Adenosine A2A receptors (A2ARs) were recently described to control synaptic plasticity and network activity in the prefrontal cortex (PFC). We now probed the role of these PFC A2AR by evaluating the behavioral performance (locomotor activity, anxiety-related behavior, cost-benefit decision making and working memory) of rats upon downregulation of A2AR selectively in the prelimbic medial PFC (PLmPFC) via viral small hairpin RNA targeting the A2AR (shA2AR). The most evident alteration observed in shA2AR-treated rats, when compared to sh-control (shCTRL)-treated rats, was a decrease in the choice of the large reward upon an imposed delay of 15 s assessed in a T-maze-based cost-benefit decision-making paradigm, suggestive of impulsive decision making. Spontaneous locomotion in the open field was not altered, suggesting no changes in exploratory behavior. Furthermore, rats treated with shA2AR in the PLmPFC also displayed a tendency for higher anxiety levels in the elevated plus maze (less entries in the open arms), but not in the open field test (time spent in the center was not affected). Finally, working memory performance was not significantly altered, as revealed by the spontaneous alternation in the Y-maze test and the latency to reach the platform in the repeated trial Morris water maze. These findings constitute the first direct demonstration of a role of PFC A2AR in the control of behavior in physiological conditions, showing their major contribution for the control of delay-based cost-benefit decisions.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Both endocannabinoids and insulin regulate peripheral and cerebral glucose homeostasis via convergent signaling pathways that are impacted by diabetes. Here we asked how glucose metabolism and important facets of insulin signaling are affected in the forebrain of cannabinoid CB1 receptor knockout mice (CB1R-KO) and their wild-type (WT) littermates, seven weeks after the induction of insulinopenia/hyperglycemia (diabetes) with intraperitoneal streptozotocin injection. Sham-injected animals served as control. Diabetes caused milder weight loss in the WT mice compared to the phenotypically ˜11% leaner CB1R-KO, while hyperglycemia was similar. Resting [3H]deoxyglucose uptake was significantly reduced by ˜20% in acute ex vivo frontocortical and hippocampal slices obtained from both the sham-injected CB1R-KO and the diabetic WT mice. Surprisingly, the third cohort, the diabetic CB1R-KO showed no further impairment in glucose uptake, as compared to the sham-injected CB1R-KO. Depolarization-induced [3H]deoxyglucose uptake was proportional to the respective resting values only in the cortex in all four cohorts. The dissipative metabolism of [14C]-U-glucose remained largely unaffected in all cohorts of animals. However, diabetes reduced cortical CB1R density by ˜20%, as assessed by Western blotting. Albeit the changes in insulin signaling did not reflect the glucose uptake profile in each cohort, there were significant interactions between diabetes and genotype. In conclusion, a chronic decrease or lack of CB1R expression reduces glucose uptake in the mouse brain. Additionally, diabetes failed to cause further impairment in cerebral glucose uptake in the CB1R-KO. These suggest that diabetic encephalopathy may be in part associated with lower CB1R expression.

The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model.

Corticosteroid and endocannabinoid actions converge on prefrontocortical circuits associated with neuropsychiatric illnesses. Corticosteroids can also modulate forebrain synapses by using endocannabinoid effector systems. Here, we determined whether corticosteroids can modulate transmitter release directly in the frontal cortex and, in doing so, whether they affect presynaptic CB1 cannabinoid receptor- (CB1R) mediated neuromodulation. By Western blotting of purified subcellular fractions of the rat frontal cortex, we found glucocorticoid receptors (GcRs) and CB1Rs enriched in isolated frontocortical nerve terminals (synaptosomes). CB1Rs were predominantly presynaptically located while GcRs showed preference for the post-synaptic fraction. Additional confocal microscopy analysis of cortical and hippocampal regions revealed vesicular GABA transporter-positive and vesicular glutamate transporter 1-positive nerve terminals endowed with CB1R immunoreactivity, apposing GcR-positive post-synaptic compartments. In functional transmitter release assay, corticosteroids, corticosterone (0.1-10 microM) and dexamethasone (0.1-10 microM) did not significantly affect the evoked release of [(3)H]GABA and [(14)C]glutamate in superfused synaptosomes, isolated from both rats and mice. In contrast, the synthetic cannabinoid, WIN55212-2 (1 microM) diminished the release of both [(3)H]GABA and [(14)C]glutamate, evoked with various depolarization paradigms. This effect of WIN55212-2 was abolished by the CB1R neutral antagonist, O-2050 (1 microM), and was absent in the CB1R KO mice. CB2R-selective agonists did not affect the release of either neurotransmitter. The lack of robust presynaptic neuromodulation by corticosteroids was unchanged upon either CB1R activation or genetic inactivation. Altogether, corticosteroids are unlikely to exert direct non-genomic presynaptic neuromodulation in the frontal cortex, but they may do so indirectly, via the stimulation of trans-synaptic endocannabinoid signaling.

Diabetes induces changes in neurotransmitter release in central nervous system, which depend on the type of neurotransmitter and region studied. In this study, we evaluated the effect of diabetes (two and eight weeks duration) on basal and evoked release of [(14)C]glutamate and [(3)H]GABA in hippocampal and retinal synaptosomes. We also analyzed the effect of diabetes on the protein content of vesicular glutamate and GABA transporters, VGluT-1, VGluT-2 and VGAT, and on the α(1A) subunit of P/Q type calcium channels, which are abundant in nerve terminals. The protein content of vesicular glutamate and GABA transporters, and of the α(1A) subunit, was differently affected by diabetes in hippocampal and retinal synaptosomes. The changes were more pronounced in the retina than in hippocampus. VGluT-1 and VGluT-2 content was not affected in hippocampus. Moreover, changes occurred early, at two weeks of diabetes, but after eight weeks almost no changes were detected, with the exception of VGAT in the retina. Regarding neurotransmitter release, no major changes were detected. After two weeks of diabetes, neurotransmitter release was similar to controls. After eight weeks of diabetes, the basal release of glutamate slightly increased in hippocampus and the evoked GABA release decreased in retina. In conclusion, diabetes induces early transient changes in the content of glutamate and/or GABA vesicular transporters, and on calcium channels subunit, in retinal or hippocampal synaptosomes, but only minor changes in the release of glutamate or GABA. These results point to the importance of diabetes-induced changes in neural tissues at the presynaptic level, which may underlie alterations in synaptic transmission, particularly if they become permanent during the later stages of the disease.

Endocannabinoids play a crucial neuromodulator role in both physiological and pathological states in various brain regions including the prefrontal cortex (PFC). We examined, whether presynaptic cannabinoid receptors are involved in the modulation of basal and electrical field stimulation-evoked [³H]norepinephrine ([³H]NE) release from rat PFC slices. WIN55,212-2, a nonselective CB₁ receptor (CB₁R) agonist, inhibited the electrical stimulation-evoked efflux of [³H]NE in a concentration-dependent fashion, which was antagonized by the CB₁R antagonist/inverse agonist, AM251 (1 μM). Idazoxan, a selective α₂-adrenoceptor antagonist, augmented the evoked [³H]NE release. In the presence of idazoxan, the effect of WIN55,212-2 was exacerbated or attenuated, depending on the applied concentration and stimulation frequency. Moreover their combined, but not individual application elicited a depressive-like phenomenon in the forced-swim test. These data were bolstered with fluorescent and confocal microscopy analysis, which revealed that CB₁R immunoreactivity co-localized with dopamine-β-hydroxylase positive (i.e. noradrenergic) fibers and the inhibitory α(2A) adrenergic autoreceptors (α(2A)R) in the PFC. Furthermore, idazoxan triggered a decrease in CB₁R density in the PFC, suggesting that high extracellular level of norepinephrine downregulates CB₁Rs.

Both the serotonergic and endocannabinoid systems modulate frontocortical glutamate release; thus they are well positioned to participate in the pathogenesis of psychiatric disorders. With the help of fluorescent and confocal microscopy, we localized the CB(1) cannabinoid receptor (CB(1)R) in VGLUT1- and 2- (i.e. glutamatergic) and serotonin transporter- (i.e. serotonergic) -positive fibers and nerve terminals in the mouse and rat frontal cortex. CB(1)R activation by the synthetic agonists, WIN55212-2 (1 μM) and R-methanandamide (1 μM) inhibited the simultaneously measured evoked Ca(2+)-dependent release of [(14)C]glutamate and [(3)H]serotonin from frontocortical nerve terminals of Wistar rats, in a fashion sensitive to the CB(1)R antagonists, O-2050 (1 μM) and LY320135 (5 μM). CB(1)R agonists also inhibited the evoked release of [(14)C]glutamate in C57BL/6J mice in a reversible fashion upon washout. Interestingly, the evoked release of [(14)C]glutamate and [(3)H]serotonin was significantly greater in the CB(1)R knockout CD-1 mice. Furthermore, CB(1)R binding experiments revealed similar frontocortical CB(1)R density in the rat and the CD-1 mouse. Still, the evoked release of [(3)H]serotonin was modulated by neither CB(1)R agonists nor antagonists in wild-type CD-1 or C57BL/6J mice. Altogether, this is the first study to demonstrate functional presynaptic CB(1)Rs in frontocortical glutamatergic and serotonergic terminals, revealing species differences.

Here we aimed to unify some previous controversial reports on changes in both cannabinoid CB1 receptor (CB1R) expression and glucose metabolism in the forebrain of rodent models of diabetes. We determined how glucose metabolism and its modulation by CB1R ligands evolve in the frontal cortex of young adult male Wistar rats, in the first 8 weeks of streptozotocin-induced type-1 diabetes (T1D). We report that frontocortical CB1R protein density was biphasically altered in the first month of T1D, which was accompanied with a reduction of resting glucose uptake ex vivo in acute frontocortical slices that was normalized after eight weeks in T1D. This early reduction of glucose uptake in slices was also restored by ex vivo treatment with both the non-selective CB1R agonists, WIN55212-2 (500 nM) and the CB1R-selective agonist, ACEA (3 μM) while it was exacerbated by the CB1R-selective antagonist, O-2050 (500 nM). These results suggest a gain-of-function for the cerebrocortical CB1Rs in the control of glucose uptake in diabetes. Although insulin and IGF-1 receptor protein densities remained unaffected, phosphorylated GSKα and GSKβ levels showed different profiles 2 and 8 weeks after T1D induction in the frontal cortex. Altogether, the biphasic response in frontocortical CB1R density within a month after T1D induction resolves previous controversial reports on forebrain CB1R levels in T1D rodent models. Furthermore, this study also hints that cannabinoids may be useful to alleviate impaired glucoregulation in the diabetic cortex.

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson's disease (PD) begin many years before the appearance of the characteristic motor symptoms. Neuropsychiatric, sensorial and cognitive deficits are recognized as early non-motor manifestations of PD, and are not attenuated by the current anti-parkinsonian therapy. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient mice do not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to dopaminergic neurotoxins such as 6-OHDA and MPTP. Here, we employed adult homozygous C57BL/6 mice with parkin gene deletion on exon 3 (parkin-/-) to further investigate the relevance of Parkin in the regulation of non-motor features, namely olfactory, emotional, cognitive and hippocampal synaptic plasticity. Parkin-/- mice displayed normal performance on behavioral tests evaluating olfaction (olfactory discrimination), anxiety (elevated plus-maze), depressive-like behavior (forced swimming and tail suspension) and motor function (rotarod, grasping strength and pole). However, parkin-/- mice displayed a poor performance in the open field habituation, object location and modified Y-maze tasks suggestive of procedural and short-term spatial memory deficits. These behavioral impairments were accompanied by impaired hippocampal long-term potentiation (LTP). These findings indicate that the genetic deletion of parkin causes deficiencies in hippocampal synaptic plasticity, resulting in memory deficits with no major olfactory, emotional or motor impairments. Therefore, parkin-/- mice may represent a promising animal model to study the early stages of PD and for testing new therapeutic strategies to restore learning and memory and synaptic plasticity impairments in PD.

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Neurodegeneration is a process transversal to neuropsychiatric diseases and the understanding of its mechanisms should allow devising strategies to prevent this irreversible step in brain diseases. Neurodegeneration caused by seizures is a critical step in the aggravation of temporal lobe epilepsy, but its mechanisms remain undetermined. Convulsions trigger an elevation of extracellular adenosine and upregulate adenosine A2A receptors (A2AR), which have been associated with the control of neurodegenerative diseases. Using the rat and mouse kainate model of temporal lobe epilepsy, we now tested whether A2AR control convulsions-induced hippocampal neurodegeneration. The pharmacological or genetic blockade of A2AR did not affect kainate-induced convulsions but dampened the subsequent neurotoxicity. This neurotoxicity began with a rapid A2AR upregulation within glutamatergic synapses (within 2 h), through local translation of synaptic A2AR mRNA. This bolstered A2AR-mediated facilitation of glutamate release and of long-term potentiation (LTP) in CA1 synapses (4 h), triggered a subsequent synaptotoxicity, heralded by decreased synaptic plasticity and loss of synaptic markers coupled to calpain activation (12 h), that predated overt neuronal loss (24 h). All modifications were prevented by the deletion of A2AR selectively in forebrain neurons. This shows that synaptic A2AR critically control synaptic excitotoxicity, which underlies the development of convulsions-induced neurodegeneration.

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212-2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB1R) and cancelled by concomitant administration of adenosine A2A receptor (A2AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by A2AR antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212-2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212-2 (300 nM), an effect partially rescued by the A2AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A2AR or CB1R binding in the hippocampus and in the prefrontal cortex. These results, showing that A2AR antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A2ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.

It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals.