Optimizing assays of zebrafish larvae swimming performance for drug discovery

Abstract

Introduction: Zebrafish larvae are one of the few vertebrates amenable to large-scale drug discovery screens. Larval swimming behavior is often used as an outcome variable and many fields of study have developed assays for evaluating swimming performance. An unintended consequence of this wide interest is that details related to assay methodology and interpretation become scattered across the literature. The aim of this review is to consolidate this information, particularly as it relates to high-throughput approaches.

Areas covered: The authors describe larval swimming behaviors as this forms the basis for understanding their experimentally evoked swimming or spontaneous activity. Next, they detail how swimming activity can serve as an outcome variable, particularly in the multi-well formats used in large-scale screening studies. They also highlight biological and technical factors that can impact the sensitivity and variability of these measurements.

Expert opinion: Careful attention to animal husbandry, experimental design, data acquisition, and interpretation of results can improve screen outcomes by maximizing swimming activity while minimizing intra- and inter-larval variability. The development of more sensitive, quantitative methods of assessing swimming performance that can be incorporated into high-throughput workflows will be important in order to take full advantage of the zebrafish model.

Keywords: Danio rerio; Zebrafish; biomechanics; drug development; locomotion; swimming.

Figure 1:. Locomotor milestones attained during zebrafish larvae development.

Note that embryos are capable of moving very early in development, well before 24 hpf. By the time they hatch, usually between 48–72 hpf, the neuromuscular system has developed to the point where larvae will swim in response to touch. After another day or two of development, larvae show proficient spontaneous and evoked swimming behaviors. Times are based on an incubation temperature of 28.5°C.

Figure 2:. Stages of an escape response.

In Stage 1, the C-start, the larvae bends to draw the head and tail together (A to B to C). Stage 2 consists of a rapid counter-bend of the tail (C to D), producing the power stroke. The power stroke is followed by cycles of burst swimming, of which only the initial cycle is illustrated here (D to E to F).

Figure 3:. Activity of zebrafish larvae in a lighted environment.

Spontaneous swimming activity was measured in an infra-red activity monitor (DanioVision, Noldus Information Technology, Wageningen, the Netherlands). Larvae (6 days post-fertilization) were studied in 48-well plates at 28°C that were equilibrated in the monitor for 20 minutes in the dark prior to data collection. Following equilibration, the illumination was switched on and the larvae filmed at 25 frames/s for 10 minutes. Two-dimensional coordinates of the larval center of mass were determined using EthoVision software. Dependent variables were derived from this data set using custom scripts written in R. A) Top: Activity tracks of 4 representative larvae during the 10 minute lights on protocol. Bottom: The average distance covered by all 48 larvae for each minute of the protocol. Values are mean ± SE. B) Cumulative distance (calculated every 500 ms) across the protocol. Each line represents an individual larvae. C) The total distance covered by each larva derived from the data in Panel B. The total distance is a function of, D) the time that larvae were engaged in activity (active time), and E) their average velocity while active (mean active velocity). F) The coefficient of variation for cumulative distance, active time, and mean active velocity. In panels C, D, and E the horizontal dashed line indicates the mean.

Figure 4:. Activity of zebrafish larvae during a 15:9 hr light-dark cycle in different diameter wells.

Spontaneous swimming activity was measured in a DanioVision infra-red activity monitor. Wild-type larvae (5 days post-fertilization, dpf) were studied in 24-, 48-, and 96-well plates. At 5 dpf, 3.30 pm (EST) the illumination was switched on for 7 hours, followed by a 9-hour period of darkness, and a 8-hour period of light. Larvae were filmed at 30 frames/s, and the two-dimensional coordinates of the larval center of mass were determined using EthoVision software. The graph represents the average distance covered by all 24 larvae per plate for each hour of the protocol. Values are mean ± SE.

Figure 5:. Activity of zebrafish when exposed to a dark environment.

Immediately upon the conclusion of the protocol outlined in Figure 3, the visible illumination in the activity tracker was switched off and the activity of the larvae recorded for another 10 minutes. Data were processed as described in the legend to Figure 3. A) Top: Activity tracks of 4 larvae during the dark protocol. The larvae shown are the same larvae illustrated at the top of Figure 3-A. Bottom: Mean (±SE) distance covered each minute during the dark protocol. B) Cumulative distance across the 10 minute dark protocol. Each line represents an individual larva. C) Total distance covered by each larva. D) The total time that each larva was active. E) The mean active velocity of each larva. F) The coefficient of variation for total distance, active time, and mean active velocity for the protocol conducted in the light and in the dark. In panels C, D, and E the horizontal dashed line indicates the mean.