TCMacro: A Simple and Robust ImageJ-Based Method for Automated Measurement of Tail Coiling Activity in Zebrafish

Abstract

Tail coiling is a reflection response in fish embryos that can be used as a model for neurotoxic analysis. The previous method to analyze fish tail coiling is largely based on third-party software. In this study, we aim to develop a simple and cost-effective method called TCMacro by using ImageJ macro to reduce the operational complexity. The basic principle of the current method is based on the dynamic change of pixel intensity in the region of interest (ROI). When the fish tail is moving, the average intensity is increasing. In time when the fish freeze, the peak of mean intensity is maintaining at a relatively low level. By using the optimized macro settings and excel VBA scripts, all the tail coiling measurement processes can be archived with few operation steps with high precision. Three major endpoints of tail coiling counts, tail coiling duration and tail coiling intervals can be obtained in batch. To validate this established method, we tested the potential neurotoxic activity of Tricaine (methanesulfonate, MS-222) and psychoactive compound of caffeine. Zebrafish embryos after Tricaine exposure displayed significantly less tail coiling activity in a dose-dependent manner, and were comparable to manual counting through the Wilcoxon test and Pearson correlation double validation. Zebrafish embryos after caffeine exposure displayed significantly high tail coiling activity. In conclusion, the TCMacro method presented in this study provides a simple and robust method that is able to measure the relative tail coiling activities in zebrafish embryos in a high-throughput manner.

The sensitivity of the zebrafish embryo coiling assay for the detection of neurotoxicity by compounds with diverse modes of action

“…Various protocols have been developed for the coiling assay, which has been designed to identify effects on early behavior of zebrafish embryos through the assessment of spontaneous tail coiling (Selderslaghs et al 2010 , 2013 ; Velki et al 2017 ; Wang et al 2018 ; Zindler et al 2019b , a ; Bachour et al 2020 ; Guo et al 2021 ; Kurnia et al 2021 ). Behavioral profiles become more complex, when exposure to neurotoxicants induces hyperactivity at low concentrations (e.g., through their ability to inhibit acetylcholine esterase) and hypoactivity at higher concentrations.…”

“…Given the comparatively short experimental duration, the most time-consuming aspect of the coiling assay remains the software-based analysis of videos and subsequent data processing. If this step could be automated as suggested by González-Fraga et al ( 2019 ), Ogungbemi et al ( 2020 , 2021 ) as well as Kurnia et al ( 2021 ), the coiling assay with zebrafish embryos might even be developed into a high throughput alternative test method for neurotoxicity testing.…”

“…The recording of coiling incidents can be accomplished either manually (Chen et al 2012 ; Abu Bakar et al 2017 ; Wang et al 2019a , b ) or automatically (Ogungbemi et al 2021 ; Zhang et al 2021 ). Some studies rely on expensive third-party software (Zindler et al 2019a ; de Oliveira et al 2021 ), while others use open-access software which require coding knowledge (González-Fraga et al 2019 ), or the use of simpler programs (Kurnia et al 2021 ). Past studies have also shown considerable variation in the experimental setup: In some protocols, exposure was initiated with fertilization (Velki et al 2017 ; Zindler et al 2019b , a ), others only began exposure during the coiling development itself (Vliet et al 2017 ).…”