RESUMEN
The zebrafish (Danio rerio) is a well-established vertebrate model organism. Its embryos are used extensively in biology and medicine to perform chemical screens to identify drug candidates or to evaluate teratogenicity and embryotoxicity of substances. Behavioral readouts are increasingly used to assess the effects of compounds on the nervous system. Early stage zebrafish show characteristic behavioral features at stages between 30 and 42 hours post fertilization (hpf) when exposed to a short and bright light flash. This so-called Photomotor Response (PMR) is a reaction of the nervous system of the fish and can be used as a marker in screenings for neuroactive chemicals. To probe a broad and diverse chemical space, many different substances have to be tested and repeated observations are necessary to warrant statistical significance of the results. Although PMR-based chemical screens must use a large number of specimens, there is no sophisticated, automated high-throughput platform available which ensures minimal human intervention. Here we report a PMR platform that was developed by combining an improved automatic sample handling with a remotely controllable microscope setup and an image analysis pipeline. Using infrared illumination during automatic sample preparation, we were able to eliminate excess amounts of visible light that could potentially alter the response results. A remotely controlled microscope setup allows us to screen entire 96-well microtiter plates without human presence that could disturb the embryos. The development of custom video analysis software, including single egg detection, enables us to detect variance among treated specimens and extract easy to interpret numerical values representing the PMR motion. By testing several neuroactive compounds we validated the workflow that can be used to analyze more than one thousand zebrafish eggs on a single 96-well plate.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/efectos de la radiación , Procesamiento de Imagen Asistido por Computador/métodos , Pruebas de Toxicidad/métodos , Animales , Humanos , Pez CebraRESUMEN
A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Frecuencia Cardíaca , Grabación de Cinta de Video , Pez Cebra/fisiología , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/fisiología , Grabación de Cinta de Video/instrumentación , Pez Cebra/embriologíaRESUMEN
Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light but does not require the eyes, pineal gland, or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical nonvisual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Using in vivo calcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.
Asunto(s)
Movimiento/fisiología , Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/fisiología , Rombencéfalo/citología , Conducta Estereotipada/fisiología , Factores de Edad , Análisis de Varianza , Animales , Fenómenos Biomecánicos , Biofisica , Calcio/metabolismo , Embrión no Mamífero , Femenino , Masculino , Microscopía Confocal , Morfolinos/farmacología , Movimiento/efectos de los fármacos , Movimiento/efectos de la radiación , Células Musculares/efectos de los fármacos , Células Musculares/efectos de la radiación , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Vías Nerviosas/efectos de la radiación , Opsinas/química , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Rombencéfalo/fisiología , Conducta Estereotipada/efectos de los fármacos , Conducta Estereotipada/efectos de la radiación , Factores de Tiempo , Pez CebraRESUMEN
Over the past decade, the zebrafish has become a key model organism in genetic screenings and drug discovery. A number of genes have been identified to affect the development of the shape and functioning of the heart, leading to zebrafish mutants with heart defects. The development of semiautomated microscopy systems has allowed for the investigation of drugs that reverse a disease phenotype on a larger scale. However, there is a lack of automated feature detection, and commercially available computer-aided microscopes are expensive. Screening of the zebrafish heart for drug discovery typically includes the identification of heart parameters, such as the frequency or fractional shortening. Until now, screening processes have been characterized by manual handling of the larvae and manual microscopy. Here, an intelligent robotic microscope is presented, which automatically identifies the orientation of a zebrafish in a micro well. A predefined region of interest, such as the heart, is detected automatically, and a video with higher magnification is recorded. Screening of a 96-well plate takes 35 to 55 min, depending on the length of the videos. Of the zebrafish hearts, 75% are recorded accurately without any user interaction. A description of the system, including the graphical user interface, is given.
Asunto(s)
Automatización de Laboratorios/métodos , Corazón/embriología , Ensayos Analíticos de Alto Rendimiento , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía por Video/métodos , Pez Cebra/embriología , Animales , Fármacos Cardiovasculares/aislamiento & purificación , Corazón/efectos de los fármacosRESUMEN
The zebrafish (danio rerio) is one of the most important model organisms in modern drug discovery and disease modeling. Handling and analyzing large numbers of zebrafish larvae require an immense manpower and involve time-consuming manual processes. A novel modular, robotic platform for high-throughput screening is being developed at BioRobotLab (KIT). In this article the fish sorter, which is a robotic device for the automation of a manual process in bio analysis, is presented. The fish sorter detects randomly spread zebrafish eggs and larvae up to an age of 120 hours post fertilization (hpf) in Petri dishes and transfers them to standard 96- or 384- well plates. The robot is controlled by an advanced algorithm with sensor-based process control. Fast and precise hardware components lead to a high working speed and success rate >= 95%.
Asunto(s)
Algoritmos , Robótica , Pez Cebra , AnimalesRESUMEN
The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost-effective and ethically acceptable animal models for drug screening as well as toxicity testing.