preparation of zebrafish embryos and chemicals for testing neuromuscular and general toxicity on embryos

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

In recent years, zebrafish have become highly popular model organisms for the evaluation of chemical compound effects, encompassing research areas from drug development to environmental toxicology1. As vertebrates, zebrafish share many aspects of their genetic makeup and overall physiology with humans2,3. Therefore, results obtained in this model often are directly relevant to human health. Several drug candidates currently in clinical trials have been identified in compound screens using zebrafish4.
Toxicity assessment is one major application where tests using zebrafish embryonic stages are of interest. Various Organisation for Economic Co-operation and Development (OECD) test guidelines exist for the use of zebrafish in environmental toxicity testing5,6. The small size and rapid development of zebrafish embryos make them highly suitable for screening approaches on a medium to high throughput scale1,3,4. Toxicological endpoints targeted by such screens include embryonic malformations and lethality7, endocrine disruption8, organ toxicity9, and behavioral assessments indicative of neural toxicity10,11. The behavioral assays are possible because zebrafish embryos show various types of locomotor responses to different stimuli depending on their stage of development. For example, 1 day post fertilization (dpf) embryos show spontaneous tail coiling12 and respond to a sequence of light pulses with a typical sequence of movements, the so-called photomotor response (PMR)10. After hatching, typically occurring around 48-72 hours post fertilization (hpf), the freely swimming eleutheroembryos13 gradually develop startle and escape responses to vibrational stimuli starting around 4 dpf14. These responses are characterized by a distinctive bend to the direction opposite the direction of the stimulus (the so-called C-bend or C-start), which is followed by a smaller counter bend and swimming behavior14,15,16,17. Notably, embryonic behaviors are governed by neural circuits using various neurotransmitter systems, allowing for probing chemical compound effects targeting these systems. For example, the PMR assay revealed the effects of compounds interfering with cholinergic, adrenergic, and dopaminergic signaling10, while the startle response involves cholinergic, glutamatergic, and glycinergic neurons16,18. Furthermore, compounds that damage the muscles or the neuro-muscular interface will also affect these behaviors, as will compounds toxic to the inner ear/lateral line hair cells19,20. Observing zebrafish locomotor behavior in response to a stimulus is thus a suitable means to assess not only neurotoxicity but equally ototoxicity and myotoxicity. Scoring locomotor behavior also serves as a proxy for general toxicity/lethality assessment since dead embryos do not move. Thereby, embryonic locomotion behaviors represent an integrative readout for a first-tier toxicity screening approach, which indicates lethal and neuromuscular compound effects in one setup. Given that the eleutheroembryos are already capable of metabolizing compounds, the approach may also detect the effects of metabolic transformation products7,21,22. Importantly, zebrafish embryos are not considered as protected life stage under some animal protection legislations until the stage of free feeding after 120 hpf13. Therefore, they are regarded as an alternative to animal toxicity testing.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66153/66153fig01.jpg" /> 
Figure 1: Vibration startle response system setup. (A) Overview of the system. Plates with embryos exposed to the test compounds are placed on the electrodynamic transducer array (&#34;loudspeakers&#34;). The camera is sequentially moved by the belt-driven linear drive into the recording position above the targeted transducer. (B) Detailed view of the transducer/loudspeaker with tissue culture dish inserted on top. The plates are illuminated from below by an LED light sheet at 4000-5000 lux. An LED light next to the speaker lights up while the stimulus is given. (C) Still image of video recorded by the camera upon stimulation of the embryos. (D) Screenshot of the configuration file. (E) Screenshot of the recording software interface. <a href="https://www.jove.com/files/ftp_upload/66153/66153fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we describe a testing protocol for the evaluation of compound effects on the vibration startle response using an in-house build simple testing device based on vibration stimuli generated by electrodynamic transducers coupled with an automated video recording of several freely moving embryos in a tissue culture dish23. The system is modular and allows for sequential recording from several tissue culture dishes in parallel. In the setup currently used, five electrodynamic transducers provide a vibrational stimulus (500 Hz, duration 1 ms) to tissue culture dishes containing 20 embryos placed on top of them (Figure 1). The plates are illuminated from below at 4000-5000 lux with LED light sheets. An LED light next to each transducer indicates periods of stimulus application, and an oscilloscope indicates waveforms and frequency of the applied stimulus (for details, see Ref. 23). The behavior of the embryos is recorded by a high-speed camera (Table of Materials) at 1000 frames per second (fps), which is moved above the targeted speaker by a belt-driven linear drive. This recording speed is required to reliably resolve the startle response. The system provides a low-cost, individually adaptable alternative to current commercial systems. The precise workflow detailed below is currently performed in the framework of the Precision Toxicology initiative24 in order to determine suitable exposure conditions for OMICS data acquisition from zebrafish embryos treated with a selected set of toxicants.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

When screening chemicals for toxicity, a current experimental challenge is the determination of toxic effects in vivo, taking into account the complexity of a whole organism, and to do this in a rapid high-throughput manner. By using a behavioral readout that monitors the escape response of zebrafish embryos to a vibration stimulus, our system allows us to identify compounds that interfere with neural or muscular function. Because dead embryos don't move, we also capture compounds that cause lethality by unspecific toxicity.The system we present can be built for a modest price and is customizable. It is also easy to maintain and all pieces can be replaced. We are currently employing the startle assay system within the PrecisionTox Consortium to determine a chemical compound dose for OMICS data acquisition.The data are generated across five model organisms and human cell lines. They will be used to derive toxicity pathways and biomarkers for human toxicity prediction.

Preparation of Zebrafish Embryos and Chemicals for Testing Neuromuscular and General Toxicity on Embryos

Research

JoVE Journal

Biology

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Evaluating Lethality and Neuromuscular Toxicity of Chemicals in Zebrafish Embryos Using Vibration Startle Assay