title: An automated and high-throughput Photomotor Response platform for chemical screens.
abstract: 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.
authors.forename: Daniel
authors.initials: D
authors.lastname: Marcato
authors.forename: Rudiger
authors.initials: R
authors.lastname: Alshut
authors.forename: Helmut
authors.initials: H
authors.lastname: Breitwieser
authors.forename: Ralf
authors.initials: R
authors.lastname: Mikut
authors.forename: Uwe
authors.initials: U
authors.lastname: Strahle
authors.forename: Christian
authors.initials: C
authors.lastname: Pylatiuk
authors.forename: Ravindra
authors.initials: R
authors.lastname: Peravali
mesh_terms: D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004625:Embryo, Nonmammalian / Q000187:drug effects* / Q000528:radiation effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods*; D015027:Zebrafish