Role of hippocampal NF-κB and GluN2B in the memory acquisition impairment of experiences gathered prior to cocaine administration in rats

Cocaine can induce severe neurobehavioral changes, among others, the ones involved in learning and memory processes. It is known that during drug consumption, cocaine-associated memory and learning processes take place. However, much less is known about the effects of this drug upon the mechanisms involved in forgetting.The present report focuses on the mechanisms by which cocaine affects memory consolidation of experiences acquired prior to drug administration. We also study the involvement of hippocampus in these processes, with special interest on the role of Nuclear factor kappa B (NF-κB), N-methyl-D-aspartate glutamate receptor 2B (GluN2B), and their relationship with other proteins, such as cyclic AMP response element binding protein (CREB). For this purpose, we developed a rat experimental model of chronic cocaine administration in which spatial memory and the expression or activity of several proteins in the hippocampus were assessed after 36 days of drug administration. We report an impairment in memory acquisition of experiences gathered prior to cocaine administration, associated to an increase in GluN2B expression in the hippocampus. We also demonstrate a decrease in NF-κB activity, as well as in the expression of the active form of CREB, confirming the role of these transcription factors in the cocaine-induced memory impairment

Cocaine promotes oxidative stress and microglial-macrophage activation in rat cerebellum

Different mechanisms have been suggested for cocaine neurotoxicity, including oxidative stress alterations. Nuclear factor kappa B (NF-κB), considered a sensor of oxidative stress and inflammation, is involved in drug toxicity and addiction. NF-κB is a key mediator for immune responses that induces microglial/macrophage activation under inflammatory processes and neuronal injury/degeneration. Although cerebellum is commonly associated to motor control, muscular tone, and balance. Its relation with addiction is getting relevance, being associated to compulsive and perseverative behaviors. Some reports indicate that cerebellar microglial activation induced by cannabis or ethanol, promote cerebellar alterations and these alterations could be associated to addictive-related behaviors. After considering the effects of some drugs on cerebellum, the aim of the present work analyzes pro-inflammatory changes after cocaine exposure. Rats received daily 15 mg/kg cocaine i.p., for 18 days. Reduced and oxidized forms of glutathione (GSH) and oxidized glutathione (GSSG), glutathione peroxidase (GPx) activity and glutamate were determined in cerebellar homogenates. NF-κB activity, CD68, and GFAP expression were determined. Cerebellar GPx activity and GSH/GSSG ratio are significantly decreased after cocaine exposure. A significant increase of glutamate concentration is also observed. Interestingly, increased NF-κB activity is also accompanied by an increased expression of the lysosomal mononuclear phagocytic marker ED1 without GFAP alterations. Current trends in addiction biology are focusing on the role of cerebellum on addictive behaviors. Cocaine-induced cerebellar changes described herein fit with previosus data showing cerebellar alterations on addict subjects and support the proposed role of cerebelum in addiction

Thyroid hormones regulate zebrafish melanogenesis in a gender-specific manner

12 páginas, 5 figuras.-- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedZebrafish embryos are treated with anti-thyroidal compounds, such as phenylthiourea, to inhibit melanogenesis. However, the mechanism whereby the thyroidal system controls melanin synthesis has not been assessed in detail. In this work, we tested the effect of the administration of diets supplemented with T3 (500μg/g food) on the pigment pattern of adult zebrafish. Oral T3 induced a pronounced skin paling in both adult female and male zebrafish that was reversible upon cessation of treatment. The number of visible melanophores was significantly reduced in treated fish. Accordingly, treatment down-regulated expression of tyrosinase-related protein 1 in both sexes. We also found sexually dimorphic regulation of some melanogenic genes, such as Dct/Tyrp2 that was dramatically up-regulated in females after T3 treatment. Thus, we demonstrated that melanogenesis is reversibly inhibited by thyroid hormones in adult zebrafish and make the discovery of gender-specific differences in the response of melanogenic gene expression. Thus, fish gender is now shown to be an important variable that should be controlled in future studies of fish melanogenesisMinisterio de Economia y Competitividad grant numbers AGL2013-46448-C3-3-R and AGL2014-52473R, Biotechnology and Biological Sciences Research Council (BBSRC) grant number BB/L00769X/1, and Medical Research Council (MRC) grant number MR/J001457/1Peer reviewe

Distribución Neuroanatómica de los Receptores de Estrógenos en la Lubina (Dicentrarchus labrax): Implicaciones en la Función Reproductora y el Comportamiento Alimenticio. Control Transcripcional de la Hormona Folículo Estimulante (FSH)

En los vertebrados, los estrógenos, además de ejercer un papel principal en la diferenciación y maduración sexual, son también responsables de otras muchas funciones metabólicas y fisiológicas. Su rango de acción es muy amplio, afectando a la expresión génica en diversos tejidos, incluido el sistema nervioso central (SNC). Las acciones fisiológicas de los estrógenos están mediadas por receptores específicos (ERs), los cuales, actúan tanto a nivel nuclear (acción genómica), como fuera del núcleo (acción no genómica). En los vertebrados, se han identificado dos subtipos, el subtipo alfa (Esr1) y el subtipo beta (Esr2). De manera adicional, los peces teleósteos presentan un tercer subtipo de ER denominado beta dos (Esr2a) en contraposición al primer subtipo descubierto beta uno (Esr2b). Estudios diversos, tanto en mamíferos como en peces, sugieren que los ERs, pueden ejercer funciones diferenciales, pero también redundantes. En el trabajo presente, se establece el patrón de distribución de los tres subtipos de ERs caracterizados en peces teleósteos, en el cerebro de la lubina, como una primera aproximación a la funcionalidad que estos pueden ejercer en el SNC. Asimismo, se demuestra su implicación en la función reproductora y el comportamiento alimenticio de la especie anterior, a partir de la presencia de los mismos en las principales áreas de control gonadotrópico y de regulación de la ingesta. También se demuestra la presencia de estos receptores en las células productoras de las hormonas folículo estimulante (FSH) y luteinizante (LH), sugiriendo una acción directa de los estrógenos sobre la producción de gonadotropinas. Finalmente, el desarrollo de estudios in vitro aporta información sobre el control transcripcional por estrógenos de la FSH en la lubina. CONCLUSIONES PRIMERA. Los experimentos de hibridación in situ demuestran que los tres subtipos de ERs caracterizados en peces teleósteos, se expresan en el cerebro de la lubina. SEGUNDA. La amplia variedad de núcleos y áreas cerebrales en la que los ERs aparecen expresados, así como, su localización conjunta en algunas de las anteriores sugiere que éstos desarrollan tanto funciones diferenciales como redundantes. TERCERA. La presencia de los tres subtipos de ERs en las áreas principales de control hipofisiotrópico sugiere que los tres están implicados en la regulación, a través de los sistemas de retroalimentación largos, de la síntesis y secreción de las gonadotropinas. Asimismo, la expresión diferencial de los tres subtipos en núcleos neuronales de las áreas anteriores sugiere una funcionalidad no redundante de los mismos en dicha regulación. CUARTA. Los estudios de localización conjunta de los ERs con los gonadotropos de FSH y LH, suministran una base neuroanatómica para una regulación, a través de los sistemas de retroalimentación cortos, de la síntesis y secreción de las gonadotropinas. QUINTA. La inhibición producida por 17ß-estradiol sobre la expresión del gen ßFSH en cultivos primarios de células de pituitaria de lubina demuestra que la acción de este esteroide sobre la expresión de la subunidad ßFSH, in vivo, podría ejercerse de manera directa sobre la pituiaria misma. SEXTA. Los experimentos de transfección con un vector de expresión portador del Esr1 de lubina demuestran que éste se expresa y es funcional en las líneas celulares de riñón humano embrionario (HEK 293) y de gonadotropos de ratón (LßT2).SEPTIMA. La caracterización farmacológica del Esr1 de la lubina señala que éste es activado por 17ß-estradiol, tanto a dosis fisiológicas como farmacológicas, demostrando que puede mediar en el control por estrógenos de la expresión génica de la especie anterior, in vivo. Dosis elevadas de testosterona son capaces de inducir efectos celulares similares a los causados por los estrógenos sin mediación de la aromatización. Conclusiones 174 OCTAVA. La detección significativa de actividad luciferasa observada a partir de experimentos de transfección con construcciones plasmídicas de la región promotora del gen ßFSH de la lubina en LßT2, así como la ausencia de la misma en HEK 293, demuestra que este gen requiere factores celulares específicos para su expresión. NOVENA. La caracterización de la actividad basal de la región promotora del gen ßFSH pone de manifiesto que las primeras 3.5 Kb de promotor contienen, al menos, una región silenciadora y una potenciadora de la actividad transcripcional. DÉCIMA. Los experimentos realizados para determinar el control transcripcional por estrógenos de la subunidad ßFSH de la lubina sugieren que los ERs no reconocen elementos de respuesta dentro de las 3.5 Kb de región promotora analizada, o bien, que necesitan de la interacción con factores celulares específicos de gonadotropos de peces. DÉCIMOPRIMERA. La presencia de los ERs en las áreas principales de control del comportamiento alimenticio en peces, como son el núcleo lateral tuberal y lóbulo inferior hipotalámico, dan soporte al efecto anorexigénico que los estrógenos ejercen en la lubina. DÉCIMOSEGUNDA. La obtención de una línea celular expresando de manera estable el Esr1 de lubina y el gen luciferasa, bajo el control de un promotor portador de elementos de respuesta a los estrógenos, ha permitido desarrollar un sistema sensible para la detección y cuantificación de sustancias estrogénicas. El sistema revela la presencia de componentes estrogénicos en los piensos comerciales utilizados para el crecimiento de lubinas y doradas. La presencia de componentes estrogénicos en dietas para peces de cultivo podría causar disrupciones en su función reproductora y comportamiento alimenticio a través de la activación de los ERs.Esta tesis doctoral ha sido financiada por el Proyecto AGL 2005-00796 del Ministerio de Educación y Ciencia, por el Proyecto PUBERTIMING QLTR-2001-01801 de la UE y por el Proyecto PROBASS QLTR-2001-01801 por la UE.Peer Reviewe

Sex steroids and food intake in fish

34 p., 4 figures, 1 table and bibliographyThis study was conducted to test the sensitivity to gonadal steroids of the systems regulating food intake in sea bass. Animals were treated with silastic implants containing 17-β-estradiol or testosterone. Self-feeding was recorded for 31 days using computerized demand feeders and unfed-pellet recovery systems. Both steroids strongly decreased selffeeding levels, feed efficiency and specific growth rates. The linear growth of fish treated with testosterone was higher than in 17-β-estradiol treated fish. In the second experiment, fish were treated with lower 17-β-estradiol doses and 11-keto-androstenedione, a precursor of the main fish androgen (11-keto-testosterone). The results demonstrated a dose-response effect of estrogen and no effect of non-aromatizable androgens on food intake or growth performance. The inhibitory effect of testosterone on food intake seems to be mediated by its aromatization to estradiol, while linear growth promotion is mediated by the androgen “per se”. Data suggest that gonadal steroids may be involved in the seasonal feeding pattern of sea bass. The results demonstrate the sensitivity of the mechanisms regulating food intake to estrogenic compounds and point to the risk of including feed containing estrogenic substances in fish diets as well as the risk involved in exposure to “estrogenic environments”.This work was supported by grants AGL2004-08137-C04-04, AGL2007-65744-C03-02 from Spanish Science and Education Ministry (MEC) to JMC-R

Characterization of sea bass FSHβ 5′ flanking region: Transcriptional control by 17β-estradiol

The sea bass follicle-stimulating hormone 5′ flanking region (sbFSHβ 5′ FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHβ gene. Analysis of the ~3.5 kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5 kb of the sbFSHβ 5′ FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LβT2) cell lines. The sbFSHβ 5′ FR was efficiently expressed under basal conditions in LβT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHβ transcriptional activity, in vitro treatments with 17β-estradiol were carried out on primary cultures of pituitary cells and LβT2 cells transiently expressing luciferase under the control of sbFSHβ 5′ FR. Overall, these results demonstrate that 17β-estradiol inhibits sbFSHβ gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHβ 5′FR (~3.5 kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article. © 2013 Springer Science+Business Media Dordrecht.This work was supported by EU (QLRT-2001-01801) and MEC (AGL-2005-00796) grants. During this work, Borja Muriach was the recipient of an I3P Predoctoral Fellowship from Consejo Superior de Investigaciones Científicas (CSIC). During part of this research, JMC-R was financed by AGL2010-22247-C03-01 grant and JMC-R and SZ by CSD 2007-00002 grant.Peer Reviewe

Distribution of estrogen receptor 2 mRNAs (Esr2a and Esr2b) in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Three different estrogen receptors (ERs) have been characterized in teleost fish, i.e. Esr1, Esr2a and Esr2b. In this study we carried out in situ hybridizations in the brain and pituitary of the sea bass (Dicentrarchus labrax) to study the putative involvement of Esr2 subtypes in the control of gonadotropin secretion in fish. Our studies demonstrated that both receptors are expressed within the main hypophysiotrophic areas of the sea bass brain thus providing neuroanatomical basis for the involvement of Esr2 subtypes in the long (or indirect) regulatory feedbacks on pituitary function in the sea bass. The results revealed that Esr2b-mRNA distribution was restricted to the preoptic area and tuberal hypothalamus. On the contrary, Esr2a presented a widespread distribution and transcripts were detected within the ventral telencephalon, preoptic area, hypothalamus, thalamus, posterior tubercle, mesencephalon and rhombencephalon. New Esr2-expressing areas were described in all of the above areas. This paper is the first demonstration of Esr2a and Esr2b expression in the follicle-stimulating hormone β-subunit (βFSH)- and luteinizing hormone β-subunit (βLH)-expressing cells in the fish pituitary, thus suggesting the participation of both receptors in the direct effect of estrogen on the control of gonadotropin hormone synthesis. © 2008 Elsevier B.V. All rights reserved.This work was supported by a research grant from the European Community under the Quality of Life and Management of Living Resources Programme (contracts Q5RS-2000-31365 and Q5RS-2002-01801 to SZ and MC) and MEC grants (AGL 2002-10024E and AGL 2002-12470-E to SZ and MC). During this work Borja Muriach was a recipient of a I3P Predoctoral Fellowship from Consejo Superior de Investigaciones Científicas (CSIC).Peer Reviewe

A cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax): The midbrain and hindbrain

This study is the third part of a comprehensive series of publications on the cytoarchitectonic organization of the brain of the European sea bass, Dicentrarchus labrax. This study provides an atlas of the brain stem based on Nissl-stained transverse sections as well as a description of cell masses and a discussion on comparative aspects of brain stem nuclei, including methodological studies in other species. By external examination, the sea bass exhibits a prominent Optic tectum and Corpus cerebelli as expected in a predator species with a highly developed visual system. However, no hypertrophy of the facial and vagal lobes was observed as reported in other non-perciform teleosts. The general organization pattern of the mesencephalon and rhombencephalon of the sea bass brain resembles that reported for other perciform teleosts. However, the Valvula cerebelli has been subdivided into anterior, central and posterior parts. In addition, the ventricular surface of the granular layer of the Valvula cerebelli appears to be in contact with those of the Torus longitudinalis. This cell apposition could be interpreted as a direct connection, but more studies demonstrating the absence of ependyma between both structures are needed. Furthermore, we have tentatively described the electro/mechano receptive pre-eminential nucleus in the rhombencephalon of the sea bass. This study completes one of the few descriptions, as well as the most complete and detailed available, of the brain of any marine perciform species. © 2008 Elsevier GmbH. All rights reserved.This study has been supported by CYTMAR MAR95-1888-C03-02 AGL2001-0593-C03-02 to JA M-C and AGL2004-08137-C04-04 and GV05/045 to JM C-RPeer Reviewe

Molecular characterization and central distribution of the estradiol receptor alpha (ERα) in the sea bass (Dicentrarchus labrax)

Three different estrogen receptors (ERs) have been cloned and characterized in teleosts fish, i.e. ERα, ERβ or ERβ1 and ERγ or ERβ2. In order to study the sea bass ER subtype involved in the regulation of gonadotropin production, as well as to elucidate the possible involved neuronal pathways, we characterized the transactivation properties of the cloned sea bass ERα (sbERα) and studied its distribution in the brain and gonadotropic cells of the sea bass by in situ hybridization. The results revealed that sbERα transactivates promoters containing estradiol responsive elements (ERE) in a dose-response manner. The sbERα showed the highest affinity for 17-β-estradiol. In situ hybridization studies demonstrated that ERα mRNA positive neurons are widely distributed within the sea bass brain, including the telencephalon, preoptic area, thalamus, hypothalamus, mesencephalic tectum and tegmentum and rhombencephalon. New estrogen dependent nuclei were described in all above areas. The sbERα was profusely expressed in the main neuroendocrine areas such as the preoptic area and hypothalamus, thus suggesting the steroidal modulation of the hypophysiotropic neurons. The presence of sbERα expression in the FSHβ and LHβ cells suggests a direct effect of estrogens in the control of gonadotropin hormone synthesis.This work was supported by research grants from the European Community under the Quality of Life and Management of Living Resources Programme (contracts Q5RS-2000-31365 and Q5RS-2002-01801 to SZ and MC) and MEC grants (AGL 2002-10024E and AGL 2002-12470-E to SZ and MC)Peer Reviewe

Reduction of sexual maturation in male Dicentrarchus labrax by continuous light both before and during gametogenesis

The objective of this study was to investigate the effect of exposure to continuous light (LL) during the pregametogenesis (4-month duration) and gametogenesis (6-month duration) periods on early male sexual maturation in the sea bass. A simulated natural photoperiod group (SNP) and a long-term exposure group to LL during 12 months were kept as controls. Somatic growth, gonadal development, plasma testosterone (T) and 11-ketotestosterone (11-KT) and the expression of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary were analysed as indicators of maturation. Growth in all treatments was independent of photoperiod. Nevertheless, all LL treatments were effective in reducing the number of early maturing males (< 3% with < 0.2% GSI versus 22% with 0.8% GSI in SNP). This inhibition was not complete but the effect was comparable among all LL treatments thus suggesting that a potential photo-labile period might exist in the sea bass. In the SNP group, the T and 11-KT plasma levels as well as the gene expression values of the three gonadotropin subunits (i.e., LHβ, FSHβ and glycoprotein (GP) α) peaked in February concomitantly with the increase of the GSI (≥ 0.51%) and the accumulation of LH protein in the pituitary (≥ 14.6 μg) and declined at post-spermiation stage (GSI ≤ 0.12% and LH protein ≤ 10.2 μg/pit). Exposure to different LL treatments induced a differential response particularly on the 11-KT levels and the gene expression of the FSHβ subunit. We suggest that the unbalanced production of this androgen regulated by FSHβ might be limiting the stimulation of germ cell proliferation at the testicular level and thus arresting early puberty male sea bass. © 2008 Elsevier B.V. All rights reserved.This research was supported by a UE project No. Q5RS-2002-01801 (PUBERTIMING), Generalitat Valenciana Grupos 04/80 and a MCYT Special Action No. AGL2002-12470-E to M.C. A.F. was supported by a research contract from PUBERTIMINGPeer Reviewe