High-Content Screening Assay for Identification of Chemicals Impacting Spontaneous Activity in Zebrafish Embryos

Abstract

Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants.

Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine

“…Nicotine did not affect the frequency of spontaneous contractions. This outcome was consistent with Raftery et al (2014), who exposed zebrafish embryos to nicotine (up to 8 μg/mL) starting at 5 hpf.…”

Age matters: Developmental stage of Danio rerio larvae influences photomotor response thresholds to diazinion or diphenhydramine

“…Parallel efforts to advance HTS efforts at the organismal level have also been undertaken and hold great promise for understanding contaminant at higher levels of biological complexity than in vitro systems (Padilla et al, 2012; Raftery et al, 2014; Truong et al, 2014). These in vivo HTS methodologies have largely examined early life stage studies with embryonic and larval zebrafish.…”

“…A number of behavioral investigations have occurred with fish models, including adaptations of mammalian pharmacology tests on anxiety such as light/dark preference (Steenbergen et al, 2011), thigmotaxis (Schnorr et al, 2012), and open field and color preference (Ahmad and Richardson, 2013) tests. Additionally photomotor responses (PMR) are increasingly used to understand baseline, stimulatory and refractory zebrafish behaviors (Kokel et al, 2010; Raftery et al, 2014). …”

Zebrafish: A Marvel of High-Throughput Biology for 21st Century Toxicology

“…Many examples of transgenic zebrafish expressing fluorescent reporters under the control of neural-specific promoters have allowed researchers unparalleled access to real-time investigations of neural architecture and function, thus paving the way towards comprehensive assessments of the effects of environmental exposures on diverse neurological endpoints, including molecular, cellular, anatomical and functional phenotypes. Over the last two years, neural-specific transgenic zebrafish have been at the forefront of chemical screens for compounds targeting the circadian clock (108), dopaminergic pathway (109), neurogenesis (110), spontaneous activity (111), pleotropic neurotoxicity (112) and environmental influences on axon growth and connectivity (113). …”