Standardized Method for the Assessment of Behavioral Responses of Zebrafish Larvae

Abstract

Zebrafish are easy to breed in a laboratory setting as they are extremely fertile and produce dozens of eggs per set. Because zebrafish eggs and the skin of the early-stage larvae are transparent, their embryos and the hearts and muscles of their larvae can be easily observed. Multiple rapid analyses of heart rate and behavior can be performed on these larvae simultaneously, enabling investigation of the influence of neuroactive substances on abnormal behavior, death, and associated pathogenetic mechanisms. Zebrafish larvae are becoming increasingly popular among researchers and are used in laboratories worldwide to study various vertebrate life phenomena; more experimental systems using zebrafish will undoubtedly be developed in the future. However, based on the available literature, we believe that the conceptualization of a protocol based on scientific evidence is necessary to achieve standardization. We exposed zebrafish larvae at 6–7 days post-fertilization to 50 repeated light–dark stimuli at either 15-min or 5-min intervals. We measured the traveled distance and habituation time through a video tracking apparatus. The traveled distance stabilized after the 16th repetition when the zebrafish were exposed to light–dark stimuli at 15-min intervals and after the 5th repetition when exposed at 5-min intervals. Additionally, at 15-min intervals, the peak of the traveled distance was reached within the first minute in a dark environment, whereas at 5-min intervals, it did not reach the peak even after 5 min. The traveled distance was more stable at 5-min intervals of light/dark stimuli than at 15-min intervals. Therefore, if one acclimatizes zebrafish larvae for 1 h and collects data from the 5th repetition of light/dark stimuli at intervals of 5 min in the light/dark test, a stable traveled distance result can be obtained. The establishment of this standardized method would be beneficial for investigating substances of unknown lethal concentration.

Cannabidiol improves haloperidol-induced motor dysfunction in zebrafish: a comparative study with a dopamine activating drug

“…Normal zebrafish display low activity levels during light periods, while in the dark, they are hyperactive (Maeda et al 2021b ). This property can be used to investigate the effects of each drug on dopamine receptors in zebrafish.…”

“…The light intensity used during the light-on period was 5000 lx, whereas under infrared illumination, the intensity was 0 lx. Fish were subjected to alternating (six times) light and dark exposure at 5-min intervals to determine differences in locomotor activities and response (Hasumi et al 2020 ; Basnet et al 2019 ; Yang et al 2017 ; Maeda et al 2021a ; Maeda et al 2021b ). Drug effects were measured for 1 h using Danio Vision XT and Ethovision XT 11.5 (Noldus Information Technology, Wageningen, The Netherlands) (Maeda et al 2018 ).…”

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

“…In effect, the activity under light was greater when light was presented last, compared to when light was presented first. In contrast, activity under dark conditions was higher when presented first, compared to presented last (S2B and S2C Fig) . While we currently have no explanation for these observations, habituation/acclimation to a novel environment [3,25] or even the startle trigger [26] may play a role. Habituation is a non-associative form of learning defined as a decrease in behavioral response after repeated stimuli [27].…”

Zebrafish: A Pharmacological Model for Learning and Memory Research

“…In addition, psychoactive drugs often impact locomotor activity. LAT is used to establish the baseline locomotor activity, as well as the effect of the drugs and environmental stimuli on locomotor activity, which may impact the cognitive test results later [ 49 ]. LAT is conducted by holding a single zebrafish in a trapezoid or rectangle water tank for a few minutes.…”

“…Considering that zebrafish are increasingly used as study models across different laboratories worldwide, standardization may be necessary to make the findings from different studies comparable. Recently, Maeda et al developed a standardized method for assessing the behavioral response (including locomotor activity/response) in zebrafish larvae (6 to 7 dpf) using a light–dark locomotion test [ 49 ]. The characterized responses were proposed to be used for screening psychoactive substances.…”