Publications by authors named "Cristina Lemos"

26 Publications

  • Page 1 of 1

Involvement of cAMP-Dependent Protein Kinase in the Nucleus Accumbens in Cocaine Versus Social Interaction Reward.

Int J Mol Sci 2020 Dec 31;22(1). Epub 2020 Dec 31.

Division of Psychiatry I, Department of Psychiatry, Psychotherapy and Psychosomatics, Medical University Innsbruck, 6020 Innsbruck, Austria.

Evidence suggests that PKA activity in the nucleus accumbens (NAc) plays an essential role in reward-related learning. In this study, we investigated whether PKA is differentially involved in the expression of learning produced by either natural reinforcers or psychostimulants. For that purpose, we inhibited PKA through a bilateral infusion of Rp-cAMPS, a specific PKA inhibitor, directly into the NAc. The effects of PKA inhibition in the NAc on the expression of concurrent conditioned place preference (CPP) for cocaine (drug) and social interaction (natural reward) in rats were evaluated. We found that PKA inhibition increased the expression of cocaine preference. This effect was not due to altered stress levels or decreased social reward. PKA inhibition did not affect the expression of natural reward as intra-NAc Rp-cAMPS infusion did not affect expression of social preference. When rats were trained to express cocaine or social interaction CPP and tested for eventual persisting preference 7 and 14 days after CPP expression, cocaine preference was persistent, but social preference was abolished after the first test. These results suggest that PKA in the NAc is involved in drug reward learning that might lead to addiction and that only drug, but not natural, reward is persistent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22010345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794935PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.brainresbull.2020.05.004DOI Listing
August 2020

Social interaction reward in rats has anti-stress effects.

Addict Biol 2021 01 26;26(1):e12878. Epub 2020 Jan 26.

Department of Psychiatry, Psychotherapy and Psychosomatics, Division of Psychiatry I, Medical University Innsbruck, Innsbruck, Austria.

Social interaction in an alternative context can be beneficial against drugs of abuse. Stress is known to be a risk factor that can exacerbate the effects of addictive drugs. In this study, we investigated whether the positive effects of social interaction are mediated through a decrease in stress levels. For that purpose, rats were trained to express cocaine or social interaction conditioned place preference (CPP). Behavioural, hormonal, and molecular stress markers were evaluated. We found that social CPP decreased the percentage of incorrect transitions of grooming and corticosterone to the level of naïve untreated rats. In addition, corticotropin-releasing factor (CRF) was increased in the bed nucleus of stria terminalis after cocaine CPP. In order to study the modulation of social CPP by the CRF system, rats received intracerebroventricular CRF or alpha-helical CRF, a nonselective antagonist of CRF receptors. The subsequent effects on CPP to cocaine or social interaction were observed. CRF injections increased cocaine CPP, whereas alpha-helical CRF injections decreased cocaine CPP. However, alpha-helical CRF injections potentiated social CPP. When social interaction was made available in an alternative context, CRF-induced increase of cocaine preference was reversed completely to the level of rats receiving cocaine paired with alpha-helical CRF. This reversal of cocaine preference was also paralleled by a reversal in CRF-induced increase of p38 MAPK expression in the nucleus accumbens shell. These findings suggest that social interaction could contribute as a valuable component in treatment of substance use disorders by reducing stress levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/adb.12878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757251PMC
January 2021

Synaptic and memory dysfunction in a β-amyloid model of early Alzheimer's disease depends on increased formation of ATP-derived extracellular adenosine.

Neurobiol Dis 2019 12 5;132:104570. Epub 2019 Aug 5.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; FMUC-Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal. Electronic address:

Adenosine A receptors (AR) overfunction causes synaptic and memory dysfunction in early Alzheimer's disease (AD). In a β-amyloid (Aβ)-based model of early AD, we now unraveled that this involves an increased synaptic release of ATP coupled to an increased density and activity of ecto-5'-nucleotidase (CD73)-mediated formation of adenosine selectively activating AR. Thus, CD73 inhibition with α,β-methylene-ADP impaired long-term potentiation (LTP) in mouse hippocampal slices, which is occluded upon previous superfusion with the AR antagonist SCH58261. Furthermore, α,β-methylene-ADP did not alter LTP amplitude in global AR knockout (KO) and in forebrain neuron-selective AR-KO mice, but inhibited LTP amplitude in astrocyte-selective AR-KO mice; this shows that CD73-derived adenosine solely acts on neuronal AR. In agreement with the concept that ATP is a danger signal in the brain, ATP release from nerve terminals is increased after intracerebroventricular Aβ administration, together with CD73 and AR upregulation in hippocampal synapses. Importantly, this increased CD73 activity is critically required for Aβ to impair synaptic plasticity and memory since Aβ-induced synaptic and memory deficits were eliminated in CD73-KO mice. These observations establish a key regulatory role of CD73 activity over neuronal AR and imply CD73 as a novel target for modulation of early AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2019.104570DOI Listing
December 2019

Emergence of the East-Central-South-African genotype of Chikungunya virus in Brazil and the city of Rio de Janeiro may have occurred years before surveillance detection.

Sci Rep 2019 02 26;9(1):2760. Epub 2019 Feb 26.

Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York, USA.

Brazil, which is hyperendemic for dengue virus (DENV), has had recent Zika (ZIKV) and (CHIKV) Chikungunya virus outbreaks. Since March 2016, CHIKV is the arbovirus infection most frequently diagnosed in Rio de Janeiro. In the analysis of 1835 syndromic patients, screened by real time RT-PCR, 56.4% of the cases were attributed to CHIKV, 29.6% to ZIKV, and 14.1% to DENV-4. Sequence analyses of CHIKV from sixteen samples revealed that the East-Central-South-African (ECSA) genotype of CHIKV has been circulating in Brazil since 2013 [95% bayesian credible interval (BCI): 03/2012-10/2013], almost a year before it was detected by arbovirus surveillance program. Brazilian cases are related to Central African Republic sequences from 1980's. To the best of our knowledge, given the available sequence published here and elsewhere, the ECSA genotype was likely introduced to Rio de Janeiro early on 2014 (02/2014; BCI: 07/2013-08/2014) through a single event, after primary circulation in the Bahia state at the Northestern Brazil in the previous year. The observation that the ECSA genotype of CHIKV was circulating undetected underscores the need for improvements in molecular methods for viral surveillance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-39406-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391440PMC
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.brainresbull.2019.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420377PMC
April 2019

Reply to commentary on Zika: An Ongoing Threat to Women and Infants.

Cad Saude Publica 2018 11 8;34(11):e00192318. Epub 2018 Nov 8.

Secretaria Municipal de Saúde, Rio de Janeiro, Brasil.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/0102-311X00192318DOI Listing
November 2018

Zika: an ongoing threat to women and infants.

Cad Saude Publica 2018 11 8;34(11):e00038218. Epub 2018 Nov 8.

Secretaria Municipal de Saúde, Rio de Janeiro, Brasil.

Recent data from the municipality of Rio de Janeiro, Brazil, shows a sharp drop in the number of reported occurrences of Zika during the summer of 2016/2017, compared to the previous summer. There is still a much higher incidence among women than men, almost certainly due to sexual transmission. An unexpected feature of the new data is that there are proportionally far more cases affecting children under 15 months than older age classes. By comparing incidence rates in 2016/2017 and 2015/2016, we were able to deduce the proportion of reported cases affecting men and women, and verify that gender disparity is still present. Women and children are still risk groups for Zika infection, even during non-epidemic seasons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/0102-311X00038218DOI Listing
November 2018

Cocaine Paired Environment Increases SATB2 Levels in the Rat Paraventricular Thalamus.

Front Behav Neurosci 2018 2;12:224. Epub 2018 Oct 2.

Experimental Psychiatry Unit, Medical University of Innsbruck, Innsbruck, Austria.

SATB2 is a DNA binding protein that specifically binds the nuclear matrix attachment region and functions as a regulator of the transcription of large chromatin domains. Unlike its well addressed role during brain development, the role of SATB2 in adult brain is under-investigated. It has been shown that deletion of SATB2 from the forebrain of adult mice significantly impaired long-term memory for contextual fear and object recognition memory. The aim of the present study was to investigate the effects of appetitive stimuli such as cocaine and social interaction (SI) on SATB2 expression in the adult rat brain. For that, we performed conditioned place preference (CPP) to cocaine (15 mg/kg) and to SI, then assessed SATB2 expression in the brain 1 h (24 h after the last conditioning) and 24 h (48 h after the last conditioning) after the CPP test. We found that SATB2 expression in the paraventricular thalamus of rats was increased 1 h after the cocaine CPP test. This increase was selective for the cocaine-paired environment since the SI-paired environment did not increase SATB2 expression in the paraventricular thalamus. Also, the cocaine paired environment-induced increase of SATB2 levels in the paraventricular thalamus was due to cocaine conditioning as the unpaired cocaine group did not show an increase of SATB2 in the paraventricular thalamus. These results suggest that SATB2 in the paraventricular thalamus appears to be involved in the association between cocaine effects and environmental context. Further studies are needed to address the functional role of SATB2 in cocaine conditioning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnbeh.2018.00224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190852PMC
October 2018

Blockade of adenosine A receptors recovers early deficits of memory and plasticity in the triple transgenic mouse model of Alzheimer's disease.

Neurobiol Dis 2018 09 31;117:72-81. Epub 2018 May 31.

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

Alzheimer's disease (AD) begins with a deficit of synaptic function and adenosine A receptors (AR) are mostly located in synapses controlling synaptic plasticity. The over-activation of adenosine A receptors (AR) causes memory deficits and the blockade of AR prevents memory damage in AD models. We now enquired if this prophylactic role of AR might be extended to a therapeutic potential. We used the triple transgenic model of AD (3xTg-AD) and defined that the onset of memory dysfunction occurred at 4 months of age in the absence of locomotor or emotional alterations. At the onset of memory deficits, 3xTg mice displayed a decreased density of markers of excitatory synapses (10.6 ± 3.8% decrease of vGluT1) without neuronal or glial overt damage and an increase of synaptic AR in the hippocampus (130 ± 22%). After the onset of memory deficits in 3xTg-AD mice, a three weeks treatment with the selective AR antagonist normalized the up-regulation of hippocampal AR and restored hippocampal-dependent reference memory, as well as the decrease of hippocampal synaptic plasticity (60.0 ± 3.7% decrease of long-term potentiation amplitude) and the decrease of global (syntaxin-I) and glutamatergic synaptic markers (vGluT1). These findings show a therapeutic-like ability of AR antagonists to recover synaptic and memory dysfunction in early AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2018.05.024DOI Listing
September 2018

Can Zika Account for the Missing Babies?

Front Public Health 2017 29;5:317. Epub 2017 Nov 29.

Prefeitura da Cidade do Rio de Janeiro, Rio de Janeiro, Brazil.

The Zika virus (ZIKV) spread rapidly in Brazil in 2015 and 2016. Rio de Janeiro was among the Brazilian cities which were hit the hardest, with more that a hundred thousand confirmed cases up to the end of 2016. Given the severity of the neurological damage caused by ZIKV on fetuses, we wondered whether it would also cause an increase in the number of miscarriages, especially very early ones. As early miscarriages are unlikely to be recorded as a health event, this effect-if it occurred-would only show up as a reduction in the number of live births. In this article, we show that there was a 15% drop in live births between September and December 2016 compared with the previous year, and that this sharp drop from epidemiological week 33 onward is strongly correlated with the number of recorded cases of Zika about 40 weeks earlier. We postulate that ZIKV is directly responsible for this drop in the birth rate. Further work is required to ascertain whether other factors such as the fear of having a microcephaly baby or the economic crisis are having a significant effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpubh.2017.00317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712541PMC
November 2017

The effects of physical exercise on nonmotor symptoms and on neuroimmune RAGE network in experimental parkinsonism.

J Appl Physiol (1985) 2017 Jul 6;123(1):161-171. Epub 2017 Apr 6.

Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;

Parkinson's disease (PD) prodromal stages comprise neuropsychiatric perturbations that critically compromise a patient's quality of life. These nonmotor symptoms (NMS) are associated with exacerbated innate immunity, a hallmark of overt PD. Physical exercise (PE) has the potential to improve neuropsychiatric deficits and to modulate immune network including receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) in distinct pathological settings. Accordingly, the present study aimed to test the hypothesis that PE ) alleviates PD NMS and ) modulates neuroimmune RAGE network in experimental PD. Adult Wistar rats subjected to long-term mild treadmill were administered intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probed for PD NMS before the onset of motor abnormalities. Twelve days after MPTP, neuroimmune RAGE network transcriptomics (real-time quantitative PCR) was analyzed in frontal cortex, hippocampus, and striatum. Untrained MPTP animals displayed habit-learning and motivational deficits without gross motor impairments (cued version of water-maze, splash, and open-field tests, respectively). A suppression of RAGE and neuroimmune-related genes was observed in frontal cortex on chemical and physical stressors (untrained MPTP: RAGE, TLR5 and -7, and p22 NADPH oxidase; saline-trained animals: RAGE, TLR1 and -5 to -11, TNF-α, IL-1β, and p22 NADPH oxidase), suggesting the recruitment of compensatory mechanisms to restrain innate inflammation. Notably, trained MPTP animals displayed normal cognitive/motivational performances. Additionally, these animals showed normal RAGE expression and neuroprotective PD-related gene upregulation in frontal cortex when compared with untrained MPTP animals. These findings corroborate PE efficacy in improving PD NMS and newly identify RAGE network as a neural substrate for exercise intervention. Additional research is warranted to unveil functional consequences of PE-induced modulation of RAGE/DJ-1 transcriptomics in PD premotor stages. This study newly shows that physical exercise (PE) corrects nonmotor symptoms of the intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental parkinsonism. Additionally, we show that suppression of neuroimmune receptor for advanced glycation end products (RAGE) network occurs in frontal cortex on chemical (MPTP) and physical (PE) interventions. Finally, PE normalizes frontal cortical RAGE transcriptomics and upregulates the neuroprotective gene in the intranasal MPTP model of experimental parkinsonism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/japplphysiol.01120.2016DOI Listing
July 2017

Higher incidence of Zika in adult women than adult men in Rio de Janeiro suggests a significant contribution of sexual transmission from men to women.

Int J Infect Dis 2016 Oct 21;51:128-132. Epub 2016 Sep 21.

Fundação Getulio Vargas, Praia de Botafogo, 190, Rio de Janeiro, Brazil; MINES Paristech, PSL Research University, CERNA Centre for Industrial Economy, i3, CNRS UMR 9217, Paris, France.

Objectives: The recent emergence of Zika in Brazil and its association with an increased rate of congenital malformations has raised concerns over its impact on the birth rate in the country. Using data on the incidence of Zika in 2015-2016 and dengue in 2013 and 2015-2016 for the city of Rio de Janeiro (population 6.4 million), a massive increase of Zika in women compared to men was documented.

Methods: The age-adjusted incidence was compared between men and women. A negative binomial Poisson generalized linear model was fitted to the Zika incidence data to determine the significance of sexual transmission statistically.

Results: Even after correcting for the bias due to the systematic testing of pregnant women for Zika, there were found to be 90% more registered cases per 100000 women than men in the sexually active age group (15-65 years); this was not the case for age groups <15 years and >65 years. Assuming that infected men transmit the disease to women in their semen, but that the converse is not true, some extra incidence in women is to be expected. An alternate hypothesis would be that women visit doctors more often than men. To test this, the incidence of dengue fever was compared in men and women in 2015 and in 2013 (before Zika reached Rio de Janeiro): in both years, women were 30% more likely to be reported with dengue.

Conclusion: Women in the sexually active age group are far more likely to get Zika than men (+90% increase); sexual transmission is the most probable cause. Women in the 15-65 years age group are also 30% more likely to be reported with dengue than men, which is probably due to women being more careful with their health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijid.2016.08.023DOI Listing
October 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cns.12649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492743PMC
February 2017

Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.

Nat Commun 2016 06 17;7:11915. Epub 2016 Jun 17.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, CNRS UMR 5297, F-33000 Bordeaux, France.

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915032PMC
June 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2016.03.015DOI Listing
November 2016

Depression as a Glial-Based Synaptic Dysfunction.

Front Cell Neurosci 2015 22;9:521. Epub 2016 Jan 22.

CNC - Center for Neuroscience and Cell Biology, University of CoimbraCoimbra, Portugal; Faculty of Medicine, University of CoimbraCoimbra, Portugal.

Recent studies combining pharmacological, behavioral, electrophysiological and molecular approaches indicate that depression results from maladaptive neuroplastic processes occurring in defined frontolimbic circuits responsible for emotional processing such as the prefrontal cortex, hippocampus, amygdala and ventral striatum. However, the exact mechanisms controlling synaptic plasticity that are disrupted to trigger depressive conditions have not been elucidated. Since glial cells (astrocytes and microglia) tightly and dynamically interact with synapses, engaging a bi-directional communication critical for the processing of synaptic information, we now revisit the role of glial cells in the etiology of depression focusing on a dysfunction of the "quad-partite" synapse. This interest is supported by the observations that depressive-like conditions are associated with a decreased density and hypofunction of astrocytes and with an increased microglia "activation" in frontolimbic regions, which is expected to contribute for the synaptic dysfunction present in depression. Furthermore, the traditional culprits of depression (glucocorticoids, biogenic amines, brain-derived neurotrophic factor, BDNF) affect glia functioning, whereas antidepressant treatments (serotonin-selective reuptake inhibitors, SSRIs, electroshocks, deep brain stimulation) recover glia functioning. In this context of a quad-partite synapse, systems modulating glia-synapse bidirectional communication-such as the purinergic neuromodulation system operated by adenosine 5'-triphosphate (ATP) and adenosine-emerge as promising candidates to "re-normalize" synaptic function by combining direct synaptic effects with an ability to also control astrocyte and microglia function. This proposed triple action of purines to control aberrant synaptic function illustrates the rationale to consider the interference with glia dysfunction as a mechanism of action driving the design of future pharmacological tools to manage depression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2015.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722129PMC
February 2016

Decreased synaptic plasticity in the medial prefrontal cortex underlies short-term memory deficits in 6-OHDA-lesioned rats.

Behav Brain Res 2016 Mar 18;301:43-54. Epub 2015 Dec 18.

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis 88049-900, SC, Brazil; Centro de Neurociências Aplicadas (CeNAp), Hospital Universitário (HU), Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil. Electronic address:

Parkinson's disease (PD) is characterized by motor dysfunction associated with dopaminergic degeneration in the dorsolateral striatum (DLS). However, motor symptoms in PD are often preceded by short-term memory deficits, which have been argued to involve deregulation of medial prefrontal cortex (mPFC). We now used a 6-hydroxydopamine (6-OHDA) rat PD model to explore if alterations of synaptic plasticity in DLS and mPFC underlie short-term memory impairments in PD prodrome. The bilateral injection of 6-OHDA (20μg/hemisphere) in the DLS caused a marked loss of dopaminergic neurons in the substantia nigra (>80%) and decreased monoamine levels in the striatum and PFC, accompanied by motor deficits evaluated after 21 days in the open field and accelerated rotarod. A lower dose of 6-OHDA (10μg/hemisphere) only induced a partial degeneration (about 60%) of dopaminergic neurons in the substantia nigra with no gross motor impairments, thus mimicking an early premotor stage of PD. Notably, 6-OHDA (10μg)-lesioned rats displayed decreased monoamine levels in the PFC as well as short-term memory deficits evaluated in the novel object discrimination and in the modified Y-maze tasks; this was accompanied by a selective decrease in the amplitude of long-term potentiation in the mPFC, but not in DLS, without changes of synaptic transmission in either brain regions. These results indicate that the short-term memory dysfunction predating the motor alterations in the 6-OHDA model of PD is associated with selective changes of information processing in PFC circuits, typified by persistent changes of synaptic plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbr.2015.12.011DOI Listing
March 2016

The Association of Amyloid-β Protein Precursor With α- and β-Secretases in Mouse Cerebral Cortex Synapses Is Altered in Early Alzheimer's Disease.

Mol Neurobiol 2016 10 26;53(8):5710-21. Epub 2015 Oct 26.

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

Amyloid-β peptides (Aβ), the proposed triggers of synaptic dysfunction in early Alzheimer's disease (AD), derive from the endoproteolytic cleavage of amyloid-β precursor protein (APP) by β-secretases (BACE1), whereas APP cleavage by α-secretases (ADAM10) abrogates Aβ formation. We now mapped the synaptic localization of APP, ADAM10, and BACE1 in the mouse cerebral cortex. All three proteins were present in cortical synapses and subsynaptic fractionation revealed that APP was located mainly in the pre-synaptic active zone (53 %) and in the post-synaptic density (37 %), whereas ADAM10 was enriched in the post-synaptic density (61 %) and BACE1 was concentrated in extra-synaptic regions (72 %). Immunocytochemistry analysis further showed that APP and BACE1 were co-localized in about 30 % of both glutamatergic and GABAergic terminals, whereas few terminals were endowed with ADAM10. This distribution is modified in a mouse model of early AD based on Aβ1-42-intracerebroventricular injection, where the synaptic levels of APP and ADAM10 increased by 30 %, whereas BACE1 levels were reduced. This suggests that, in early AD, there are compensatory mechanisms to avoid Aβ overload in cortical synapses favoring the non-amyloidogenic processing of APP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-015-9491-9DOI Listing
October 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/nbm.3355DOI Listing
September 2015

Temporal Dissociation of Striatum and Prefrontal Cortex Uncouples Anhedonia and Defense Behaviors Relevant to Depression in 6-OHDA-Lesioned Rats.

Mol Neurobiol 2016 08 12;53(6):3891-3899. Epub 2015 Jul 12.

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

The dorsolateral striatum (DLS) processes motor and non-motor functions and undergoes extensive dopaminergic degeneration in Parkinson's disease (PD). The nigrostriatal dopaminergic degeneration also affects other brain areas including the pre-frontal cortex (PFC), which has been associated with the appearance of anhedonia and depression at pre-motor phases of PD. Using behavioral, neurochemical, and electrophysiological approaches, we investigated the temporal dissociation between the role of the DLS and PFC in the appearance of anhedonia and defense behaviors relevant to depression in rats submitted to bilateral DLS lesions with 6-hydroxydopamine (6-OHDA; 10 μg/hemisphere). 6-OHDA induced partial dopaminergic nigrostriatal damage with no gross motor impairments. Anhedonic-like behaviors were observed in the splash and sucrose consumption tests only 7 days after 6-OHDA lesion. By contrast, defense behaviors relevant to depression evaluated in the forced swimming test and social withdrawal only emerged 21 days after 6-OHDA lesion when anhedonia was no longer present. These temporally dissociated behavioral alterations were coupled to temporal- and structure-dependent alterations in dopaminergic markers such as dopamine D1 and D2 receptors and dopamine transporter, leading to altered dopamine sensitivity in DLS and PFC circuits, evaluated electrophysiologically. These results provide the first demonstration of a dissociated involvement of the DLS and PFC in anhedonic-like and defense behaviors relevant to depression in 6-OHDA-lesioned rats, which was linked with temporal fluctuations in dopaminergic receptor density, leading to altered dopaminergic system sensitivity in these two brain structures. This sheds new light to the duality between depressive and anhedonic symptoms in PD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-015-9330-zDOI Listing
August 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ejn.12851DOI Listing
April 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jneumeth.2011.11.028DOI Listing
March 2012