Doing the locomotion: Insights and potential pitfalls associated with using locomotor activity as a readout of the circadian rhythm in larval zebrafish

Abstract

Background: Zebrafish have been used as a model to study circadian rhythms (CRs) for over 20 years by analyzing various endpoints including locomotor activity. Such studies often utilize high-throughput analysis monitoring activity of larvae placed in well plates numbering >48 wells per plate. Although the CR can be influenced by numerous factors, it is not clear if such effects are permanent. Here, we investigated the variability of CRs of larvae analyzed in different types of well plates and determined the permanency of experimentally-induced aberrations in CRs.

New method: Utilized the tracking software Ethovision XT to investigate how different well plate sizes influence the CR. Re-tested subjects for recovery from long-term CR disruptions and evaluated CR patterns at the individual level.

Results: CR tracking using locomotion as a readout is best in 24 well plates. CR consistency is not maintained in larvae tracked in 48 or 96 well plates. A perturbed CR due to constant light recovered after just 3 days of a normal light/dark cycle.

Comparison with existing methods: Unlike other CR locomotor-based assays, our approach allowed for a medium-throughput analysis of individual CRs, minimized variability and allowed for the re-evaluation of larval CRs 4-5 days later.

Conclusions: This medium-throughput locomotor CR analysis allows for a standardized, less variable approach whereby larvae can be re-tested to identify potential long-term changes after experimental manipulations. Long-term behavioral experiments in 48 or 96 well plates may impart stress on the larvae due to space constraints which could impact nervous system function and/or behavior.

Keywords: Circadian rhythm; EthoVision; High speed recording; Larval zebrafish; Locomotor activity.