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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.

SUBMITTER: Maeda H

PROVIDER: S-EPMC8389650 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The zebrafish (Danio rerio) is an important model organism in the study of the neurobiological basis of human mental disorders. Yet the utility of this species is limited by the quality of the phenotypical characterization tools available. Here, we present a complex testing environment for the quantification of explorative behavior in adult zebrafish, the zebrafish Multivariate Concentric Square Field™ (zMCSF), adapted from the rodent equivalent that has been used in > 40 studies. The apparatus consists of a central open area which is surrounded by a dark corner with a roof (DCR), corridors, and an inclined ramp. These areas differ in illumination, water depth, and are sheltered or exposed to different degrees. We quantified behavior of male and female wild-caught and AB strain zebrafish in the zMCSF (day 1) and cross-validated these results using the novel tank diving test (NTDT) (day 2). To assess the effect of repeated testing, AB zebrafish we tested a second time in both tests 1 week later (on days 7 and 8). We detected strong differences between the strains, with wild zebrafish swimming faster and spending more time in the corridors and on the ramp, while they avoided the open area in the center. AB zebrafish were less hesitant to enter the center but avoided the ramp, and often left one or more zones unexplored. No major sex differences in exploratory behavior were detected in either strain, except for a slightly higher velocity of AB males which has been reported before. Importantly, the zMCSF was largely resilient to repeated testing. The diving test revealed only one difference confined to one sex; wild females paid more visits to the top third than AB females. In isolation, this finding could lead to the conclusion that wild zebrafish are more risk-taking, which is incorrect given this strain's avoidance of open areas. To conclude, our results suggest that the zMCSF presents a sophisticated behavioral tool that can distinguish between different magnitudes and types of risk, allowing the user to create an intricate behavioral profile of individual adult zebrafish.

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Standardized Method for the Assessment of Behavioral Responses of Zebrafish Larvae.

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