Exposure to Morphine and Cocaine Modify the Transcriptomic Landscape in Zebrafish Embryos.

Morphine and other opioid analgesics are the drugs of election to treat moderate-to-severe pain, and they elicit their actions by binding to the opioid receptors. Cocaine is a potent inhibitor of dopamine, serotonin, and noradrenaline reuptake, as it blocks DAT, the dopamine transporter, causing an increase in the local concentration of these neurotransmitters in the synaptic cleft. The molecular effects of these drugs have been studied in specific brain areas or nuclei, but the systemic effects in the whole organism have not been comprehensively analyzed. This study aims to analyze the transcriptomic changes elicited by morphine (10 uM) and cocaine (15 uM) in zebrafish embryos. An RNAseq assay was performed with tissues extracts from zebrafish embryos treated from 5 hpf (hours post fertilization) to 72 hpf, and the most representative deregulated genes were experimentally validated by qPCR. We have found changes in the expression of genes related to lipid metabolism, chemokine receptor ligands, visual system, hemoglobins, and metabolic detoxification pathways. Besides, morphine and cocaine modified the global DNA methylation pattern in zebrafish embryos, which would explain the changes in gene expression elicited by these two drugs of abuse.

Quantitative and qualitative evaluation of a learning model based on workstation activities.

BACKGROUND: Moving towards a horizontal and vertical integrated curriculum, Work-Station Learning Activities (WSLA) were designed and implemented as a new learning instrument. Here, we aim to evaluate whether and how this specific learning model affects academic performance. To better understand how it is received by medical students, a mixed methods research study was conducted. METHODS: In the quantitative strand, two cohorts of first year students were compared: academic year 2015-2016 n = 320 with no exposure to WSLA, and academic year 2016-2017 n = 336 with WSLA. Learning objectives at different levels of Bloom's taxonomy were identified and performance evaluated from multiple-choice questions. In the qualitative strand, a total of six students were purposely selected considering academic performance and motivation, and submitted to semistructured interviews. RESULTS: Performance at both cohorts for learning objectives at lower levels of Bloom's taxonomy was similar (38.8 vs. 39.0%; p = 0.955). In contrast, students in the WSLA group outperformed significantly those not exposed for learning objectives involving upper levels (68.5 vs. 54.2%; p <0.001). A multivariate analysis confirmed that the probability of mastering the second (more complex) objective is 1.64 times higher in students with WSLA methodology (OR 95% CI, 1.15-2.34; p = 0.007) than with traditional methodology. In the interviews, students perceived the clinical scenario of WSLA as a motivator and recognized this methodology as a more constructive framework for understanding of complicated concepts. CONCLUSIONS: In summary, our mixed methods research supports WSLA as a strategy that promotes deep learning and has a positive impact on academic performance for learning objectives involving higher order thinking skills in medical curricula.

Liderando el cambio: hacia un currículo integrado para ciencias biomédicas. Experiencia de la Universidad Europea de Madrid / Leading change: moving towards an integrated curriculum suitable for biomedical sciences. Experience from Universidad Europea de Madrid

Las ciencias biomédicas han experimentado una gran revolución en un corto período. Este avance es posible a través del estudio continuado de los mecanismos moleculares, genéticos y fisiológicos de los procesos biológicos, y ello contribuye a una mejor comprensión del funcionamiento normal de nuestro cuerpo y establece el conocimiento de las bases de la patología. Esto implica que los profesionales de ciencias de la salud deben desarrollar competencias y capacidades especiales que les permitan establecer nexos dinámicos entre las ciencias básicas y su práctica profesional. El diseño curricular más adecuado para la formación en estas competencias y capacidades se logra a través del currículo integrado. El aprendizaje integrado es un proceso centrado en el alumno, mediante el cual se adquieren conocimientos de manera flexible e individualizada a largo plazo. En la Universidad Europea de Madrid hemos afrontado esta nueva necesidad utilizando un modelo de aprendizaje integrado de materias básicas indicado para abordar la integración curricular progresiva, y que hemos denominado WSLA (Work Stations Learning Activities). Se basa en una modificación del aprendizaje basado en equipos adaptada a las directrices europeas y españolas, especialmente indicada para los grados de ciencias de la salud. Utilizando el modelo WSLA podemos crear módulos de actividades de aprendizaje integrado adaptables a distintas situaciones, desde clases magistrales hasta gamificación o prácticas de laboratorio. Proponemos nuestro modelo WSLA como una opción flexible y escalable para adoptar la integración de manera escalonada como paso previo a la integración curricular completa

Biomedical sciences have faced a strong developmental shift in a short period of time. This advance has been boosted by the study of the molecular, genetic and physiological mechanisms of the biological processes. This has a direct effect on the better understanding of the normal functioning of our body and establishes the bases of pathology knowledge. Thus, health science professionals must develop new skills and abilities that allow them to establish links between basic sciences and their professional practice. The most appropriate curricular design for competency and capacity building is achieved through the integrated curriculum. Integrated learning is a student-centered process, through which knowledge is developed lifelong in a flexible and individualized manner. At the Universidad Europea de Madrid we have faced new demands by using a model of integrated learning of basic subjects especially suitable to achieve progressive curricular integration. We have named this new model WSLA (Work Stations Learning Activities) and it is based on a modification of the team based learning adapted to the European and Spanish guidelines especially indicated for the Degrees of Health Sciences. Using the WSLA model different modules of integrated learning activities can be created and adapted to different situations, including master classes, gamification or laboratory practices. We propose our WSLA model as a scalable and flexible option to adopt the stepwise integration as a stage prior to the complete curricular integration

Tracing Gene Expression Through Detection of ß-galactosidase Activity in Whole Mouse Embryos.

The Escherichia coli LacZ gene, encoding ß-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, ß-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of ß-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for ß-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Developmental expression of membrane type 4-matrix metalloproteinase (Mt4-mmp/Mmp17) in the mouse embryo.

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development.

Whole-genome expression profile in zebrafish embryos after chronic exposure to morphine: identification of new genes associated with neuronal function and mu opioid receptor expression.

BACKGROUND: A great number of studies have investigated changes induced by morphine exposure in gene expression using several experimental models. In this study, we examined gene expression changes during chronic exposure to morphine during maturation and differentiation of zebrafish CNS. RESULTS: Microarray analysis showed 254 genes whose expression was identified as different by at least 1.3 fold change following chronic morphine exposure as compared to controls. Of these, several novel genes (grb2, copb2, otpb, magi1b, grik-l, bnip4 and sox19b) have been detected for the first time in an experimental animal model treated with morphine. We have also identified a subset of genes (dao.1, wls, bnip4 and camk1γb) differentially expressed by chronic morphine exposure whose expression is related to mu opioid receptor gene expression. Altered expression of copb2, bnip4, sox19b, otpb, dao.1, grik-l and wls is indicative of modified neuronal development, CNS patterning processes, differentiation and dopaminergic neurotransmission, serotonergic signaling pathway, and glutamatergic neurotransmission. The deregulation of camk1γb signaling genes suggests an activation of axonogenesis and dendritogenesis. CONCLUSIONS: Our study identified different functional classes of genes and individual candidates involved in the mechanisms underlying susceptibility to morphine actions related to CNS development. These results open new lines to study the treatment of pain and the molecular mechanisms involved in addiction. We also found a set of zebrafish-specific morphine-induced genes, which may be putative targets in human models for addiction and pain processes.

In vivo UVB-photoprotective activity of extracts from commercial marine macroalgae.

The photoprotective potential against UVB radiation of extracts obtained from 21 commercial macroalgae from the Phyla Ochrophyta and Rhodophyta, was evaluated in vivo, using the zebrafish embryo as a whole model organism. Our results showed that the phenolic extracts from Macrocystis pyrifera and Porphyra columbina exhibited the highest photoprotective activity, close to complete photoprotection (100%), similar to that obtained for the carrageenophytes Sarcothalia radula and Gigartina skottsbergii. Under the assayed conditions, the extracts were safe and non-toxic to the embryos at a concentration of 0.04 mg/ml PGE.

Methadone: does it really have low efficacy at micro-opioid receptors?

There is confusion in the literature concerning the relative agonist efficacy of methadone at micro-opioid receptors (MOPrs). Here, we confirm that methadone is a full agonist in guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS) binding studies. Methadone, however, seems to have low efficacy in studies of MOPr activation of G-protein-gated potassium (GIRK) channels, but this is because it directly inhibits the GIRK channels. Methadone also inhibits alpha2-adrenoceptor-activated GIRK channels. Methadone is not a specific GIRK channel blocker. It also inhibits small conductance Ca2+-activated K+ (SK2) channels. We conclude that methadone is a full agonist at MOPrs that, as we and others have shown, induces MOPr desensitization and internalization.

C-terminal splice variants of the mu-opioid receptor: existence, distribution and functional characteristics.

The distribution of the mRNA of different C-terminal splice variants of the mu-opioid receptor in rat CNS was assessed by RT-PCR. The mRNA species for MOR1, MOR1A and MOR1B were readily detectable and distributed widely throughout the rat CNS, with levels of MOR1 and MOR1A mRNA being overall greater than for MOR1B. We did not find convincing evidence that significant levels of MOR1C, MOR1C1, MOR1C2 and MOR1D are present in rat CNS. To examine possible differences in the agonist-induced regulation of MOR1, MOR1A and MOR1B, we expressed these constructs in HEK293 cells along with G-protein-coupled inwardly rectifying K+ channel subunits and measured the rate and extent of desensitisation of (d-Ala2,N-Me-Phe4,glycinol5)-enkephalin (DAMGO)- and morphine-induced G-protein-coupled inwardly rectifying K+ currents. Morphine-induced desensitisation was rapid for all three splice variants (t1/2: 1.2-1.7 min) but DAMGO-induced desensitisation was significantly slower for MOR1B (t1/2 4.2 min). Inhibition of endocytosis by expression of a dynamin-dominant negative mutant increased the rate of DAMGO-induced desensitisation of MOR1B. These data show that some splice variants of mu-opioid receptor are widely expressed in rat CNS but question the existence of others that have been reported in the literature. In addition, whereas the rate of desensitisation of MOR1 and MOR1A is agonist-independent, that for MOR1B is agonist-dependent.

Characterization of cannabinoid-binding sites in zebrafish brain.

We present here the pharmacological characterization of cannabinoid-binding sites in zebrafish brain homogenates using radiolabeled binding techniques. The nonselective agonist [3H]-CP55940 binds with high affinity (KD = 0.50+/-0.06 nM and a Bmax = 1047+/-36.01 fmol/mg protein), displaying one binding site. The slightly CB2 selective agonist [3H]-WIN55212-2 also binds with high affinity to zebrafish brain membranes displaying two different binding sites with affinities KD1 = 0.35+/-0.09 nM and KD2 = 105.81+/-66.36 nM. Competition binding assays using [3H]-WIN55212-2 and several unlabeled ligands were performed. WIN55212-2 significantly displaced the tritiated ligand binding showing the two binding sites observed with its tritiated homologous, while the slightly selective CB1 cannabinoid ligand HU-210, the nonselective cannabinoid ligand CP55940 and the endogenous cannabinoid ligand anandamide presented one binding site. Also, the functionality of these cannabinoid sites was analyzed using the known [35S]GTPgammaS assay. All the agonist used presented an agonist profile and the rank order for potency was HU-210 > WIN55212-2 > CP55940 >anandamide. Our results provide evidence that, although some of the typical cannabinoid ligands for mammalian receptors do not fully recognize the cannabinoid-binding sites in zebrafish brain, the activity of the endogenous zebrafish cannabinoid system might not significantly differ from that displayed by the cannabinoid system described in other species. Hence the study of zebrafish cannabinoid activity may contribute to an understanding of the endogenous cannabinoid system in higher vertebrates.

New kappa opioid receptor from zebrafish Danio rerio.

A cDNA that encodes a kappa opioid receptor like from zebrafish (ZFOR3) has been cloned and characterized. The encoded protein is 377 residues long and presents 70% identity with the mammalian kappa receptors, although less homology is found in the amino- and carboxyl-terminus as well as in the extracellular loops. In situ hybridization studies have revealed that ZFOR3 mRNA is highly expressed in particular brain areas that coincide with the expression of the kappa opioid receptor in other species. When ZFOR3 is stably expressed in HEK293 cells, [(3)H]-diprenorphine binds with high affinity (K(D)=1.05+/-0.26 nM), being this value on the same range as those reported for mammalian kappa opioid receptors. On the other hand, the selective agonist for mammalian kappa receptors U69,593 does not bind to ZFOR3. [(3)H]-diprenorphine binding is readily displaced by the peptidic ligand dynorphin A and by the non-endogenous compounds bremazocine, naloxone and morphine, although with different affinities. Our results demonstrate that ZFOR3 is a unique model to study the kappa opioid receptor functionality.