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1.
Artículo en Inglés | MEDLINE | ID: mdl-38218564

RESUMEN

Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos.


Asunto(s)
Síndromes de Neurotoxicidad , Pez Cebra , Animales , Pez Cebra/fisiología , Embrión no Mamífero
2.
Zebrafish ; 21(1): 48-52, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38193768

RESUMEN

Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.


Asunto(s)
Perciformes , Pez Cebra , Animales , Pez Cebra/fisiología , Conducta Animal/fisiología , Larva/fisiología , Programas Informáticos
3.
Environ Sci Technol ; 57(48): 19453-19462, 2023 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-37956114

RESUMEN

Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.


Asunto(s)
Artrópodos , Programas Informáticos , Animales , Conducta Animal
4.
Zebrafish ; 19(1): 32-35, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-35100037

RESUMEN

Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.


Asunto(s)
Embrión no Mamífero , Sistema Nervioso/efectos de los fármacos , Pruebas de Toxicidad , Pez Cebra , Animales , Embrión no Mamífero/efectos de los fármacos , Pez Cebra/fisiología
5.
Stat Med ; 41(4): 625-644, 2022 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-34866221

RESUMEN

Network meta-analysis (NMA) simultaneously estimates multiple relative treatment effects based on evidence that forms a network of treatment comparisons. Heterogeneity in treatment definitions, such as dose, can lead to a violation of the consistency assumption that underpins NMA. Model-based NMA (MBNMA) methods have been proposed that allow functional dose-response relationships to be estimated within an NMA, which avoids lumping different doses together and thereby reduces the likelihood of inconsistency. Dose-response MBNMA relies on appropriate specification of the dose-response relationship as well as consistency of relative effects. In this article we describe methods to check for inconsistency in dose-response MBNMA models. Global and local (node-splitting) tests for inconsistency are described that account for studies with ≥3 arms that are typical in dose-finding trials. We show that consistency needs to be assessed with respect to the choice of dose-response function. We illustrate the methods using a network comparing biologics for moderate-to-severe psoriasis. By comparing results from an Emax and an exponential dose-response function we show that failure to correctly characterise the dose-response can introduce apparent inconsistency. The number of comparisons for which node-splitting is possible is also shown to be dependent on the complexity of the selected dose-response function. We highlight that the nature of dose-finding studies, which typically compare multiple doses of the same agent, provide limited scope to assess inconsistency, but these study designs help guard against inconsistency in the first place. We demonstrate the importance of assessing consistency to obtain robust relative effects to inform drug-development and policy decisions.


Asunto(s)
Metaanálisis en Red , Humanos
6.
J Clin Epidemiol ; 95: 45-54, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29217451

RESUMEN

OBJECTIVE: We investigated the associations between risk of bias judgments from Cochrane reviews for sequence generation, allocation concealment and blinding, and between-trial heterogeneity. STUDY DESIGN AND SETTING: Bayesian hierarchical models were fitted to binary data from 117 meta-analyses, to estimate the ratio λ by which heterogeneity changes for trials at high/unclear risk of bias compared with trials at low risk of bias. We estimated the proportion of between-trial heterogeneity in each meta-analysis that could be explained by the bias associated with specific design characteristics. RESULTS: Univariable analyses showed that heterogeneity variances were, on average, increased among trials at high/unclear risk of bias for sequence generation (λˆ 1.14, 95% interval: 0.57-2.30) and blinding (λˆ 1.74, 95% interval: 0.85-3.47). Trials at high/unclear risk of bias for allocation concealment were on average less heterogeneous (λˆ 0.75, 95% interval: 0.35-1.61). Multivariable analyses showed that a median of 37% (95% interval: 0-71%) heterogeneity variance could be explained by trials at high/unclear risk of bias for sequence generation, allocation concealment, and/or blinding. All 95% intervals for changes in heterogeneity were wide and included the null of no difference. CONCLUSION: Our interpretation of the results is limited by imprecise estimates. There is some indication that between-trial heterogeneity could be partially explained by reported design characteristics, and hence adjustment for bias could potentially improve accuracy of meta-analysis results.


Asunto(s)
Metaanálisis como Asunto , Proyectos de Investigación/normas , Teorema de Bayes , Sesgo , Interpretación Estadística de Datos , Diseño de Investigaciones Epidemiológicas , Humanos
7.
Am J Epidemiol ; 187(5): 1113-1122, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29126260

RESUMEN

Flaws in the design of randomized trials may bias intervention effect estimates and increase between-trial heterogeneity. Empirical evidence suggests that these problems are greatest for subjectively assessed outcomes. For the Risk of Bias in Evidence Synthesis (ROBES) Study, we extracted risk-of-bias judgements (for sequence generation, allocation concealment, blinding, and incomplete data) from a large collection of meta-analyses published in the Cochrane Library (issue 4; April 2011). We categorized outcome measures as mortality, other objective outcome, or subjective outcome, and we estimated associations of bias judgements with intervention effect estimates using Bayesian hierarchical models. Among 2,443 randomized trials in 228 meta-analyses, intervention effect estimates were, on average, exaggerated in trials with high or unclear (versus low) risk-of-bias judgements for sequence generation (ratio of odds ratios (ROR) = 0.91, 95% credible interval (CrI): 0.86, 0.98), allocation concealment (ROR = 0.92, 95% CrI: 0.86, 0.98), and blinding (ROR = 0.87, 95% CrI: 0.80, 0.93). In contrast to previous work, we did not observe consistently different bias for subjective outcomes compared with mortality. However, we found an increase in between-trial heterogeneity associated with lack of blinding in meta-analyses with subjective outcomes. Inconsistency in criteria for risk-of-bias judgements applied by individual reviewers is a likely limitation of routinely collected bias assessments. Inadequate randomization and lack of blinding may lead to exaggeration of intervention effect estimates in randomized trials.


Asunto(s)
Sesgo , Evaluación de Resultado en la Atención de Salud/métodos , Ensayos Clínicos Controlados Aleatorios como Asunto/normas , Teorema de Bayes , Estudios Epidemiológicos , Humanos , Metaanálisis como Asunto , Oportunidad Relativa
8.
Res Synth Methods ; 8(1): 43-52, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27259973

RESUMEN

In meta-analysis, the random-effects model is often used to account for heterogeneity. The model assumes that heterogeneity has an additive effect on the variance of effect sizes. An alternative model, which assumes multiplicative heterogeneity, has been little used in the medical statistics community, but is widely used by particle physicists. In this paper, we compare the two models using a random sample of 448 meta-analyses drawn from the Cochrane Database of Systematic Reviews. In general, differences in goodness of fit are modest. The multiplicative model tends to give results that are closer to the null, with a narrower confidence interval. Both approaches make different assumptions about the outcome of the meta-analysis. In our opinion, the selection of the more appropriate model will often be guided by whether the multiplicative model's assumption of a single effect size is plausible. Copyright © 2016 John Wiley & Sons, Ltd.


Asunto(s)
Investigación Empírica , Metaanálisis como Asunto , Modelos Estadísticos , Algoritmos , Intervalos de Confianza , Interpretación Estadística de Datos , Bases de Datos Factuales , Humanos , Infarto del Miocardio/terapia , Sesgo de Publicación , Literatura de Revisión como Asunto
9.
J Neurosci ; 34(9): 3402-12, 2014 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-24573296

RESUMEN

Myelin membrane, which ensheaths axons, has an unusually high amount of cholesterol. Cholesterol influences membrane fluidity and assembles lipid-rich microdomains within membranes, and some studies have shown that cholesterol is important for myelination. How cholesterol influences the development and differentiation of oligodendrocytes, glial cells that make myelin, is not known nor is clear whether isoprenoids, which also are products of the cholesterol biosynthetic pathway, contribute to myelination. Through a forward genetic screen in zebrafish we discovered that mutation of hmgcs1, which encodes an enzyme necessary for isoprenoid and cholesterol synthesis, causes oligodendrocyte progenitor cells (OPCs) to migrate past their target axons and to fail to express myelin genes. Drawing on a combination of pharmacological inhibitor and rescue experiments, we provide evidence that isoprenoids and protein prenylation, but not cholesterol, are required in OPCs to halt their migration at target axons. On the other hand, cholesterol, but not isoprenoids, is necessary both for axon ensheathment and myelin gene expression. Our data reveal that different products of the cholesterol biosynthetic pathway have distinct roles in oligodendrocyte development and that they together help to coordinate directed migration, axon wrapping, and gene expression.


Asunto(s)
Axones/fisiología , Movimiento Celular/genética , Colesterol/metabolismo , Hidroximetilglutaril-CoA Sintasa/genética , Mutación/genética , Vaina de Mielina/metabolismo , Oligodendroglía/fisiología , Animales , Animales Modificados Genéticamente , Axones/efectos de los fármacos , Axones/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Embrión no Mamífero , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Vaina de Mielina/genética , Oligodendroglía/efectos de los fármacos , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Células Madre/efectos de los fármacos , Células Madre/fisiología , Imagen de Lapso de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genética
10.
Dev Biol ; 286(1): 114-35, 2005 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16125164

RESUMEN

Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.


Asunto(s)
Intestinos/embriología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Diferenciación Celular , Polaridad Celular , Proliferación Celular , ADN Recombinante/genética , Endodermo/citología , Endodermo/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Mucosa Intestinal/metabolismo , Intestinos/citología , Morfogénesis , Proteínas Recombinantes/genética , Pez Cebra/genética , Pez Cebra/metabolismo
11.
Dev Biol ; 269(1): 302-15, 2004 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-15081375

RESUMEN

The Netrin receptor Deleted in colon cancer (Dcc) has been shown to play a pivotal role in the guidance of nascent axons towards the ventral midline in the developing nervous systems of both vertebrates and invertebrates. In contrast, the function during embryogenesis of a second Dcc-like Netrin receptor Neogenin has not yet been defined. We used antisense morpholino oligonucleotides to knockdown Neogenin activity in zebrafish embryos and demonstrate that Neogenin plays an important role in neural tube formation and somitogenesis. In Neogenin knockdown embryos, cavitation within the neural rod failed to occur, producing a neural tube lacking a lumen. Somite formation was also defective, implicating Neogenin in the migration events underlying convergent extension during gastrulation. These observations suggest a role for Neogenin in determining cell polarity or migrational directionality of both neuroectodermal and mesodermal cells during early embryonic development.


Asunto(s)
Sistema Nervioso Central/embriología , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Somitos/metabolismo , Animales , Sistema Nervioso Central/anomalías , Sistema Nervioso Central/metabolismo , Proteínas Hedgehog , Receptores de Netrina , Transactivadores/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo
12.
Methods Cell Sci ; 25(1-2): 7-14, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-14739582

RESUMEN

We describe here simple methods for producing transgenic zebrafish reporter lines using BAC clones. The use of BAC clones facilitates creation of useful transgenics as the large amounts of genomic DNA they contain increase the likelihood that reporter gene expression will be properly regulated. Combined with recent advances in live embryo image analysis, this strategy has the potential to greatly advance the investigation of neural cell behavior during development.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas Motoras/metabolismo , Sistema Nervioso Periférico/metabolismo , Células de Purkinje/metabolismo , Pez Cebra/genética , Animales , Clonación Molecular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Genes Reporteros/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microinyecciones , Neuronas Motoras/citología , Sistema Nervioso Periférico/citología , Células de Purkinje/citología , Pez Cebra/anatomía & histología , Pez Cebra/metabolismo
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