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

Abstract

Tests with zebrafish embryos have gained wide acceptance as an alternative test model for drug development and toxicity testing. In particular, the behavioral response of the zebrafish embryo is currently seen as a useful endpoint to diagnose neuroactive substances. Consequently, several behavioral test methods have been developed addressing various behavioral endpoints such as spontaneous tail coiling (STC), photomotor response (PMR), locomotor response (LMR) and alternating light/dark-induced locomotor response (LMR-L/D). Although these methods are distinct in their application, most of their protocols differ quite strongly in the use of experimental parameters and this is usually driven by different research questions. However, if a single mode of action is to be diagnosed, then varying experimental parameters may cause incoherent behavioral responses (hypo-or hyperactivity) of zebrafish during toxicity assessment. This could lead to inconclusiveness of behavioral test results for use within a prospective and diagnostic risk assessment framework. To investigate the influence of these parameters, we conducted a review of existing behavioral assays to address the following two questions: (1) To what extent do varying experimental parameters influence observed effects in published behavioral test methods? (2) Is the observed behavior change (hypo-or hyperactivity) of zebrafish embryos consistent with the expected mode of action of a chemical? We compiled a set of 18 substances which are anticipated to be neuroactive. We found that behavioral changes are not only affected by chemicals but also variation in the use of experimental parameters across studies seems to have a high impact on the outcome and thus comparability between studies. Four parameters, i.e., exposure concentration, exposure duration, endpoint parameter and developmental stage were the most influential parameters. Varying combinations of these parameters caused a non-reproducible outcome for the hyperactivity expected for the organophosphates; chlorpyrifos and diazinon. We highlighted that the STC test shows a higher capacity to predict the hyperactivity of organophosphates, while PMR and LMR-L/D were more suitable to predict the hypoactivity expected for anticonvulsants. We provide a list of recommendations which, when implemented, may help to exclude the risk of bias due to experimental parameters if similar goals are desired.

AEO-7 surfactant is “super toxic” and induces severe cardiac, liver and locomotion damage in zebrafish embryos

“…However, considering the increasing numbers of environmental contaminants with neurotoxic potential, we believe that eco-neurotoxicity should also be considered in risk assessment [76]. The behavioral response of the zebrafish embryo is currently seen as a useful endpoint for identifying neurotoxic chemicals [49,76,77]. The locomotor response for various neurotoxicants can be evaluated by different type of assays such as locomotion or tail flicking assays [37].…”

Zebrafish as a Successful Animal Model for Screening Toxicity of Medicinal Plants

“…The creation of behavioral tests that can employ repeated tests in a short window (i.e., within 24 h) may prove to be the perfect compromise, and some researchers have begun designing tasks that use multiple tests to measure their acquisition during a single session [ 38 ]. If one particular mode of action is to be detected, various experimental parameters can contribute to incoherent actions (hypo-or hyperactivity) responses of zebrafish when determining toxicity [ 122 ]. As a consequence, several behavioral evaluation methods have been developed to address different behavioral endpoints, including spontaneous tail coiling, photomotor response (PMR), locomotor response (LMR), and the alternating light/dark-induced locomotor response (LMR-L/D) [ 122 ], which we will not discuss further in this review.…”

“…If one particular mode of action is to be detected, various experimental parameters can contribute to incoherent actions (hypo-or hyperactivity) responses of zebrafish when determining toxicity [ 122 ]. As a consequence, several behavioral evaluation methods have been developed to address different behavioral endpoints, including spontaneous tail coiling, photomotor response (PMR), locomotor response (LMR), and the alternating light/dark-induced locomotor response (LMR-L/D) [ 122 ], which we will not discuss further in this review.…”

“…The zebrafish model is vital in evaluating the effects on embryonic/larval activity of developmental exposure to harmful compounds [ 38 ]. Comprehension and monitoring of these considerations and future revision/harmonization of procedures will reduce the uncertainty of the outcomes for chemical hazard assessments [ 122 ].…”

Evaluation of the effects of acetylcholinesterase inhibitors in the zebrafish touch-evoked response: quantitative vs. qualitative assessment

“…Although many different behavioural tests in zebrafish have been proposed, there is still no harmonization on the experimental parameters to perform them or on how to evaluate them, which hinders inter-laboratory comparison of results [5,11]. One of the proposed tests, which is quite simple but very informative, is the touch-evoked response (TER) assay.…”