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

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.