Behavioral screening of the LOPAC1280 library in zebrafish embryos

Abstract

Spontaneous activity represents an early, primitive form of motor activity within zebrafish embryos, providing a potential readout for identification of neuroactive compounds. However, despite use as an endpoint in chemical screens around the world, the predictive power and limitations of assays relying on spontaneous activity remain unclear. Using an improved high-content screening assay that increased throughput from 384 to 3,072 wells per week, we screened a well-characterized library of 1,280 pharmacologically active compounds (LOPAC1280) – 612 of which target neurotransmission – to identify which targets are detected using spontaneous activity as a readout. Results from this screen revealed that (1) 8% of the LOPAC1280 library was biologically active; (2) spontaneous activity was affected by compounds spanning a broad array of targets; (3) only 4% of compounds targeting neurotransmission impacted spontaneous activity; and (4) hypoactivity was observed for 100% of hits detected, including those that exhibit opposing mechanisms of action for the same target. Therefore, while this assay was able to rapidly identify potent neuroactive chemicals, these data suggest that spontaneous activity may lack the ability to discriminate modes of action for compounds interfering with neurotransmission, an issue that may be due to systemic uptake following waterborne exposure, persistent control variation, and/or interference with non-neurotransmission-related mechanisms.

Hypo- or hyperactivity of zebrafish embryos provoked by neuroactive substances: a review on how experimental parameters impact the predictability of behavior changes

“…They found an effect concentration for the spontaneous tail coiling (STC, conducted in 384-well plates) which is 12-fold higher than that reported in a subsequent study [87] in which embryos were exposed in glass beakers (both used nominal concentrations). Similarly, Vliet et al [118] who also exposed embryos to abamectin in 384-well plates found an effect concentration which is 6 fold higher than that observed in Raftery and Volz [87]. Beside the test container material, the ratio of surface area to volume could also induce variability.…”

“…This event is assumed to be important for the hatching of the embryo from its chorion but evidence for a role of STC for hatching has not yet been provided [58,98]. Frequency changes of the STC have been used as a tool to detect the effects of neuroactive chemicals in developing embryo [118,121].…”

“…It is assumed that neuroactive substances are able to modulate nervous receptors leading to hypo-or hyperactivity behavior [114]. For example, Vliet et al [118] used the STC response as a metric to screen a library of 1280 pharmacologically active compounds for neuroactivity. Reif et al [89] used the hypo-or hyperactivity observed in the different phases of the PMR to characterize a suite of 1060 chemicals; and Bugel and Tanguay [14] were able to negate a GABA mode of action for a suite of 24 flavonoids in the LMR based on the induction of hypo-or hyperactivity.…”

“…In a STC study by Vliet et al [118], exposure duration was reduced from 23 h (2-25 hpf ) to 2 h (23-25 hpf ) to eliminate false positives associated with malformations. This decreased the number of STC hits from 43 to 15.…”

Dynamic Alterations in DNA Methylation Precede Tris(1,3-dichloro-2-propyl)phosphate-Induced Delays in Zebrafish Epiboly

“…Adult 5D zebrafish ( Danio rerio ) were maintained and bred on a recirculating system as previously described by Vliet et al 24 . All embryos were collected immediately after spawning and staged according to previously described methods 25 .…”