Antonio Vargas

Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2′-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion but not with the prevention of cocaine-induced sensitization.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca2+ and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca2+-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB+1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin+ cells (granular and pyramidal neurons), and calretinin+ and parvalbumin+ interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin+ principal cells in the dentate gyrus and CA1, and in the calretinin+ and parvalbumin+ interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL+ terminals were only observed around CA1 calbindin+ pyramidal cells, CA1/3 calretinin+ interneurons and CA3 parvalbumin+ interneurons localized in the pyramidal cell layers. Interestingly, calbindin+ pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that lead to addiction.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle.

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB 1 -/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca 2+ -binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca2+ fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca2+-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα+/calbindin+ cells were closely surrounded by NAPE-PLD+ fiber varicosities. No pyramidal PPARα+/calbindin+ cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD+/calretinin+ cells were specifically detected in CA3. NAPE-PLD+ puncta surrounded the calretinin+ cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Cocaine-induced behavioral sensitization decreases the expression of endocannabinoid signaling-related proteins in the mouse hippocampus.

In the reward mesocorticolimbic circuits, the glutamatergic and endocannabinoid systems are implicated in neurobiological mechanisms underlying cocaine addiction. However, the involvement of both systems in the hippocampus, a critical region to process relational information relevant for encoding drug-associated memories, in cocaine-related behaviors remains unknown. In the present work, we studied whether the hippocampal gene/protein expression of relevant glutamate signaling components, including glutamate-synthesizing enzymes and metabotropic and ionotropic receptors, and the hippocampal gene/protein expression of cannabinoid type 1 (CB1) receptor and endocannabinoid metabolic enzymes were altered following acute and/or repeated cocaine administration resulting in conditioned locomotion and locomotor sensitization. Results showed that acute cocaine administration induced an overall down-regulation of glutamate-related gene expression and, specifically, a low phosphorylation level of GluA1. In contrast, locomotor sensitization to cocaine produced an up-regulation of several glutamate receptor-related genes and, specifically, an increased protein expression of the GluN1 receptor subunit. Regarding the endocannabinoid system, acute and repeated cocaine administration were associated with an increased gene/protein expression of CB1 receptors and a decreased gene/protein expression of the endocannabinoid-synthesis enzymes N-acyl phosphatidylethanolamine D (NAPE-PLD) and diacylglycerol lipase alpha (DAGLα). These changes resulted in an overall decrease in endocannabinoid synthesis/degradation ratios, especially NAPE-PLD/fatty acid amide hydrolase and DAGLα/monoacylglycerol lipase, suggesting a reduced endocannabinoid production associated with a compensatory up-regulation of CB1 receptor. Overall, these findings suggest that repeated cocaine administration resulting in locomotor sensitization induces a down-regulation of the endocannabinoid signaling that could contribute to the specifically increased GluN1 expression observed in the hippocampus of cocaine-sensitized mice.

Cooperative role of the glucagon-like peptide-1 receptor and β3-adrenergic-mediated signalling on fat mass reduction through the downregulation of PKA/AKT/AMPK signalling in the adipose tissue and muscle of rats

To explore the cooperation of GLP‐1 receptor and β3‐adrenergic receptor (β3‐AR)‐mediated signalling in the control of fat mass/feeding behaviour by studying the effects of a combined therapy composed of the GLP‐1R agonist liraglutide and the β3‐AR agonist CL316243.

Evaluation of four commercial virological assays for early infant HIV-1 diagnosis using dried blood specimens

Background:Early infant diagnosis (EID) of HIV-1 is necessary to reduce HIV-related mortality. As maternal antibodies transferred across the placenta may persist up to 18 mo, commercial virological assays (CVAs) are needed. This study compares four CVAs for EID using dried blood specimens (DBS) from HIV-1-exposed infants.Methods:DBS from 68 infants born to HIV-1-infected women were collected from November 2012 to December 2013 in Equatorial Guinea. Four CVAs were performed: Siemens VERSANT HIV-1 RNA 1.0 kPCR assay, Roche CAP/CTM Quantitative Test v2.0, CAP/CTM Qualitative Tests v1.0 and v2.0. Definitive diagnosis was established following World Health Organization (WHO) recommendations.Results:Two HIV-1-infected infants (2.9%) were detected by the four CVAs while 49 (72%) resulted negative. Discordant results were observed in 17 (25%) infants and HIV-1 infection was excluded in 14 patients when virological and serological testing was performed in additional DBS. Different false-positive rates HIV-1 were observed with Roche assays.Conclusion:CVAs using DBS were useful for EID, although discrepant results were common. Further research is required to reduce false-positive results that could result in wrong diagnosis and unneeded treatment. We propose caution with low viral load (VL) values when using VL assays. Clear guidelines are required for EID of HIV-exposed infants with discrepant virological results.

Acetaminophen-induced liver injury alters the Acyl ethanolamine-based anti-inflammatory signaling system in liver

Protective mechanisms against drug-induced liver injury are actively being searched to identify new therapeutic targets. Among them, the anti-inflammatory N-acyl ethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system has gained much interest after the identification of its protective role in steatohepatitis and liver fibrosis. An overdose of paracetamol (APAP), a commonly used analgesic/antipyretic drug, causes hepatotoxicity, and it is being used as a liver model. In the present study, we have analyzed the impact of APAP on the liver NAE-PPARα system. A dose-response (0.5–5–10–20 mM) and time-course (2–6–24 h) study in human HepG2 cells showed a biphasic response, with a decreased PPARα expression after 6-h APAP incubation followed by a generalized increase of NAE-PPARα system-related components (PPARα, NAPE-PLD, and FAAH), including the NAEs oleoyl ethanolamide (OEA) and docosahexaenoyl ethanolamide, after a 24-h exposure to APAP. These results were partially confirmed in a time-course study of mice exposed to an acute dose of APAP (750 mg/kg). The gene expression levels of Pparα and Faah were decreased after 6 h of treatment and, after 24 h, the gene expression levels of Nape-pld and Faah, as well as the liver levels of OEA and palmitoyl ethanolamide, were increased. Repeated APAP administration (750 mg/kg/day) up to 4 days also decreased the expression levels of PPARα and FAAH, and increased the liver levels of NAEs. A resting period of 15 days completely restored these impairments. Liver immunohistochemistry in a well-characterized human case of APAP hepatotoxicity confirmed PPARα and FAAH decrements. Histopathological and hepatic damage (Cyp2e1, Caspase3, αSma, Tnfα, and Mcp1)-related alterations observed after repeated APAP administration were aggravated in the liver of Pparα-deficient mice. Our results demonstrate that the anti-inflammatory NAE-PPARα signaling system is implicated in liver toxicity after exposure to APAP overdose, and may contribute to its recovery through a long-term time-dependent response.

The Pertinence of Applying Qualitative Investigation Strategies in the Design and Evaluation of HIV Prevention Policies

In the last decade, the incidence of HIV has globally diminished by 19% (Joint United Nations Programme on HIV/Aids, [Unaids], 2010). Likewise, new cases of children infected by HIV have diminished due to the spread of vertical mother-child prevention among pregnant women. The percentage of people who received anti-retroviral treatment increased by 30% due to improvements in the accessibility of therapeutic treatment. Also, the annual percentage of death caused by Aids has decreased (Unaids, 2010). Nevertheless, in spite of this progress, HIV/Aids pandemic continues to be one of the main threats to global health. On a world scale, pandemic epidemiological data shows alarming numbers: there are more than 33 million people around the world living with HIV. Approximately, 7,000 new cases of infection arise every day. Every year, an average of 1,800,000 people die as a consequence of Aids. It is estimated that 12 million children have been orphaned (Unaids, 2010). However, such numbers are distributed in unequal proportions throughout the world. Thus, since not every country presents the same level of prevalence, big differences can be found. Currently, most of the countries suffering from HIV pandemic are developing countries. Also, these countries show low or medium levels of human development. The Table 1 shows the relation between Human Development Index in some countries with a higher pandemic level. As seen on table 1, the distribution of the pandemic tendency throughout these countries reveals a clear correlation: The less socio-economically developed a country is, the higher the HIV prevalence is. This being so, it is hardly surprising that sub-Saharan African or Asian countries present widespread epidemic HIV levels. On a world scale, these countries hold the highest Multidimensional Poverty Index (United Nations Development Programme, [Unpd], 2010). For instance, the worst Human Development Index in the world belongs to