Characterization of sea bass FSHß 5' flanking region: transcriptional control by 17ß-estradiol.

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHß 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHß 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 sbFSHß 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LßT2) cell lines. The sbFSHß 5' FR was efficiently expressed under basal conditions in LßT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHß transcriptional activity, in vitro treatments with 17ß-estradiol were carried out on primary cultures of pituitary cells and LßT2 cells transiently expressing luciferase under the control of sbFSHß 5' FR. Overall, these results demonstrate that 17ß-estradiol inhibits sbFSHß 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 sbFSHß 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.

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.

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

Three different estrogen receptors (ERs) have been cloned and characterized in teleosts fish, i.e. ERalpha, ERbeta or ERbeta1 and ERgamma or ERbeta2. 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 ERalpha (sbERalpha) and studied its distribution in the brain and gonadotropic cells of the sea bass by in situ hybridization. The results revealed that sbERalpha transactivates promoters containing estradiol responsive elements (ERE) in a dose-response manner. The sbERalpha showed the highest affinity for 17-beta-estradiol. In situ hybridization studies demonstrated that ERalpha 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 sbERalpha 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 sbERalpha expression in the FSHbeta and LHbeta cells suggests a direct effect of estrogens in the control of gonadotropin hormone synthesis.

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 beta-subunit (betaFSH)- and luteinizing hormone beta-subunit (betaLH)-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.

Thyroid Hormones Regulate Zebrafish Melanogenesis in a Gender-Specific Manner.

Zebrafish 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 melanogenesis.

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.

Sex steroid-induced inhibition of food intake in sea bass (Dicentrarchus labrax).

This 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-beta-estradiol or testosterone. Self-feeding was recorded for 31 days using computerized demand feeders and unfed-pellet recovery systems. Both steroids strongly decreased self-feeding levels, feed efficiency and specific growth rates. The linear growth of fish treated with testosterone was higher than in 17-beta-estradiol treated fish. In the second experiment, fish were treated with lower 17-beta-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".

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.