Abstract
Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.
Highlights
Evaluation of embryonic and larval behaviour is an important tool for toxicological, pharmacological and biomedical studies as it enables the noninvasive investigation of sub-lethal effects on the embryonic phenotype [1,2,3]
We combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, followed by spectral analysis, as a first step towards automating data extraction and analysis for such screenings
Zebrafish embryos have recently been cultured successfully in a microfluidic flow-through system constructed from borosilicate glass (BioWell plate) [6]
Summary
Evaluation of embryonic and larval behaviour is an important tool for toxicological, pharmacological and biomedical studies as it enables the noninvasive investigation of sub-lethal effects on the embryonic phenotype [1,2,3]. Behavioural screening assays bring major challenges in terms of data collection and data evaluation, both of which are highly time-consuming activities that could prohibit the use of high throughput systems compared to in vitro assays. These limitations are being addressed by technological innovations in imaging, culture techniques and drug delivery. Zebrafish (Danio rerio) embryos have recently been proposed as an in vivo model to bridge the gap between simple in vitro assays and biological validation in whole animals such as rodents [5,6,7] Their small size, transparency during the embryonic period and rapid, external development make zebrafish suitable for high throughput behavioural assays [8, 9]. This makes it easier to image large numbers of individuals, because it reduces the seek time and means that more pixels coincide with the embryo itself rather than with dead space
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