Characterization of locomotor phenotypes in zebrafish larvae requires testing under both light and dark conditions

Abstract

Despite growing knowledge, much remains unknown regarding how signaling within neural networks translate into specific behaviors. To pursue this quest, we need better understanding of the behavioral output under different experimental conditions. Zebrafish is a key model to study the relationship between network and behavior and illumination is a factor known to influence behavioral output. By only assessing behavior under dark or light conditions, one might miss behavioral phenotypes exclusive to the neglected illumination setting. Here, we identified locomotor behavior, using different rearing regimes and experimental illumination settings, to showcase the need to assess behavior under both light and dark conditions. Characterization of free-swimming zebrafish larvae, housed under continuous darkness or a day/night cycle, did not reveal behavioral differences; larvae were most active during light conditions. However, larvae housed under a day/night cycle moved a shorter distance, had lower maximum velocity and maximum acceleration during the startle response under light conditions. Next, we explored if we could assess behavior under both dark and light conditions by presenting these conditions in sequence, using the same batch of larvae. Our experiments yielded similar results as observed for naïve larvae: higher activity during light conditions, regardless of order of illumination (i.e. dark-light or light-dark). Finally, we conducted these sequenced illumination conditions in an experimental setting by characterizing behavioral phenotypes in larvae following neuromast ablation. Depending on the illumination during testing, the behavioral phenotype following ablation was characterized differently. In addition, the results indicate that the order in which the light and dark conditions are presented has to be considered, as habituation may occur. Our study adds to existing literature on illumination-related differences in zebrafish behavior and emphasize the need to explore behavioral phenotypes under both light and dark condition to maximize our understanding of how experimental permutations affect behavior.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression

“…This assay allows to analyze the larvae locomotor behavior in different light conditions, with the aim of evaluating the sensorimotor function of zebrafish larvae, and to characterize neurobehavioral responses to chemicals. Typically, untreated zebrafish larvae exhibit higher locomotor activities during the dark phase than in the light phase, because a dark environment offers a better chance of survival for the larvae [ 42 ]. Indeed, calculation of average locomotor activity per minute revealed that the zebrafish larvae covered a greater distance and moved at greater speed during dark than light phases, regardless of the experimental treatment ( Supplementary Figure S1 ).…”

A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

“…They did not however observe this with the five-minute protocol of the same length. A possible reason for the reduced locomotion observed could be habituation to the dark (Tuz-Sasik et al, 2022).…”

“…They did not however observe this with the five-minute protocol of the same length. A possible reason for the reduced locomotion observed could be habituation to the dark (Tuz-Sasik et al, 2022). Habituation is non-associative learning defined here as a decrease in dark phase locomotion following repeated exposure to the light/dark transitions (Tuz-Sasik et al, 2022).…”

“…A possible reason for the reduced locomotion observed could be habituation to the dark (Tuz-Sasik et al, 2022). Habituation is non-associative learning defined here as a decrease in dark phase locomotion following repeated exposure to the light/dark transitions (Tuz-Sasik et al, 2022). In the wild, repeated exposure to harmless stimuli causes high energy expenditure and motor fatigue, therefore, habituation ensures the fish conserves energy for survival (Tuz-Sasik et al, 2022).…”

“…Habituation is non-associative learning defined here as a decrease in dark phase locomotion following repeated exposure to the light/dark transitions (Tuz-Sasik et al, 2022). In the wild, repeated exposure to harmless stimuli causes high energy expenditure and motor fatigue, therefore, habituation ensures the fish conserves energy for survival (Tuz-Sasik et al, 2022). Another possible reason for the reduced dark phase locomotion may be exhaustion brought-on by excessive energy use (Tuz-Sasik et al, 2022).…”