodulator or gliotransmitter, by neurons and/or glial cells, J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 3 undergoes rapid enzymatic catabolism originating adenosine diphosphate (ADP), AMP, and adenosine [67,68]. This catabolic process involves multiple ectonucleotidases such as E-NTPDases (ectonucleoside triphosphate diphosphohydrolases) – which include CD39, also known as NTPDase 1 or ecto-apyrase – E-NPPs (ectonucleotide pyrophosphatase and/or phosphodiesterases), alkaline phosphatases (APs), and ecto-5′-nucleotidase (CD73) [69,70]. These enzymes vary in several important aspects, most notably their specific substrates and end-products, but also in their coupling to the plasma membrane [70]. E-NTPDases, which possess transmembranar domains, are nucleotide-specific and hydrolyze nucleoside triand diphosphates, resulting in their respective monophosphates [69,70]. E-NPPs, the vast Fig. 1. Schematic representation of the adenosinergic system. Depending on energy requirements, intracellular ATP and adenosine are constantly recycled through a series of dephosphorylation and phosphorylation steps, mediated by enzymes such as 5 ′-nucleotidase (5′-NT), adenosine kinase (ADK), AMP kinase (AMPK), and S-adenosil-homocysteine (SAH) hydrolase. Extracellular adenosine is synthetized through the hydrolysis of ATP and ADP. Ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) and alkaline phosphatase (AP) hydrolyse ATP to AMP. Ecto-nucleoside triphosphate diphosphydrolase (E-NTDPase/CD39) hydrolyses of ATP to ADP and ADP to AMP. AMP is further hydrolyzed by Ecto-5′NT/CD73 promoting the formation of adenosine, which can bind to four adenosine receptors. Canonically, activation of adenosine receptors either inhibits (A 1 R and A 3 R) or stimulates (A 2A R and A 2B R) adenylate cyclase activity, and cyclic AMP (cAMP) production. Changes in phospholipase C (PLC) activity have been described after A 1 R and A 3 R activation, and it has also been shown that A 2A R, A 2B R and A 3 R can couple to G q -protein. A 1 R activation also leads to respective increases and decreases in K + and Ca 2+ conductance, leading to neuronal hyperpolarization. Adenosine can be transported through the plasma membrane by two families of nucleoside transporters: the equilibrative nucleoside transporter (ENT) which allows bidirectional flux by diffusion, and the concentrative nucleoside transporter (CNT) which uses Na + -coupling to transport adenosine to the intracellular space against the concentration gradient. Lastly, adenosine can be irreversibly deaminated into inosine by the adenosine deaminase (ADA), with inosine also being a bioactive molecule in brain, through A 1 R, A 2A R and A 3 R binding. Abbreviations: 5′-NT, 5′-nucleotidase; A 1 R, adenosine A 1 receptor; A 2A R, adenosine A 2A receptor; A 2B R, adenosine A 2B receptor; A 3 R, adenosine A 3 receptor; ADA, adenosine deaminase; ADK, adenosine kinase; ADP, adenosine diphosphate; AMP, adenosine monophosphate AMPK, AMP kinase; ATP, adenosine triphosphate; cAMP, cyclic AMP; CNT, concentrative nucleoside transporter; DAG, diacylglycerol; Ecto-5′NT/CD73, Ecto-5′nucleotidase/cluster of differentiation 73; E-NPP, Ectonucleotide pyrophosphatase/phosphodiesterase; ENT, equilibrative nucleoside transporter; E-NTDPase/CD39, Ecto-nucleoside triphosphate diphosphydrolase/ cluster of differentiation 39; IP3, inositol trisphosphate; PIP 2 , Phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein, kinase C; PLC, phospholipase C; SHA, S-adenosil-homocysteine. J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 4 majority of which also possess transmembrane domains, do not hydrolyze AMP, but do hydrolyze nucleoside triand diphosphates, in addition to also hydrolyzing ADP ribose, dinucleoside polyphosphates, and NAD + [69,70]. Conversely, both APs and CD73 are bound to the plasma membrane through glycosylphosphatidylinositol (GPI) anchoring, being primarily involved in local catalysis, and autocrine and paracrine signaling. CD73 is a plasma membrane-bound nucleotide-specific homodimer, found in both neuronal [71–76] and glial cells [77,78], which is primarily responsible for the conversion of extracellular AMP to adenosine. APs, on the other hand, are known to hydrolyze nucleoside tri-, diand monophosphates, in addition to also hydrolyzing pyrophosphate. Furthermore, in addition to their membrane bound forms, soluble CD73 and AP forms do exist, being released by the breakdown of GPI anchors, extending the range of action of these enzymes [69,70]. As mentioned above, in addition to being synthetized extracellularly, adenosine is also synthetized intracellularly, after which it can be released to the extracellular space by bidirectional equilibrative nucleoside transporters (ENTs). The existing ENT subtypes (ENT1−4) transport both purine and pyrimidine nucleosides across the plasma membrane, in a concentration-gradient dependent manner, thus also being involved in the uptake of extracellular adenosine [79,80]. Radioligand binding studies, have reported ENT1 as being most expressed in the thalamus and superior colliculus, with reduced hippocampal, cortical and cerebellar expression [80–82]. However, it must be noted that studies using in situ hybridization have found evidence for marked ENT1 mRNA expression in both the hippocampus and the cerebellum of rats [83]. ENT2 expression extensively overlaps that of ENT1 [84], with mRNA expression being observed in cortical, striatal, thalamic, hippocampal, and cerebellar neurons [85]. Unlike ENTs 1 and 2, ENT3 is primarily located in the intracellular space, having an especially relevant role in lysosomal functioning [86]. Furthermore, while ENT3 has been reported to have CNS expression [87], it is thought to be most abundant in the placenta [86]. Lastly, ENT4 mRNA has been found to be expressed across the brain of multiple mammalian species [88–91], with studies finding ENT4 protein expression in the mouse cerebral cortex, hippocampus, basal ganglia, cerebellum, thalamus, and hypothalamus [91]. Contrastingly, concentrative nucleoside transporters (CNTs) – of which three known subtypes exist (CNT1-3) [79,80] – exclusively uptake extracellular nucleosides, using the force of the transmembrane Na + gradient. The highest CNT1 transcript levels were observed in the brain stem and cortex, with intermediate expression in the choroid plexus, hypothalamus, hippocampus, and cerebellum. On the other hand, CNR2 mRNA was found to be highly expressed in most brain regions [92], but in situ hybridization studies found evidence for greater expression in the dentate gyrus of the hippocampus, and the periaqueductal grey, with intermediate expression in the basal ganglia, hypothalamus, cortex and cerebellum [92,93]. Furthermore, while some evidence suggests that CNT2 may be expressed in astrocytes [94,95], in tissue sections it was exclusively found in neuronal cells [93]. Lastly, CNT3 expression has been found to be extremely reduced in rat and mouse neuronal cells [87], and to be absent, or below the detection threshold, in astrocytes [94]. The K m values for adenosine vary between transporter subtypes (for a review see [79]), with CNTs having higher affinity than ENTs. Within ENTs, ENTs 1 and 2 can be considered of high affinity vis a vis ENTs 3 and 4 [79]. As such, the overall type of translocation processes implicated in adenosine transport (i.e., “concentrative” versus “equilibrative”) will be contingent upon transporter distribution and affinity. Importantly, transport direction varies as a function of ENT activity and of the extraand intracellular concentrations of adenosine, which depend on intraand extracellular purine metabolism. Adenosine can be catabolized in both extraand intracellularly. Extracellularly, adenosine is primarily converted to inosine by adenosine deaminase (ADA), though ecto-ADA expression is usually relatively low. It is important to note that inosine, once believed to be an inert metabolite, is now known to be bioactive, with significant A 1 R, A 2A R and A 3 R interactions in the CNS [96–98]. Given the generally reduced expression of ecto-ADA, most extracellular adenosine is taken up by the aforementioned ENTs and CNTs, and then catabolized. This occurs mainly through phosphorylation into AMP by adenosine kinase (ADK), but also by conversion to inosine by intracellular ADA. Due to ENT activity, both ADK and intracellular ADA indirectly regulate extracellular adenosine concentration: studies in the hippocampus showed that extracellular adenosine concentrations increase with the inhibition of both ADK [65] and ADA [99,100]. However, it should be notated that basal adenosine concentrations depend not only on the activity of those enzymes, but also on adequate oxygen and/or glucose levels [100]. Furthermore, it is possible that the relative contribution of each particular enzyme to adenosine level oscillations may be cell-specific and, as referred, dependent upon the tissue microenvironment. Nevertheless, much remains to be determined regarding the factors contributing to the regulation of adenosine levels, despite attempts having already been made [101]. 2.2. Adenosine receptors Currently, four different adenosine receptors have been identified and cloned, namely the A 1 , A 2A , A 2B and A 3 receptors (A 1 R, A 2A R, A 2B R and A 3 R, respectively) [102]. All four receptors are G protein-coupled receptors: A 1 R and A 3 R are negatively coupled to adenylyl cyclase through G i/o protein α -subunits, whereas A 2A R and A 2B R are positively coupled to adenylyl cyclase through G s proteins. Changes in the activity of phospholipase C activity have been described after A 1 R [103,104] and A 3 R [105] activation, and it has also been shown that A 2A R, A 2B R and A 3 R can couple to G q11 [102]. In the striatum, A 2A R are mainly coupled to G olf [106], a G-protein abundant in this brain area, that also activates adenylyl cyclase [107]. Adenosine receptors also have significantly different densities and affinities for adenosine: A 1 R and A 2A R are widespread and have high affinity for adenosine, whereas A 2B R and A 3 R, which are less expressed and have lower affinity [57,108]. However, it must be noted that the affinity of A 3 R for adenosine is species-dependent, being high in humans and low in rodents [109]. Adenosine receptors are differentially expressed in different areas of CNS. A 1 R is highly expressed in the cortex, cerebellum, hippocampus, and dorsal horn of spinal cord, whereas A 2A R is highly expressed in the striatum and olfactory bulb, being expressed to a lower extent in other brain regions [110], such as the amygdala, hippocampus or prefrontal cortex [111–113]. The overall modulatory role of adenosine in the CNS is mainly contingent upon on the balance between A 1 R and A 2A R activity [59]. Both receptors can be present in the same synapse, with approximately 80 % of hippocampal nerve terminals expressing both A 1 R and A 2A R [114]. Furthermore, in the hippocampus, neuronal A 1 R and A 2A R expression is predominantly, but not exclusively, presynaptic [55, 114–118]. A 1 R are most abundant in neurons, both preand postsynaptically [115,116,119], but are also expressed in glial cells, such in astrocytes [120] and microglia [121]. At the presynaptic level, A 1 R activation decreases the release of several neurotransmitters, including glutamate, acetylcholine, serotonin (5-HT) and dopamine (DA), leading to the inhibition of synaptic transmission [122,123]. At the postsynaptic level, A 1 R activation is associated with the inhibition of glutamate N-methyl-D-Aspartate receptor (NMDAR) mediated currents [124], inhibition of voltage sensitive Ca 2+ channels [122], increased K + conductance [125], and disinhibition of inhibitory neurons through a decrease of GABA A receptor-mediated tonic inhibition [126]. These actions make it so that A 1 R plays a key role in the regulation of synaptic transmission and plasticity, with its activation inhibiting hippocampal long-term potentiation (LTP), [127] and long-term depression (LTD) [128]. On the other hand, despite having relatively little hippocampal expression [111,129], A 2A R are readily activated by extracellularly generated adenosine, to facilitate neurotransmitter release [130], and J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 5 can have significant impact in hippocampal synaptic plasticity. Presynaptically, A 2A R stimulation is triggers the release of several neurotransmitters, including glutamate [131,132], acetylcholine [133], DA [134], 5-HT [135], and GABA [131,136]. In addition, presynaptic A 2A R play a critical role in supressing cannabinoid type 1 receptor- (CB 1 R), and A 1 R-mediated inhibition [137,138]. Postsynaptically, A 2A R modulate the activation of NMDAR [118,139,140], CB 1 R [141], metabotropic glutamate receptor 5 (mGluR5) [142], DA type 2 receptors (D 2 R) [143]. Moreover, insofar postsynaptic A 2A R are necessary for the co-activation of NMDAR [140], they are required for the maintenance of LTP [144]. Additionally, A 2A R activity is known the facilitate the effects of BDNF upon synaptic transmission and plasticity [144–146]. As previously described, there are two major sources of extracellular adenosine. On the one hand, under rest or low-frequency stimulation, A 1 R is primarily activated by ENT-released adenosine [147]. On the other hand, conditions of increased presynaptic stimulation favor the release of ATP, which is then metabolized to adenosine. This pathway leads to preferential A 2A R activation [130,147]. Thus, adenosine induced suppression of neuronal activity, preservation of ATP stores, and neuroprotection [148] are mainly attributable to A 1 R activation, while A 2A R activation, may exacerbate excitotoxicity in several brain areas [149–152]. Importantly, however, despite A 2A R being mainly associated with pathological situations, these receptors also gate the neuroprotective actions of some molecules, such as neurotrophic factors [145,146,153]. Unlike A 1 R and A 2A R, A 2B R has a low level of expression in the brain, being primarily expressed in peripheral tissues [154]. Lastly, A 3 R is reported to have intermediate levels of expression in the human cerebellum and hippocampus, and low levels in most of the remaining regions [102]. Besides their reduced expression in the brain, A 2B R and A 3 R also have low-affinity for adenosine [155]. Nonetheless, these receptors might be activated during conditions of hypoxia or ischemia, in which extracellular adenosine levels rise [58,156–158]. Lastly it must be noted that, in addition to their neuronal expression, all four adenosine receptors are detected in astrocytes [159], and have been reported to be expressed in microglial cells or microglial cell lines [160,161]. 3. The adenosinergic system in MDD pathophysiology and treatment 3.1. Adenosine synthesis The impact of MDD-like conditions on the levels and activity of enzymes involved in adenosine synthesis have been investigated in multiple animal studies. ATPase, ADPase and CD73 activity were assessed in synaptosomes derived from the spinal cords of male and female SpragueDawley rats, that had undergone a chronic restraint stress procedure [162]. In this work, the authors found male-specific decreases and increases in ADPase and CD73 activity, respectively, with no changes in ATPase activity [162]. Interestingly, in this study no effects were observed in females [162], likely due to the effects of estradiol, as evidenced by a posterior study in ovariectomized female rats, where chronic restraint stress induced a decrease in spinal cord CD73 (but not ATPase or ADPase) activity, which was not observed in similarly stressed 17β-estradiol-treated animals [163]. In another study from these authors, neither ATPase, ADPase, nor CD73 activity were observed to be altered in synaptosomes derived from the hypothalamus and cortex derived of male Sprague-Dawley rats exposed to a similar chronic restraint stress protocol [164]. Moreover, in these animals, a decrease in blood serum ADPase activity was observed [164]. Furthermore, both 15and 40-day restraint stress exposures were found to induce increased ATPase activity in hippocampal synaptosomes obtained from male Sprague-Dawley rats, without affecting ADPase or CD73 activity [165]. In recent years there has been an increase in the number of studies using zebrafish as model organism for the study of depression, and of putative treatments for it. This is likely due to the fact that not only do zebrafish allow rapid, low-cost, high throughput manipulation and testing, but also due to their remarkably high physiological and genetic homology to mammals [166,167]. Moreover, zebrafish models have been demonstrated to have considerable predictive value, in regards to detecting the effects of known antidepressant compounds [166,167]. Interestingly, a recent study with zebrafish exposed to an unpredictable chronic stress protocol, found no differences in the hydrolysis of either ATP, ADP or AMP, suggesting no changes in enzymatic activity [168]. However, it should be noted that, despite this lack of changes in the activity of hydrolytic enzymes, when ATP metabolism was measured directly, a significant increase in ATP-derived adenosine levels was observed in the brains of stress-exposed zebrafish [168]. A recent study found that chronic social defeat stress (CSDS) exposure resulted in increased hippocampal CD39 expression and activity, as well as a marked depressive-like state, as evidenced by increased immobility time in the tail suspension (TST) and forced swim tests (FST), and decreased sucrose intake in the sucrose preference test (SPT) [169] (for a brief overview of the most common behavioral tests used in the assessment of depressiveand anxiety-like behaviors see Table 1). Furthermore, it was also found that CSDS induces an equally strong anxiogenic effect in the elevated plus maze (EPM) and the Open Field Test (OFT) [169]. Most notably, these authors found that both pharmacological and genetic silencing of hippocampal CD39, resulted in a partial reversal of CSDS-induced depressive-like behaviors, and associated molecular alterations, including increased hippocampal neurogenesis, spinogenesis, and extracellular ATP level [169]. Congruently, administration of the CD39-analog apyrase resulted in CSDS-like increases in depressive-like behaviors [169]. Conversely, in nonCSDS-exposed mice, genetic or pharmacologic silencing of hippocampal CD39 resulted in antidepressant-like effects, with animals performing better than controls [169]. Thus, hippocampal CD39 seems to have a key role in the regulation of mood states, and may represent an important target for future drug-development. However, these results must be considered in light of the fact that CD39 silencing may imply other undesirable effects, as evidenced by the fact that CD39-KO mice have been reported to be highly seizure-prone [196]. Nonetheless, in agreement with the data of Cui et al. [169], CD39-KO mice were reported to show decreased anxiety-like behavior in the EPM, without alterations of in the social interaction test (SIT), the OFT, or the fear conditioning paradigm [196]. On the other hand, the impact of CD73 manipulations was assessed in a pair of studies with CD73-KO mice. In the first of these studies, CD73-KO mice were found to have wild type (WT)-like performance in the EPM, OFT and the light-dark box test (LDBT), as well as in the FST, and the fear conditioning paradigm, thus suggesting unaltered emotional function [197]. In line with this data, another study reported the LDBT and OFT performance of CD73-KO mice to be similar to that of controls [198]. Intriguingly, regarding cognitive parameters known to be impacted in MDD, it was first reported that neither working memory, nor spatial memory and learning were affected in CD73-KO mice [197]. However, a subsequent study observed an improvement in working memory, assessed in the Spontaneous Alternation Y-Maze, in CD73-KO mice [198]. Furthermore, CD73-KO mice evidenced altered social behavior, with decreased social dominance, and preference for social novelty in the social motivation and social novelty test, despite presenting increased social activity [197]. Interestingly, there is some evidence that conventional antidepressants may significantly impact the enzymes involved in adenosine synthesis (see Table 2 for an overview of the effects of classical antidepressant treatments upon the adenosinergic system). In the blood serum, acute treatment with the tricyclic antidepressant (TCA) nortriptyline – but not the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine – led to a decrease in ATP hydrolysis, without affecting ADP or AMP hydrolysis [199]. Similarly, acute nortriptyline, but not J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 6 Table 1 Overview of the most widely used behavioral tests for the assessment of anxietyand depressive-like behaviors. Test Description Construct Modelled Main Measures Limitations/Biases Key Refs. Differential Reinforcement of Low-rate 72-s (DRL72-s) Animals are trained to acquire a stable pattern of operant behavior, whereby lever presses are only reinforced if performed at least 72 s after the last reinforcement. Decreased motivation Number of responses (index of depressive-like behavior) Biased by impairments in learning processes; [170] Number of reinforcers obtained (inverse index of depressive-like behavior) Cumulative inter-response time distribution (rightward shift =inverse index of depressive-like behavior) Elevated Plus Maze (EPM) Animals are placed in the center of an elevated maze shapes like a plus – with two open arms and two arms enclosed on three sides by high walls – and are left to explore for a defined period of time. Generalized anxiety Time spent and number of entries in the open arms (inverse indexes of anxiety-like behavior); Biased by alterations in locomotor function; [171, 172] May not be an adequate measure of anxiety-like behavior; Limited predictive validity; Fear Conditioning Paradigm Animals are conditioned to associate conditioned stimuli (cue) with an aversive unconditioned stimulus (shock), and emit a conditioned freezing response. Aversive associative memory Cue-evoked freezing responses (index of cued conditioned fear); Biased by alterations in nociception; [173, 174] Subsequently animals are exposed to the cue in a novel environment (cued fear conditioning), and/or to the context where original conditioning took place but in the absence of the cue itself (contextual fear conditioning). (e.g., phobia) Context-evoked freezing responses (index of contextual of conditioned fear); Forced Swim Test (FST) Animals are placed into a water filled cylinder from which they cannot escape for a single 6minute session (mice) or for 2 sessions – lasting 15 and 5 min respectively – spaced 24 h apart (rats). Impaired stresscoping Time spent in immobility (index of depressive-like behavior); Biased by alterations in locomotor function; [175, 176,177] Time spent climbing (inverse index of depressive-like behavior); Highly stress inducing; Time spent swimming (inverse index of depressive-like behavior); Light-Dark Box Test (LDBT) Uses an apparatus composed by two chambers – one dark, and fully covered, the other open and brightly lit – connected by a small passage. Animals are placed in the dark chamber and allowed to freely explore the entire apparatus for a defined period of time. Generalized anxiety Latency to first entry in the light chamber (index of anxiety-like behavior); Biased by alterations in locomotor function; [178, 179] Time spent in the light chamber (inverse index of anxiety-like behavior); Marble Burying Test (MBT) Animals are placed in a cage with lightly tamped bedding, on top of which an array of glass marbles has been disposed, and left to explore/ interact for a defined period of time. Unclear (anxiety, compulsion, neither?) Number of marbled buried (index of anxiety-like behavior) Interpretation is highly contentious; [180, 181,182, 183,184] Open Field Test (OFT) Animals are placed in an enclosure whose center region is brightly lit, and are allowed to freely explored for a defined period of time. Generalized anxiety Time spent in a virtually defined center zone (inverse index of anxiety-like behavior); Biased by alterations in locomotor function; [185, 186] May not be an adequate measure of anxiety-like behavior; Locomotor measures (distance travelled, average speed); Limited predictive validity; Shuttle Box Escape Animals are first exposed to inescapable shocks on one of two compartments in a conditioning chamber, with no way to access the other compartment. After a defined period of time, animals are again placed in the shock-paired compartment, with open access to the remaining compartment, allowing them to escape shock exposure. Apathy/learned helplessness Number of shocks received when escape is possible (escape failures; index of depressive-like behavior); Biased by alterations in locomotion and nociception; [187, 188,189] Latency to escape shock (inverse index of depressive-like behavior); Social Interaction Test (SIT) Animals are placed in the open field with a unknown social partner of the same sex, weight and age, and allowed to freely explore and interact for a defined period of time. Social anxiety Time spent in active social interaction (inverse index of anxiety-like behavior); Biased by alterations in locomotor function, and social motivation/reward; [190, 191] Limited predictive validity; Sucrose Preference Test (SPT) Animals are allowed free access to two bottles – one containing water, and the other a low concentration sucrose solution – for a defined period of time. Anhedonia Relative sucrose preference (inverse index of depressive-like behavior); Biased by alterations in gustatory perception; [192, 193] Sucrose intake (inverse index of depressive-like behavior); Tail Suspension Test (TST) Mice are hung upside down by their tails for a defined period of time (typically 6 min), and behavior is scored. Impaired stresscoping Time spent in immobility (index of depressive-like behavior); Biased by alterations in locomotor function; [194, 195] J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 7 fluoxetine, treatment decreased hippocampal and cortical ATP hydrolysis, while also increasing ADP hydrolysis in the hippocampus [200]. However, chronic treatment with these compounds led to different results. In blood serum, both fluoxetine and nortriptyline-treatments led to a decrease ATP, ADP, and AMP hydrolysis [199]. On the other hand, the hippocampus, both compounds decreased ATP hydrolysis, without affecting ADP or AMP metabolism [200]. Moreover, in the cortex, fluoxetine treatment decreased ATP hydrolysis, while increasing ADP and AMP metabolism, whereas nortriptyline increased hydrolysis of both ATP, ADP and AMP [200]. However, it should be noted that another study found fluoxetine treatment to have no impact upon whole brain apyrase mRNA expression [201]. Relatedly, a recent study with zebrafish found that pre-treatment with the CD73 inhibitor AMPCP did not diminish or increase the antidepressant-like actions of the NMDAR antagonist MK-801 [202]. Like classical antidepressant compounds, electroconvulsive shock (ECS) treatment has also been shown to modulate adenosine synthesis. In the short term, 12 h after a single ECS session, blood serum ADP and AMP hydrolysis was found to be decreased [203]. On the other hand, when similar assessments were performed 7 days after the last of an 8-session ECS treatment protocol, persistent increases in ATP and ADP hydrolysis were observed in both serum and cerebral spinal fluid samples, suggesting a role for increased adenosine synthesis in the antidepressant effects of ECS [203,204]. Finally, it must be noted that alterations in the intracellular adenosine precursor SAM, have been shown to translate into alterations in methyltransferase activity, resulting in abnormal methylation patterns. Importantly, such aberrant methylation patterns have been implicated Table 2 Impact of pharmacological and non-pharmacological antidepressant treatments on adenosine synthesis, transport, catabolism, levels and P1 receptors. Adenosine Synthesis Adenosine Transport Adenosine Catabolism Adenosine Levels Receptors A 1 R A 2A R A 2B R A 3 R DBS – – – ↑hipp A,B – – – – ECS/ECT ↓ Serum ADP/AMP hydrolysis A ↑ Str density A – ↑ wholebrain A ↑ Ctx density C ↔ Ctx, Hipp, Cbl, Str density A,C – – ↑ Serum & CSF ATP/ADP hydrolysis C ↔ Ctx, Hipp, Cbl density A ↔ Hipp, Cbl, Str density C ↔ Ctx, Hipp, Cbl, Str, density C ↔ Ctx, Hipp, Cbl, Str density A MAOI – – – – – – – – MRA – – – – – – – – NaSSA – – – – – – – – NDRI – – – – – – – – NMDARA – – – – – – – – NRI – – – – – – – – RIM-A – – – – – – – – SARI – – ↓ ADA activity E – – – – – SD – – – ↑BF A ↑ BF density A,C,F ↓ OT density C – – ↑ BF mRNA A ↓ OT mRNA A SMS – – – – – – – – SNRI – – – – – – – – SSRI ↔ Serum ATP/ADP/AMP hydrolysis A ↑ whole-brain CNT2 mRNA B ↑ Serum ADA activity C,F ↑ Plasma A,C,F – – – – ↓ Serum ATP/ADP/AMP hydrolysis C ↔ whole-brain ENT1−3, CNT3 mRNA B ↔ whole-brain ADK mRNA B ↔ Hipp & Ctx ATP/ADP/ AMP hydrolysis A ↓ Hipp ATP hydrolysys C ↔ Hipp ADP/AMP hydrolysis C ↑ Ctx ADP/AMP hydrolysis C ↓ Ctx ATP hydrolysis C TCA ↓ Serum ADP hydrolysis A ↓ activity D ↔ ADA & ADK activity D – – – – – ↔ Serum ATP/AMP hydrolysis A ↓ Hipp & Ctx ATP hydrolysis A ↑ Hipp ADP hydrolysis A ↔ Hipp & Ctx AMP hydrolysis A ↓ Serum ATP/ADP/AMP hydrolysis C ↓ Hipp ATP hydrolysys C ↔ Hipp ADP/AMP hydrolysis C ↑ CTX ATP/ADP/AMP hydrolysis C Abbreviations: -, effects not studied; ↔ no effects; ↓ decrease; ↑ increase; A 1 R, Adenosine A 1 receptor; A 2A R, Adenosine A 2A receptor; A 2B R, Adenosine A 2B receptor; A 3 R, Adenosine A 3 receptor; ADA, adenosine deaminase; ADK, adenosine kinase; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BF, basal forebrain; Cbl, cerebellum; CNT, concentrative nucleoside transporter; CSF, cerebral spinal fluid; Ctx, Cortex; DBS, deep brain stimulation; ECS/ ECT, electroconvulsive shock/therapy; ENT, equilibrative nucleoside transporter; Hipp, hippocampus; MAOI, monoamine oxidase inhibitor; MRA, melatonin receptor agonist; NaSSA, noradrenaline and serotonin specific antidepressant; NDRI, noradrenaline and dopamine reuptake inhibitor; NMDAR-A, NMDA receptor antagonist; NRI, noradrenaline reuptake inhibitor; OT, olfactory tubercle; RIM-A, reversible inhibitor of monoamine oxidase A; SARI, serotonin antagonist and reuptake inhibitor; SD, sleep deprivation; SMS, serotonin modulator and stimulator; SNRI, serotonin and noradrenaline reuptake inhibitor; Str, striatum; TCA, tricyclic antidepressant. a, acute administration; b, sub-chronic administration; c, chronic administration; d, cell line study; e, ex-vivo application; f, human MDD patients. J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 8 in the pathogenesis of multiple neuropsychiatric disorders, including MDD [64,205]. In sum, evidence appears to largely support the existence of an important role for altered adenosine synthesis in the emergence of MDD symptoms. Moreover, it seems to be the case that targeting those dysfunctions in adenosine synthesis has a significant beneficial effect over symptoms, and thus may hold significant therapeutic potential. 3.2. Adenosine transport Given that sleep disruption and fatigue are common symptoms of MDD, and considering the role of adenosine in sleep regulation, a possible relationship between adenosinergic dysfunction and MDDassociated sleep disruption has been proposed. In line with this, a study assessed the association between a number of single nucleotide polymorphisms (SNPs) – in the genes coding for ADA, ADK, ENTs, CNTs, and CD73 – and MDD diagnosis with or without sleep disturbances, in patients of both sexes [206]. While in initial analyses these authors found multiple significant associations between specific SNPs and MDD, only one survived correction for multiple comparisons [206]. Specifically, a significant female-specific association was found between the SLC29A3 rs12256138 SNP and MDD diagnosis, suggesting a possible involvement between ENT3 alterations and this disorder [206]. The role of nucleoside transport in depressive-like symptoms in rodents has also been studied by both genetic and pharmacological approaches. Pharmacological studies found that administration of the nonspecific transporter inhibitor papaverine, led to an anxiolytic-like effect in the EPM [207]. Similarly, microinjection of the specific ENT1 antagonist NBMPR into the amygdala, but not the caudate-putamen, of C57BL6 mice, resulted in an anxiolytic-like effect in both the EPM and the OFT [208]. Likewise, when male ENT1-KO mice were assessed, a significant decrease in anxiety-like behavior was observed in the EPM, OFT and LDBT [208,209], but not in the marble burying test (MBT) [209]. Moreover, female ENT1-KO mice tested in the OFT and the MBT, also evidenced decreased anxiety in those tests [209]. Furthermore, when ENT1-KO mice of both sexes were assessed for altered depressive-like behaviors in the FST, a significant antidepressant-like effect was observed, as evidence by reduced immobility time as compared with WT littermates [209]. One interesting discrepancy, however, comes from the fact that acute administration of the ENT inhibitor NBTI resulted in a dose-dependent impairment in shuttle box escape performance, similar to that observed after inescapable shock exposure [210], suggesting a prodepressant impact of transporter blockade. Moreover, when NBTI was combined with a sub-effective shock exposure, a synergistic effect was observed [210]. A few studies have assessed how adenosine transporters are impacted and impact antidepressant treatment. Specifically, 3-day fluoxetine treatment led to an increase in whole-brain CNT2 mRNA expression, without altering that of ENT1−3, CNT3, or ADK [201]. Furthermore, there is some evidence that the TCA amitriptyline may inhibit adenosine transport [211]. Interestingly, while it has been demonstrated that a single ECS exposure rapidly (30 min) induces marked increases in striatal adenosine uptake, these appear to be short-lasting, not being detectable 24 h later [212]. Similarly, repeated ECS treatment had no significant effect upon adenosine uptake – in either the striatum, the cortex, the hippocampus, or the cerebellum – when measurements were performed 24 h after the last session [212]. Lastly, recent study with zebrafish found that NBTI co-treatment had no impact upon the antidepressant-like action of the NMDAR antagonist MK-801 [202], but in contrast, dipyridamole, a non-specific nucleoside transport inhibitor, synergistically acted with MK-801, increasing its antidepressant-like effects [202]. Accordingly, acute dipyridamole administration potentiated the antidepressant-like effect of sub-effective zinc chloride doses, in the FST [213]. Altogether, the presently available evidence cannot firmly support a conclusion regarding the possible involvement of adenosine transporters in the pathophysiology of MDD, nor the possible relevance of nucleoside transport inhibitors in MDD therapeutics. The differences between acute and prolonged administration of nucleoside inhibitors, the short-lasting influence of intense neuronal activity upon extracellular levels of adenosine, and the different responses to selective and non-selective nucleoside inhibitors are indicative of a strong capability of the nucleoside transporters to adapt to changes in the extracellular levels of adenosine and to drug manipulations. Whether this might explain some of the apparent contradictory findings on the role of the transporters on depression or the action of antidepressant drugs awaits further studies. It is also important to keep in mind that ENTs are bidirectional and therefore take up or release adenosine as a function of the relative intraand extracellular concentrations of the nucleoside. As such, ENTs inhibitors may lead to either increases or decreases in the extracellular concentrations of adenosine as a function of the neuronal activity and metabolic state of neuronal cells, including neurons, astrocytes and microglia. 3.3. Adenosine catabolism To the best of our knowledge, all the published work regarding the association between MDD and altered adenosine catabolism, focused on ADA, with results being somewhat contradictory. In the blood serum of rats exposed sub-chronic (3-day) restraint stress, an increase was observed in ADA activity, which was not observed after a single restraint session [214]. Similarly, in a study with MDD patients, blood ADA activity was found to be increased in the MDD group [215]. Contrastingly, however, a previous in a previous study with MDD patients, an association was observed between decreased – rather than increased – blood serum ADA activity and MDD diagnosis [216]. In line with this, a recent paper using zebrafish exposed to a unpredictable chronic stress protocol, found a decrease in the activity of brain ecto-ADA activity, but not in that of cytosolic ADA [168]. Moreover, these authors assessed how chronic stress impacted the expression of several ADA genes, and found no significant alterations [168]. Regarding ADA manipulations, a recent study assessed the impact of ADA abolishment on behavioral outcomes [217]. These authors found that ADA-KO mice presented increased anxiety-like behaviors in the OFT and LDBT [217]. Results in the OFT have, however, to be evaluated in light of the fact that these animals also presented clear signs of locomotor hypoactivity [217]. Importantly, enzyme replacement therapy had no effect upon these behavioral alterations [217]. Interestingly, pharmacological inhibition of ADA has been contradictorily reported to induce both proand antidepressant-like effects. Specifically, a 1998 study showed that acute intracerebroventricular administration of EHNA induced escape deficits – similar to those observed after exposure to inescapable shocks – in the shuttle box escape paradigm [218]. Furthermore, a sub-active EHNA dose, in combination with sub-effective shock exposure, resulted in escape deficits similar to those observed by active dose EHNA or effective shock exposure, suggesting a synergistic interaction [218]. Conversely, in a more recent study, acute intraperitoneal EHNA administration, resulted in dose-dependent decreases in FST immobility time – suggesting an antidepressant-like effect – without altering OFT performance [219]. The effects of ADK manipulation were assessed in a single study, where a transgenic mouse line overexpressing ADK (Adk-tg) was assessed for alterations in anxiety-like behaviors, and fear conditioning, as well as working-, and reference memory [220]. Performance in the EPM was found to be WT-like, suggesting normal anxiety-like behavior [220]. On the other hand, in the fear conditioning, Adk-tg mice presented impaired acquisition and expression of the response to conditioned stimuli [220]. Furthermore, these animals evidenced severe deficits in both working and reference memory [220]. The effects of antidepressant treatment on the enzymes involved in adenosine catabolism were first assessed in 1985 paper, which found J.I. Gomes et al. Pharmacological Research 163 (2021) 105363 9 that the serotonin antagonist and reuptake inhibitior (SARI) trazodone acted as an inhibitor of brain ADA [221]. On the other hand, a subsequent in vitro study, where amitriptyline was found to not impact the activity of either ADK or ADA [211]. In contrast, two subsequent studies have found that in both MDD and panic disorder patients, 8-week SSRI treatment was associated with significant increases in blood ADA activity [215,222]. Curiously, a recent study found that sub-chronic (3-day) fluoxetine administration had no impact upon whole-brain ADK mRNA expression [201]. Finally, in zebrafish, acute EHNA pretreatment did not significantly alter the antidepressant-like effects of MK-801 administration [202]. Overall, ADA alterations have been consistently observed to be related to depressive and anxious symptomatology, and may represent fruitful targets for future development. However, it is important to keep in mind that ADA manipulations are likely to have markedly unselective and widespread effects, some of which may be undesirable. On the other hand, there is a clear dearth of research on the role of ADK in the pathophysiology and/or treatment of MDD symptoms, which is surprising given the key role of this enzyme in the regulation of intraand extracellular adenosine levels (see section 2.1), and its relevance as a therapeutic target multiple other pathologies [223], including neuropsychiatric ones [224]. This is, thus, an area which undoubtedly deserves future research. 3.4. Adenosine levels Changes in adenosine synthesis, transport and catabolism, found in MDD patients and/or animal models, are likely to impact adenosine levels, resulting in the non-selective changes in the activation state of adenosine receptors. However, to the best of our knowledge, no published work has assessed the possibility that adenosine levels may be altered in MDD patients or animal models. Nonetheless, a few studies have assessed the impact of manipulations aimed at altering adenosine levels. Acute intraperitoneal administration of adenosine was reported to induce marked increases in FST immobility time, which were prevented by co-administration of caffeine, theophylline, and some – albeit not all – classical antidepressant compounds [225]. In a subsequent study, acute intraperitoneal administration of the non-selective adenosine receptor agonist NECA, resulted in a dose-dependent impairment of escape behavior in the shuttle escape paradigm, similar to that induced by inescapable shock [226]. However, more recent publications have reported adenosine administration to induce markedly different effects than those originally reported. Indeed, acute adenosine administration has been consistently shown to induce reductions in immobility time in both the FST and the TST, without altering locomotor activity [227–232]. Furthermore, this effect has been demonstrated to be reliant on a number of interactions with other – non-adenosinergic – targets, including the nitric oxide–cGMP pathway [228], the 5-HT type 1A [231] and NMDA [227] receptors, K + channels [230], and the opioid system [229]. One possible explanation for the discrepancy of results vis a vis those originally reported, related to the differences adenosine doses used [225]. Specifically, whereas antidepressant effects are observed at doses of 1−10 mg/kg [227–232], the report finding a prodepressant effect of adenosine, administered a 100 mg/kg dose [225], which is likely to induce a strong sedating and/or locomotion impairing effect. Nonetheless, it must be noted that in a recent study with rats submitted to bilateral olfactory bulbectomy (OBX), 14-day adenosine treatment had no effect upon altered FST, OFT or SPT performance [233]. The impact of antidepressant drug treatment upon adenosine levels has been assessed in a single study, where MDD patients underwent a 40day treatment with daily doses of the SSRI citalopram, and plasma adenosine and 5-HT levels were assessed at multiple time-points [234]. As expected, plasma levels of 5-HT increased soon after citalopram administration, peaking after 12 h, and then gradually decreased [234]. This temporal progression remained stable across the 40-day treatment, but there was evidence of an increase in the magnitude of the effect, in response to prolonged treatment. Interestingly, adenosine levels consistently followed the same temporal progression, with a statistically significant association being observed [234]. Given this, it is curious that the co-administration of adenosine either with the SSRI fluoxetine, or the antipsychotic 5-HT type 2A receptor antagonist ketanserin, did not lead to a synergistic effect in the FST [231]. On the other hand, a synergistic antidepressant effect was observed after co-administration of adenosine and the TCA imipramine [227]. Likewise, sub-active – but not active – doses of adenosine and the NMDAR antagonists ketamine and MK-801, resulted in a synergistic antidepressant-like effect in the FST [227]. Concluding, evidence supports the notion that increasing adenosine levels may have a significantly beneficial impact upon depressive and anxious symptomatology. This is fully in line with the therapeutic effects reported while targeting adenosine synthesis, catabolism, and transport, all of which can lead to a similar increase in adenosine levels. 3.5. Adenosine receptors 3.5.1. A 1 R Given the known role of A 1 R in the regulation of sleep, and the relation between altered sleep and MDD, it is interesting to note that in a study assessing the association of multiple SNPs in genes coding for elements of the adenosinergic system, no significant effects were found for ADORA1 polymorphisms [206]. Furthermore, to the best of our knowledge there are no published reports assessing changes in A 1 R levels in the brains of human MDD patients. Interestingly, in animal studies, chronic stress exposure has been shown to impact hippocampal A 1 R expression, albeit with contradictory results. Specifically, it has been shown that sub-chronic restraint stress exposure results in a decrease in hippocampal A 1 R binding and protein levels [235]. Contrastingly, however, a subsequent study using rats exposed to either chronic restraint stress or chronic mild stress protocols, showed the opposite effect, whereby both protocols resulted in an increase – rather than a decrease – in hippocampal A 1 R binding/protein levels [236]. Pharmacological studies with A 1 R agonists have largely consistent findings. Specifically, it was found that acute administration of the A 1 R agonist CHA led to a decrease in FST immobility times [232]. Likewise, another report found weekly CHA administration to have antidepressant-like effects in the differential reinforcement of low rate 72-s (DRL-72 s) schedule assay [237]. Moreover, in addition to decreasing FST immobility, acute CCPA administration was also shown to lead to a sustained (36 h) reduction of anhedonic-like behavior in the SPT [238]. Relatedly, in a recent study, the selective A 1 R agonist MRS5474 was shown to acutely decrease FST immobility times for non-stressed mice, as well as to decrease TST immobility in mice exposed to a repeated swim stress protocol [239]. Importantly, an early study suggested that acute administration of the A 1 R agonist R-PIA mimicked the effects of pre-exposure to inescapable shock on escape shuttle performance [226]. However, it must be noted that these authors did not control for the possibility that this decrease in performance may be attributable to possible hypolocomotion-inducing effects of A 1 R agonists, rather than a true prodepressant-like effect, despite reporting visual evidence of such possible effects. Finally, it must be noted that in one study no evidence was found for an antidepressant-like effect of acute CHA administration in the FST [213]. 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