Publications by authors named "Attila Köfalvi"

40 Publications

An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer's disease.

Eur J Neurosci 2020 Aug 19. Epub 2020 Aug 19.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2-arachidonoyl glycerol (2-AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2-AG, also accepts the surrogate chromogenic substrate 4-nitrophenyl butyrate (4-NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2-AG. The initial velocity of 4-NPB hydrolysis was dependent on protein, substrate, and Ca concentrations, and was sensitive to the non-selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2-AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4-NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin - an established model of sporadic AD -, both CB R levels and 4-NPB hydrolysis and its DAGL- and MAGL-dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples.
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http://dx.doi.org/10.1111/ejn.14944DOI Listing
August 2020

Transient gain of function of cannabinoid CB receptors in the control of frontocortical glucose consumption in a rat model of Type-1 diabetes.

Brain Res Bull 2020 08 16;161:106-115. Epub 2020 May 16.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal; Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal. Electronic address:

Here we aimed to unify some previous controversial reports on changes in both cannabinoid CB receptor (CBR) expression and glucose metabolism in the forebrain of rodent models of diabetes. We determined how glucose metabolism and its modulation by CBR 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 CBR 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 CBR agonists, WIN55212-2 (500 nM) and the CBR-selective agonist, ACEA (3 μM) while it was exacerbated by the CBR-selective antagonist, O-2050 (500 nM). These results suggest a gain-of-function for the cerebrocortical CBRs 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 CBR density within a month after T1D induction resolves previous controversial reports on forebrain CBR levels in T1D rodent models. Furthermore, this study also hints that cannabinoids may be useful to alleviate impaired glucoregulation in the diabetic cortex.
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http://dx.doi.org/10.1016/j.brainresbull.2020.05.004DOI Listing
August 2020

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BMC Biol 2020 01 23;18(1). Epub 2020 Jan 23.

Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, 21224, USA.

Background: It has been hypothesized that heteromers of adenosine A receptors (A2AR) and cannabinoid CB 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.

Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A receptors or dopamine D receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level.

Conclusions: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.
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http://dx.doi.org/10.1186/s12915-020-0739-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979073PMC
January 2020

Memory deficits induced by chronic cannabinoid exposure are prevented by adenosine AR receptor antagonism.

Neuropharmacology 2019 09 16;155:10-21. Epub 2019 May 16.

Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Portugal; Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal. Electronic address:

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 (CBR) and cancelled by concomitant administration of adenosine A receptor (AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by AR 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 AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect AR or CBR binding in the hippocampus and in the prefrontal cortex. These results, showing that AR 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 ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.
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http://dx.doi.org/10.1016/j.neuropharm.2019.05.003DOI Listing
September 2019

Chronic insulinopenia/hyperglycemia decreases cannabinoid CB receptor density and impairs glucose uptake in the mouse forebrain.

Brain Res Bull 2019 04 2;147:101-109. Epub 2019 Feb 2.

CNC, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal; Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal. Electronic address:

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 CB receptor knockout mice (CBR-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 CBR-KO, while hyperglycemia was similar. Resting [H]deoxyglucose uptake was significantly reduced by ˜20% in acute ex vivo frontocortical and hippocampal slices obtained from both the sham-injected CBR-KO and the diabetic WT mice. Surprisingly, the third cohort, the diabetic CBR-KO showed no further impairment in glucose uptake, as compared to the sham-injected CBR-KO. Depolarization-induced [H]deoxyglucose uptake was proportional to the respective resting values only in the cortex in all four cohorts. The dissipative metabolism of [C]-U-glucose remained largely unaffected in all cohorts of animals. However, diabetes reduced cortical CBR 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 CBR expression reduces glucose uptake in the mouse brain. Additionally, diabetes failed to cause further impairment in cerebral glucose uptake in the CBR-KO. These suggest that diabetic encephalopathy may be in part associated with lower CBR expression.
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http://dx.doi.org/10.1016/j.brainresbull.2019.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420377PMC
April 2019

Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions.

eNeuro 2018 Nov-Dec;5(6). Epub 2018 Dec 26.

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

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 A receptors (AR), 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 AR control convulsions-induced hippocampal neurodegeneration. The pharmacological or genetic blockade of AR did not affect kainate-induced convulsions but dampened the subsequent neurotoxicity. This neurotoxicity began with a rapid AR upregulation within glutamatergic synapses (within 2 h), through local translation of synaptic AR mRNA. This bolstered AR-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 AR selectively in forebrain neurons. This shows that synaptic AR critically control synaptic excitotoxicity, which underlies the development of convulsions-induced neurodegeneration.
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http://dx.doi.org/10.1523/ENEURO.0385-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325550PMC
March 2019

Adenosine A Receptors in the Rat Prelimbic Medial Prefrontal Cortex Control Delay-Based Cost-Benefit Decision Making.

Front Mol Neurosci 2018 20;11:475. Epub 2018 Dec 20.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Adenosine A receptors (ARs) were recently described to control synaptic plasticity and network activity in the prefrontal cortex (PFC). We now probed the role of these PFC AR by evaluating the behavioral performance (locomotor activity, anxiety-related behavior, cost-benefit decision making and working memory) of rats upon downregulation of AR selectively in the prelimbic medial PFC (PLmPFC) via viral small hairpin RNA targeting the AR (shAR). The most evident alteration observed in shAR-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 shAR 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 AR in the control of behavior in physiological conditions, showing their major contribution for the control of delay-based cost-benefit decisions.
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http://dx.doi.org/10.3389/fnmol.2018.00475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306464PMC
December 2018

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

Cell Death Dis 2018 02 20;9(3):297. Epub 2018 Feb 20.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.

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 Ca entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.
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http://dx.doi.org/10.1038/s41419-018-0351-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833818PMC
February 2018

Boosting brain glucose metabolism to fight neurodegeneration?

Oncotarget 2017 Feb;8(9):14273-14274

CNC-Center for Neurosciences and Cell Biology of Coimbra and Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal.

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http://dx.doi.org/10.18632/oncotarget.15131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362400PMC
February 2017

Methamphetamine Induces Anhedonic-Like Behavior and Impairs Frontal Cortical Energetics in Mice.

CNS Neurosci Ther 2017 Feb 19;23(2):119-126. Epub 2016 Oct 19.

Laboratory of Pharmacology and Experimental Therapeutics/Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Introduction: We recently showed that a single high dose of methamphetamine (METH) induces a persistent frontal cortical monoamine depletion that is accompanied by helpless-like behavior in mice. However, brain metabolic alterations underlying both neurochemical and mood alterations remain unknown.

Aims: Herein, we aimed at characterizing frontal cortical metabolic alterations associated with early negative mood behavior triggered by METH. Adult C57BL/6 mice were injected with METH (30 mg/kg, i.p.), and their frontal cortical metabolic status was characterized after probing their mood and anxiety-related phenotypes 3 days postinjection.

Results: Methamphetamine induced depressive-like behavior, as indicated by the decreased grooming time in the splash test and by a transient decrease in sucrose preference. At this time, METH did not alter anxiety-like behavior or motor functions. Depolarization-induced glucose uptake was reduced in frontocortical slices from METH-treated mice compared to controls. Consistently, astrocytic glucose transporter (GluT1) density was lower in the METH group. A proton high rotation magic angle spinning (HRMAS) spectroscopic approach revealed that METH induced a significant decrease in N-acetyl aspartate (NAA) and glutamate levels, suggesting that METH decreased neuronal glutamatergic function in frontal cortex.

Conclusions: We report, for the first time, that a single METH injection triggers early self-care and hedonic deficits and impairs frontal cortical energetics in mice.
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http://dx.doi.org/10.1111/cns.12649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492743PMC
February 2017

Cannabis: A Treasure Trove or Pandora's Box?

Mini Rev Med Chem 2017 ;17(13):1223-1291

Laboratory of Neuromodulation and Metabolism, CNC - Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal.

Background & Objective: Cannabis is one of the earliest cultivated plants. Cannabis of industrial utility and culinary value is generally termed as hemp. Conversely, cannabis that is bred for medical, spiritual and recreational purposes is called marijuana. The female marijuana plant produces a significant quantity of bio- and psychoactive phytocannabinoids, which regained the spotlight with the discovery of the endocannabinoid system of the animals in the early 90's. Nevertheless, marijuana is surrounded by controversies, debates and misconceptions related to its taxonomic classification, forensic identification, medical potential, legalization and its long-term health consequences.

Method: In the first part, we provide an in-depth review of the botany and taxonomy of Cannabis. We then overview the biosynthesis of phytocannabinoids within the glandular trichomes with emphasis on the role of peculiar plastids in the production of the secreted material. We also compile the analytical methods used to determine the phytocannabinoid composition of glandular trichomes. In the second part, we revisit the psychobiology and molecular medicine of marijuana.

Results & Conclusion: We summarize our current knowledge on the recreational use of cannabis with respect to the modes of consumption, short-term effects, chronic health consequences and cannabis use disorder. Next, we overview the molecular targets of a dozen major and minor bioactive cannabinoids in the body. This helps us introduce the endocannabinoid system in an unprecedented detail: its up-todate molecular biology, pharmacology, physiology and medical significance, and beyond. In conclusion, we offer an unbiased survey about cannabis to help better weigh its medical value versus the associated risks.
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http://dx.doi.org/10.2174/1389557516666161004162133DOI Listing
September 2017

Hierarchical glucocorticoid-endocannabinoid interplay regulates the activation of the nucleus accumbens by insulin.

Brain Res Bull 2016 06 18;124:222-30. Epub 2016 May 18.

CNC, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal; Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal. Electronic address:

Here we asked if insulin activation of the nucleus accumbens in vitro is reflected by an increase in (3)H-deoxyglucose ([(3)H]DG) uptake, thus subserving a new model to study molecular mechanisms of central insulin actions. Additionally, we investigated the dependence of this insulin effect on endocannabinoids and corticosteroids, two major culprits in insulin resistance. We found that in acute accumbal slices, insulin (3 and 300nM but not at 0.3nM) produced an increase in [(3)H]DG uptake. The synthetic cannabinoid agonist, WIN55212-2 (500nM) and the glucocorticoid dexamethasone (10μM), impaired insulin (300nM) action on [(3)H]DG uptake. The glucocorticoid receptor (GcR) antagonist, mifepristone (10μM) prevented dexamethasone from inhibiting insulin's action. Strikingly, this anti-insulin action of dexamethasone was also blocked by two CB1 cannabinoid receptor (CB1R) antagonists, O-2050 (500nM) and SR141716A (500nM), as well as by tetrahydrolipstatin (10μM), an inhibitor of diacylglycerol lipases-the enzymes responsible for the synthesis of the endocannabinoid, 2-arachidonoyl-glycerol (2-AG). On the other hand, the blockade of the post-synaptic 2-AG metabolizing enzymes, α,β-serine hydrolase domain 6/12 by WWL70 (1μM) also prevented the action of insulin, probably via increasing endogenous 2-AG tone. Additionally, an anti-insulin receptor (InsR) antibody immunoprecipitated CB1Rs from accumbal homogenates, indicating a physical complexing of CB1Rs with InsRs that supports their functional interaction. Altogether, insulin stimulates glucose uptake in the nucleus accumbens. Accumbal GcR activation triggers the synthesis of 2-AG that in turn binds to the known CB1R-InsR heteromer, thus impeding insulin signaling.
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http://dx.doi.org/10.1016/j.brainresbull.2016.05.009DOI Listing
June 2016

Stimulation of brain glucose uptake by cannabinoid CB2 receptors and its therapeutic potential in Alzheimer's disease.

Neuropharmacology 2016 11 11;110(Pt A):519-529. Epub 2016 Mar 11.

Neurodegeneration Group, Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, CSIC, Doctor Arce, 37, 28002 Madrid, Spain; CIBERNED; Centre for Biomedical Research on Neurodegenerative Diseases, Spain. Electronic address:

Cannabinoid CB2 receptors (CB2Rs) are emerging as important therapeutic targets in brain disorders that typically involve neurometabolic alterations. We here addressed the possible role of CB2Rs in the regulation of glucose uptake in the mouse brain. To that aim, we have undertaken 1) measurement of (3)H-deoxyglucose uptake in cultured cortical astrocytes and neurons and in acute hippocampal slices; 2) real-time visualization of fluorescently labeled deoxyglucose uptake in superfused hippocampal slices; and 3) in vivo PET imaging of cerebral (18)F-fluorodeoxyglucose uptake. We now show that both selective (JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2R agonists, but not the CB1R-selective agonist, ACEA, stimulate glucose uptake, in a manner that is sensitive to the CB2R-selective antagonist, AM630. Glucose uptake is stimulated in astrocytes and neurons in culture, in acute hippocampal slices, in different brain areas of young adult male C57Bl/6j and CD-1 mice, as well as in middle-aged C57Bl/6j mice. Among the endocannabinoid metabolizing enzymes, the selective inhibition of COX-2, rather than that of FAAH, MAGL or α,βDH6/12, also stimulates the uptake of glucose in hippocampal slices of middle-aged mice, an effect that was again prevented by AM630. However, we found the levels of the endocannabinoid, anandamide reduced in the hippocampus of TgAPP-2576 mice (a model of β-amyloidosis), and likely as a consequence, COX-2 inhibition failed to stimulate glucose uptake in these mice. Together, these results reveal a novel general glucoregulatory role for CB2Rs in the brain, raising therapeutic interest in CB2R agonists as nootropic agents.
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http://dx.doi.org/10.1016/j.neuropharm.2016.03.015DOI Listing
November 2016

Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A Receptor in Hippocampal Glutamate Synapses.

Mol Neurobiol 2017 03 9;54(2):1552-1563. Epub 2016 Feb 9.

CNC-Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal.

Caffeine prophylactically prevents mood and memory impairments through adenosine A receptor (AR) antagonism. AR antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher AR density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the AR agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the AR antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal AR controlling synaptic glutamatergic function.
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http://dx.doi.org/10.1007/s12035-016-9774-9DOI Listing
March 2017

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain.

Purinergic Signal 2015 Dec 7;11(4):561-9. Epub 2015 Oct 7.

CNC, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504, Coimbra, Portugal.

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A2B receptor (A(2B)R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A(2B)R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A(2B)R antagonist, MRS1754 (200 nM), and was also absent in A(2B)R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased (3)H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A(2B)R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A(2B)R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.
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http://dx.doi.org/10.1007/s11302-015-9474-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648789PMC
December 2015

Lack of presynaptic interaction between glucocorticoid and CB1 cannabinoid receptors in GABA- and glutamatergic terminals in the frontal cortex of laboratory rodents.

Neurochem Int 2015 Nov 18;90:72-84. Epub 2015 Jul 18.

CNC, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal; Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuint.2015.07.014DOI Listing
November 2015

Ketone bodies effectively compete with glucose for neuronal acetyl-CoA generation in rat hippocampal slices.

NMR Biomed 2015 Sep 15;28(9):1111-6. Epub 2015 Jul 15.

CNC-Center for Neurosciences and Cell Biology, University of Coimbra, Portugal.

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1-(13)C]glucose and 1.0 mM [2-(13)C]lactate + 0.1 mM pyruvate (combined [2-(13)C]lactate + [2-(13)C]pyruvate) to acetyl-CoA production were measured both without and with 5.0 mM [U-(13)C]3-hydroxybutyrate in superfused rat hippocampal slices by (13)C NMR non-steady-state isotopomer analysis of tissue glutamate and GABA. Without [U-(13)C]3-hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl-CoA, respectively. With [U-(13)C]3-hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U-(13)C]3-hydroxybutyrate became the major acetyl-CoA contributor (68 ± 3%)--about three-times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U-(13)C]3-hydroxybutyrate contributed approximately the same acetyl-CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1-(13)C]glucose did not decrease significantly in the presence of 3-hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3-hydroxybutyrate. This indicates that, under these conditions, 3-hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase.
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http://dx.doi.org/10.1002/nbm.3355DOI Listing
September 2015

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus.

Eur J Neurosci 2015 Apr 19;41(7):878-88. Epub 2015 Feb 19.

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

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A(2B)R) are G(s/q)--protein-coupled receptors with low expression in the brain. As A(2B)R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A(2B)R-knockout (A(2B)R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A(2B)R agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A(2B)R antagonist MRS 1754 (200 nM) and abrogated in A(2B)R-KO mice. Accordingly, A(2B)R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A(1)R control of synaptic transmission, both the A(1)R inhibition caused by 2-chloroadenosine (0.1-1 μM) and the disinhibition caused by the A(1)R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A(2B)R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A(1)R inhibition (CPA 100 nM). A(2B)R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A(2B)R are present in hippocampal glutamatergic terminals where they counteract the predominant A(1)R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.
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http://dx.doi.org/10.1111/ejn.12851DOI Listing
April 2015

Behavioral phenotyping of Parkin-deficient mice: looking for early preclinical features of Parkinson's disease.

PLoS One 2014 8;9(12):e114216. Epub 2014 Dec 8.

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, 88049-900, SC, Brazil.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259468PMC
August 2015

Presynaptic TRPV1 vanilloid receptor function is age- but not CB1 cannabinoid receptor-dependent in the rodent forebrain.

Brain Res Bull 2013 Aug 4;97:126-35. Epub 2013 Jul 4.

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, 1089 Budapest, Hungary.

Neocortical and striatal TRPV1 (vanilloid or capsaicin) receptors (TRPV1Rs) are excitatory ligand-gated ion channels, and are implicated in psychiatric disorders. However, the purported presynaptic neuromodulator role of TRPV1Rs in glutamatergic, serotonergic or dopaminergic terminals of the rodent forebrain remains little understood. With the help of patch-clamp electrophysiology and neurochemical approaches, we mapped the age-dependence of presynaptic TRPV1R function, and furthermore, we aimed at exploring whether the presence of CB1 cannabinoid receptors (CB1Rs) influences the function of the TRPV1Rs, as both receptor types share endogenous ligands. We found that the major factor which affects presynaptic TRPV1R function is age: by post-natal day 13, the amplitude of capsaicin-induced release of dopamine and glutamate is halved in the rat striatum, and two weeks later, capsaicin already loses its effect. However, TRPV1R receptor function is not enhanced by chemical or genetic ablation of the CB1Rs in dopaminergic, glutamatergic and serotonergic terminals of the mouse brain. Altogether, our data indicate a possible neurodevelopmental role for presynaptic TRPV1Rs in the rodent brain, but we found no cross-talk between TRPV1Rs and CB1Rs in the same nerve terminal.
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http://dx.doi.org/10.1016/j.brainresbull.2013.06.007DOI Listing
August 2013

Presynaptic α₂-adrenoceptors control the inhibitory action of presynaptic CB₁ cannabinoid receptors on prefrontocortical norepinephrine release in the rat.

Neuropharmacology 2012 Oct 18;63(5):784-97. Epub 2012 Jun 18.

Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences-IEM-HAS, H-1083 Budapest, Szigony U. 43, Hungary.

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.
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http://dx.doi.org/10.1016/j.neuropharm.2012.06.003DOI Listing
October 2012

Presynaptic CB(1) cannabinoid receptors control frontocortical serotonin and glutamate release--species differences.

Neurochem Int 2012 Jul 17;61(2):219-26. Epub 2012 May 17.

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

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.
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http://dx.doi.org/10.1016/j.neuint.2012.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408788PMC
July 2012

Caffeine regulates frontocorticostriatal dopamine transporter density and improves attention and cognitive deficits in an animal model of attention deficit hyperactivity disorder.

Eur Neuropsychopharmacol 2013 Apr 4;23(4):317-28. Epub 2012 May 4.

Department of Pharmacology, Biological Science Center, Federal University of Santa Catarina, Florianopolis, Brazil.

Attention deficit hyperactivity disorder (ADHD) likely involves dopaminergic dysfunction in the frontal cortex and striatum, resulting in cognitive and motor abnormalities. Since both adenosine and dopamine modulation systems are tightly intertwined, we tested if caffeine (a non-selective adenosine receptor antagonist) attenuated the behavioral and neurochemical changes in adolescent spontaneously hypertensive rats (SHR, a validated ADHD animal model) compared to their control strain (Wistar Kyoto rats, WKY). SHR were hyperactive and had poorer performance in the attentional set-shifting and Y-maze paradigms and also displayed increased dopamine transporter (DAT) density and increased dopamine uptake in frontocortical and striatal terminals compared with WKY rats. Chronic caffeine treatment was devoid of effects in WKY rats while it improved memory and attention deficits and also normalized dopaminergic function in SHR. Additionally, we provide the first direct demonstration for the presence of adenosine A2A receptors (A2AR) in frontocortical nerve terminals, whose density was increased in SHR. These findings underscore the potential for caffeine treatment to normalize frontocortical dopaminergic function and to abrogate attention and cognitive changes characteristic of ADHD.
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http://dx.doi.org/10.1016/j.euroneuro.2012.04.011DOI Listing
April 2013

Impaired hippocampal glucoregulation in the cannabinoid CB1 receptor knockout mice as revealed by an optimized in vitro experimental approach.

J Neurosci Methods 2012 Mar 6;204(2):366-73. Epub 2011 Dec 6.

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

Several techniques exist to study the rate of glucose uptake and metabolism in the brain but most of them are not sufficiently robust to permit extensive pharmacological analysis. Here we optimized an in vitro measurement of the simultaneous accumulation of the metabolizable and non-metabolizable (3)H and (14)C d-glucose analogues; permitting convenient large-scale studies on glucose uptake and metabolism in brain slices. Next, we performed an extensive pharmacological characterization on the putative glucoregulator role of the endocannabinoid system in the hippocampal slices of the rat, and the wild-type and the CB(1) cannabinoid receptor (CB(1)R) knockout mice. We observed that (3)H-3-O-methylglucose is a poor substrate to measure glucose uptake in the hippocampus. (3)H-2-deoxyglucose is a better substrate but its uptake is still lower than that of (14)C-U-d-glucose, from which the slices constantly metabolize and dissipate (14)C atoms. Thus, uptake and the metabolism values are not to be used as standalones but as differences between a control and a treatment. The CB(1)R knockout mice exhibited ∼10% less glucose uptake and glucose carbon atom dissipation in comparison with the wild-type mice. This may represent congenital defects as acute treatments of the rat and mouse slices with cannabinoid agonists, antagonists and inhibitors of endocannabinoid uptake/metabolism failed to induce robust changes in either the uptake or the metabolism of glucose. In summary, we report here an optimized technique ideal to complement other metabolic approaches of high spatiotemporal resolution. This technique allowed us concluding that CB(1)Rs are at least indirectly involved in hippocampal glucoregulation.
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http://dx.doi.org/10.1016/j.jneumeth.2011.11.028DOI Listing
March 2012

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus.

Neurochem Int 2012 Jan 9;60(1):1-8. Epub 2011 Nov 9.

Center for Neurosciences and Cell Biology of Coimbra, University of Coimbra, Coimbra, Portugal.

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (-11.6±2.0%) and [U-¹³C]glucose (-11.2±3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (-11.7±4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus. In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.
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http://dx.doi.org/10.1016/j.neuint.2011.10.019DOI Listing
January 2012

Molecular reorganization of endocannabinoid signalling in Alzheimer's disease.

Brain 2011 Apr;134(Pt 4):1041-60

European Neuroscience Institute at Aberdeen, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.

Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.
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http://dx.doi.org/10.1093/brain/awr046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069704PMC
April 2011

Diabetes induces early transient changes in the content of vesicular transporters and no major effects in neurotransmitter release in hippocampus and retina.

Brain Res 2011 Apr 28;1383:257-69. Epub 2011 Jan 28.

Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal.

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.
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http://dx.doi.org/10.1016/j.brainres.2011.01.071DOI Listing
April 2011

Cannabinoids inhibit the synaptic uptake of adenosine and dopamine in the rat and mouse striatum.

Eur J Pharmacol 2011 Mar 23;655(1-3):38-45. Epub 2011 Jan 23.

Department of Pharmacology, Center of Biological Sciences, Federal University of Santa Catarina (UFSC), Florianópolis, SC, Brazil.

Adenosine, dopamine and endocannabinoids strictly modulate the release of one another in the dorsolateral striatum thereby controlling synaptic plasticity. As a second level of interaction, they regulate the action of one another via receptor heteromer formation. Here we investigated a putative third level of interaction, i.e. the possible control by cannabinoids of synaptic dopamine and adenosine reuptake. We found that a large number of endo- and exogenous cannabinoid ligands inhibit the uptake of [(3)H]adenosine and [(3)H]dopamine in rat sriatal nerve terminals. Maximal effects were often comparable to those of the dopamine transporter inhibitor, GBR12783 and the equilibrative nucleoside transporter inhibitor, dipyridamole. Cannabinoid ligands were generally more potent to inhibit the uptake of adenosine than that of dopamine. The inhibitory effect was: (1) unrelated to the pharmacological profile(s) of the ligands at the cannabinoid CB(1), CB(2), GPR55 and at the vanilloid TRPV(1) receptors; (2) not prevented by the cannabinoid CB(1) receptor antagonist/inverse agonist, LY320135; and (3) maintained in the cannabinoid CB(1) receptor knockout mice. In the same experiments, only O-2050, cannabidiol, and WIN55212-3 inhibited the simultaneously measured DL-TBOA-sensitive uptake of [(14)C]glutamate. In summary, many cannabinoid ligands are able to inhibit the synaptic uptake of adenosine and dopamine. These effects are not mediated by cannabinoid CB(1) receptors, and should be an additional mechanism to consider when interpreting synaptic effects of cannabinoids.
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http://dx.doi.org/10.1016/j.ejphar.2011.01.013DOI Listing
March 2011

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

J Neurochem 2011 Jan 2;116(2):273-80. Epub 2010 Dec 2.

Section of Central Nervous System Pharmacology, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.
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http://dx.doi.org/10.1111/j.1471-4159.2010.07101.xDOI Listing
January 2011

N-acyldopamines control striatal input terminals via novel ligand-gated cation channels.

Neuropharmacology 2009 Mar 7;56(3):676-83. Epub 2008 Dec 7.

Center for Neuroscience and Cell Biology of Coimbra, Department of Zoology, Faculty of Medicine, University of Coimbra, 1 Rua Larga, 3004-504 Coimbra, Portugal.

Endogenous analogues of capsaicin, N-acyldopamines, were previously identified from striatal extracts, but the putative presynaptic role of their receptor, the TRPV(1)R (formerly: vanilloid or capsaicin receptor) in the caudate-putamen is unclear. We found that the endogenous TRPV(1)R agonists, N-arachidonoyldopamine (NADA) and oleoyldopamine (OLDA) with EC(50) values in the nanomolar range, as well as the synthetic TRPV(1)R activator 2-aminoethoxydiphenylborane (2APB), and palmytoyldopamine (PALDA, another endogenous N-acyldopamine inactive at the TRPV(1)R), but not capsaicin or other endogenous and synthetic cannabinoids, triggered a rapid Ca(2+) entry with the concomitant stimulation of glutamate and dopamine release. These effects persisted in the TRPV(1)R null-mutant mice, and were insensitive to antagonists of common ionotropic receptors, to several TRPV(1)R antagonists and to the absence of K(+). Furthermore, these N-acyldopamine receptors in glutamatergic and dopaminergic terminals are different based on their different sensitivity to anandamide, capsazepine and Gd(3+) at nanomolar concentrations. Altogether, novel ion channels instead of the TRPV(1)R mediate the presynaptic action of N-acyldopamines in the striatum of adult rodents.
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http://dx.doi.org/10.1016/j.neuropharm.2008.12.001DOI Listing
March 2009