O zebrafish (Danio rerio) é uma espécie utilizada como modelo experimental em diversas áreas, tais como neurociências toxicologia. Seu genoma já está praticamente sequenciado e estudos demonstraram que muitos genes deste peixe são similares aos de mamíferos. Além disso, o zebrafish é um excelente modelo para estudar a função de diferentes sistemas de neurotransmissão. O consumo do etanol exerce diversas mudanças na coordenação motora, percepção sensorial e cognição promovendo um amplo espectro de alterações bioquímicas e fisiológicas nas células nervosas. Aqui, nós investigamos o efeito promovido pela exposição crônica de etanol nos sistemas purinérgico e glutamatérgico, e níveis de BDNF no SNC de zebrafish. Os transportadores de alta afinidade de aminoácidos (EAAT) regulam os níveis extracelulares de glutamato. Nós identificamos e descrevemos o padrão de expressão dos genes relacionados aos transportadores e as propriedades de captação de glutamato nas três importantes estruturas cerebrais de zebrafish (telencéfalo, tecto óptico e cerebelo). As pesquisas nos bancos de dados do seu genoma através de análise filogenética confirmaram a presença de diversos EAATs (EAAT2, EAAT3, três EAAT1 parálogos e duas sequências parálogas para EAAT5). Também...

Ornamental fish may be severely affected by a stressful environment. Stressors impair the immune response, reproduction and growth rate; thus, the identification of possible stressors will aid to improve the overall quality of ornamental fish. The aim of this study was to determine whole-body cortisol of adult zebrafish, Danio rerio, following visual or direct contact with a predator species. Zebrafish were distributed in three groups: the first group, which consisted of zebrafish reared completely isolated of the predator, was considered the negative control; the second group, in which the predator, Parachromis managuensis was stocked together with zebrafish, was considered the positive control; the third group consisted of zebrafish stocked in a glass aquarium, with direct visual contact with the predator. The mean whole-body cortisol concentration in zebrafish from the negative control was 6.78 +/- 1.12 ng g(-1), a concentration statistically lower than that found in zebrafish having visual contact with the predator (9.26 +/- 0.88 ng g(-1)) which, in turn, was statistically lower than the mean whole-body cortisol of the positive control group (12.35 +/- 1.59 ng g(-1)). The higher whole-body cortisol concentration found in fish from the positive control can be attributed to the detection...

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish...

O uso de modelos animais tem trazido grandes benefícios para o conhecimento dos processos biológicos normais, bem como para uma maior compreensão das doenças humanas, inclusive as epilepsias. Embora os modelos de roedores sejam os mais usados para explorar os mecanismos que permeiam a epileptogênese, recentemente, um novo modelo animal foi proposto para o estudo das epilepsias, o Daniorerio. Popularmente conhecido como zebrafish, este pequeno peixe possui muitas vantagens para a experimentação científica, principalmente no que se refere à manipulação genética.O Fator Neurotrófico Derivado do Cérebro (BDNF, do inglês, BrainDerivedNeurotrophicFactor) é a neurotrofina de maior abundância no sistema nervoso central e que está relacionada à plasticidade neuronal. Sabe-se que os níveis de transcritos e protéicos desta neurotrofina estão alterados nas epilepsias, tanto em humanos quanto em modelos animais; porém, o seu papel nesta condição ainda é controverso. Os principais objetivos deste estudo foram (i) investigar o perfil temporal de transcritos dos genes bdnfe seus receptores ntrk2a e ntrk2b após crise epiléptica aguda e (ii) após crises epilépticas repetitivas por meio da técnica da PCR quantitativa (qPCR) usando-se o sistema TaqManTM (AppliedBiosystems...

O Danio rerio é um peixe teleósteo popularmente conhecido como zebrafish que têm se destacado como modelo animal favorável para investigações genéticas devido à facilidade de manipulação in vivo, por sua transparência nas fases embrionária e larval e por seu desenvolvimento externo. Recentemente, foi demonstrado que o zebrafish é capaz de exibir padrão comportamental e alteração da atividade eletrográfica durante crise epiléptica como visto em roedores, tornando-o um modelo promissor para as investigações moleculares das epilepsias. Os estudos sobre os diferentes eventos já conhecidos das epilepsias no zebrafish estão apenas começando e, portanto, há ainda muito que ser investigado para uma melhor caracterização deste modelo para estudos em epilepsia. Estudos clínicos e em modelos animais mostraram que a crise epiléptica eleva os níveis da interleucina-1 beta e induz morte neuronal. Nesse sentido, o presente trabalho se propôs a investigar (i) o perfil temporal de expressão do transcrito do gene da interleucina-1 beta (il1b) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol por transcriptase reversa-PCR quantitativa e também, sua relação da expressão com a idade em que é realizada a indução da crise no cérebro imaturo e (ii) a morte neuronal pela histoquímica do Fluoro-Jade B (FJB) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol. Neste trabalho conseguimos estabelecer com sucesso as condições ideais para o acasalamento desta espécie e obtenção de embriões e...

Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b) are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

The aim of this study was to evaluate the effects of growth hormone (GH)overexpression on the gene expression profile of multiple components of the antioxidant defense system(ADS)of different genotypes of a GH transgenic zebrafish (Danio rerio)model. Several ADS-related genes were analyzed by semiquantitative reverse transcription???PCR in the liver of hemizygous (HE) and homozygous (HO)transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotypedependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse,which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

Dentre as v?rias esp?cies de plantas medicinais, encontra-se a esp?cie Spilanthes acmella, conhecida popularmente como jamb? que se destaca por apresentar in?meras aplica??es na ?rea da medicina popular. A medicina tradicional recomenda suas folhas e flores na elabora??o de infus?es no tratamento de anemia, dispepsia, mal?ria, afec??es da boca (dor de dente) e da garganta, contra escorbuto e tamb?m como antibi?tico e anest?sico. Sendo seus principais efeitos atribu?dos ao espilantol, que ? um representante importante das subst?ncias presentes nessas plantas. Alguns estudos j? foram realizados utilizando o espilantol, possibilitando algumas informa??es da a??o dessa subst?ncia, como seu efeito e imunomodulador devido sua intera??o funcional com mon?citos, granul?citos e c?lulas killers. Por?m, ainda n?o existem estudos eletrofisiol?gicos acerca de sua a??o ictiot?xica, utilizando, por exemplo, o eletroencefalograma para demonstrar sua a??o ao n?vel de Sistema Nervoso Central ou eletromiograma para verificar a ocorr?ncia de sua a??o a n?vel muscular no Zebrafish, evocando a necessidade dessa pesquisa a respeito do assunto. Com base nisso, o presente trabalho objetivou investigar a a??o ictiot?xica do extrato etan?lico da raiz de Spilanthes acmella em Zebrafish atrav?s da an?lise eletrofisiol?gica e comportamental. Os resultados mostraram que o extrato etan?lico de Spilanthes acmella ? um potente indutor de excitabilidade central no zebrafish...

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water...

The human fragile X mental retardation syndrome is caused by expansions of a CGG repeat in the FMR1 gene. FXR1 and FXR2 are autosomal paralogs of FMR1. The products of the three genes, FMRP, FXR1P, and FXR2P, respectively, belong to a family of RNA-binding proteins. While the FMR1-related gene family is well described in human, mouse and Drosophila, little is known about zebrafish (Danio rerio) orthologs of these genes. Here we collate the known FMR1-related gene sequences from zebrafish, examine their regions of structural conservation, and define their orthologies with the human genes. We demonstrate that zebrafish possess only three FMR1-related genes, FMR1, FXR1 and FXR2, and these are orthologous to the human FMR1, FXR1 and FXR2 genes respectively. We examine the spatiotemporal pattern of transcription of the zebrafish genes from 0 hours post fertilisation (hpf) until 24 hpf. Expression of FMR1, FXR1 and FXR2 is widespread throughout this time. However, relative to surrounding tissues, expression of FXR2 is raised in adaxial and somitic cells by 12 hpf while FXR1 expression is high in the anterior of the embryo, and is raised in adaxial cells by 12 hpf. Distinct patterns (and levels) of expression are seen for the different genes later in development. At 24 hpf...

Klotho is a multi-functional anti-ageing protein. Its deletion causes accelerated ageing in mice while its overexpression increases mouse longevity. Klotho is a single pass transmembrane protein and its extracellular domain is cleaved at the cell surface by proteolytic enzymes and subsequently shed into the blood and cerebrospinal fluid, thereby functioning as a humoral factor. The transmembrane form of Klotho is a co-receptor for fibroblast growth factor 23 (Fgf23). Fgf23 acts on the kidney to induce phosphate excretion into urine. However, in the absence of Klotho, Fgf23 cannot bind to its receptor. Therefore, Klotho deficient mice show hyperphosphataemia. The extracellular domain of Klotho released by proteolytic cleavage inhibits the insulin/insulin-like signaling pathway. The inhibition of the insulin pathway enhances protection of cells from oxidative stress and this is an evolutionarily conserved mechanism for extending life span. In addition to this function, secreted Klotho is known to activate calcium ion channels and inhibit the Wnt pathway. Taken together, Klotho acts as an ageing suppressor, but the precise mechanism of how Klotho exerts these functions is not fully understood. In particular, the function of Klotho against oxidative stress needs to be closely investigated as oxidative stress is a major contributor to ageing as well as many diseases associated with ageing...

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with pathologies such as neuron loss, glial cell proliferation, extracellular deposition of senile plaques from the accumulation of amyloid beta (Aβ) peptides and deposition of intracellular neurofibrillary tangles. Aβ is created from the cleavage of the Amyloid Precursor Protein (APP) by two different types of aspartyl proteases, β- and γ-secretase. The majority of AD cases are sporadic and have a late onset.Mutations in the genes encoding APP, PRESENILIN1 and 2 (PSEN1 and PSEN2) genes cause an autosomal dominant inherited form of the disease with an early onset known as familial AD. In some sporadic cases an aberrant splice variant of PSEN2named PS2V is formed that can be found in inclusion bodies in the brain. PS2V results from the binding of the High Mobility Group A1a (HMGA1a) protein close to the splice donor site of exon 5 of PSEN2. The High Mobility Group A1 protein,HMGA1, is widely expressed during embryo development but not in adults. Its expression can be induced in adult neurons by hypoxia/oxidative stress and it is commonly reactivated in many types of cancer. Hypoxia can be a direct consequence of hypoperfusion, a common vascular component among Alzheimer’s disease risk factors and may play an important role in AD pathogenesis. BETA-SITE AMYLOID BETA A4 PRECURSOR PROTEIN-CLEAVING ENZYME 1...

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.; Avdesh Avdesh...

The zebrafish is a versatile model organism with the potential to contribute to our understanding of the molecular pathological mechanisms underlying Alzheimer's disease (AD). An early characteristic of AD brain pathology is lipid peroxidation resulting from oxidative stress. However, changes in lipid peroxidation have not yet been assessed in zebrafish brains, and an earlier attempt to observe changes in F₂-isoprostane levels in the brains of zebrafish exposed to hypoxia was unsuccessful. In this article, we examine the utility of various assays of lipid peroxidation and more general assays of intracellular oxidative stress to detect the changes in oxidative stress in the brains of adult zebrafish exposed to hypoxia or explanted into a sodium azide solution for chemical mimicry of hypoxia. Levels of F₂-isoprostanes and F₄-neuroprostanes were low and variable in zebrafish brains such that statistically significant changes due to hypoxia or chemical mimicry of hypoxia could not be observed. However, measurement of lipid hydroperoxides did reveal significant changes in lipid peroxidation under these conditions, while analyses of catalase gene expression and an assay based on 2',7'-dicholorofluorescein oxidation also revealed changes in oxidative stress levels.; Seyyed Hani Moussavi Nik...

Reliable animal models are required to facilitate the understanding of neurodegenerative pathways in Alzheimer's disease. Animal models can also be employed to search for disease-modifying drugs. The embryos and larvae of zebrafish are particularly advantageous for this purpose. For Alzheimer's disease, drugs that can ameliorate amyloidβ (Aβ) toxicity have therapeutic and/or prophylactic potential. We attempted to generate a zebrafish model of Aβ toxicity that would be viable and fertile but have a highly visible pigmentation phenotype in larvae. The larvae could then be arrayed in microtiter plates to screen compound libraries for drugs acting to reduce Aβ toxicity. We used the promoter of the zebrafish mitfa (nacre) gene to drive expression of the pathological 42 amino acid species of human Aβ, Aβ42, specifically in the highly visible melanophores (melanocytes) of transgenic zebrafish. However, the transgenic fish only showed an aberrant pigment phenotype in adults at the advanced age of 16 months. Nevertheless, our results show that alteration of zebrafish pigment pattern may be useful for analysis of toxic peptide action.; Morgan Newman, Lachlan Wilson, Esther Camp, Giuseppe Verdile, Ralph Martins and Michael Lardelli

The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008).

There is growing interest in using zebrafish (Danio rerio) as a model of neurodegenerative disorders such as Alzheimer’s disease. A zebrafish model of tauopathies has recently been developed and characterized in terms of presence of the pathological hallmarks (i.e., neurofibrillary tangles and cell death). However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess memory and learning in animal models involve visual stimuli, including color preference. The color preference of zebrafish has received little attention. To validate zebrafish as a model for color-associated-learning and memory, it is necessary to evaluate its natural preferences or any pre-existing biases towards specific colors. In the present study, we have used four different colors (red, yellow, green, and blue) to test natural color preferences of the zebrafish using two procedures: Place preference and T-maze. Results from both experiments indicate a strong aversion toward blue color relative to all other colors (red, yellow, and green) when tested in combinations. No preferences or biases were found among reds, yellows, and greens in the place preference procedure. However, red and green were equally preferred and both were preferred over yellow by zebrafish in the T-maze procedure. The results from the present study show a strong aversion towards blue color compared to red...

Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. In this process, while the essential role for Hedgehog (Hh) signaling is well established in the adult zebrafish caudal fin system, its involvement in juvenile tissue regeneration is still unknown. The aim of this Master thesis was therefore to evaluate the contribution of the Hh signaling pathway to the larval zebrafish fin fold regeneration process. Accordingly, we analyzed the expression of several Hh signaling components through in situ hybridization. Here, we showed that several of these genes are effectively expressed in the larval regenerating fin tissue, suggesting a role for Hh signaling also during larval regeneration. However, divergence in the regulation of few Hh signaling components appears to exist between the adult and larval zebrafish fin regeneration processes. Nevertheless, similarly to adult caudal fin regeneration, when Hh signaling was blocked, by using cyclopamine, the larval fin fold regenerative outgrowth is severely impaired. Since larval zebrafish fin fold is ciliated...