A Simple Setup to Perform 3D Locomotion Tracking in Zebrafish by Using a Single Camera

Abstract

Generally, the measurement of three-dimensional (3D) swimming behavior in zebrafish relies on commercial software or requires sophisticated scripts, and depends on more than two cameras to capture the video. Here, we establish a simple and economic apparatus to detect 3D locomotion in zebrafish, which involves a single camera capture system that records zebrafish movement in a specially designed water tank with a mirror tilted at 45 degrees. The recorded videos are analyzed using idTracker, while spatial positions are calibrated by ImageJ software and 3D trajectories are plotted by Origin 9.1 software. This easy setting allowed scientists to track 3D swimming behavior of multiple zebrafish with low cost and precise spatial position, showing great potential for fish behavioral research in the future.Keywords: 3D locomotion; behavior; zebrafish; idTracker; ImageJ Zebrafish is well-known as an ideal experimental animal model in biomedical research, especially in the fields of developmental and genetic studies and drug discovery approaches [1][2][3][4]. It has become widely used within the field of pharmaceutical research and toxicology, in which ideally thousands of chemicals can be screened rapidly in vivo for therapeutic and toxic potential that are related to human disease susceptibility and risk [5]. In addition, zebrafish is also an excellent model for behavioral research because of their consistency and the rational refection of their mental and physical changes to new environments, and a comparable neural circuit system with high vertebrate counterparts [6][7][8]. Therefore, zebrafish has emerged as a promising new organism for research on anxiety due to their robust cortisol stress response, behavioral strain differences, and sensitivity to drug treatments or predators as well as their change in alarm pheromones [9][10][11][12]. There are various models commonly used to assess zebrafish behavior, include shoaling test [13][14][15], social preference [16,17], light-dark box [18][19][20], open-field [21][22][23], and novel tank [24,25] models. Among these models, they required accurate, reliable, and reproducible detection of the subject's spatiotemporal location. Previously, the manual quantification of animal behavior may have suffered systematic errors, leading to data misinterpretation [26]. In contrast, computational video-tracking technologies can record and analyze movements and optimize observation on multiple behavioral endpoints to reduce internal influence [27]. Moreover, another advantage of using the video-tracking approach is its ability to repetitively store, replay, and analyze recorded videos, instead of observing every behavioral endpoint with only human eyes [28,29].Based on the position of fish, computer-vision tracking can be classified into two-dimensional (2D) and three-dimensional (3D). Even though 2D tracking was feasible to analyze fish behavior,

Chronic Exposure to Low Concentration Lead Chloride-Induced Anxiety and Loss of Aggression and Memory in Zebrafish

“…Three-dimensional (3D) locomotion is a sensitive tool for the risk assessment of the alteration of swimming behavior after exposure to chemicals or pollutants [24]. It was found that 50 ppb PbCl 2 -treated zebrafish had the lowest average speed ( Figure 2B) and average angular velocity ( Figure 2C).…”

“…Three-dimensional locomotor activity was tracked at four time points, including 1, 4, 7, and 14 days after the exposure to PbCl 2 , according to a previously described protocol [24]. The 3D locomotion test of six fish was performed using an acrylic tank (20 cm × 20 cm × 20 cm dimension with 15 cm water level).…”

“…An infrared-sensitive Charged Coupled Device (CCD) (700-1000 nm detection window) with a maximum resolution of 1920 × 1080 pixels and 30 fps frame rate was used for video recording (3206_1080P module, Shenzhen, China). In this experiment, we recorded the average speed, average angular velocity, and meandering for one minute every hour and used idTracker software [61] to track fish movement trajectories according to our previously published method [24].…”

A Novel Function of the Lysophosphatidic Acid Receptor 3 (LPAR3) Gene in Zebrafish on Modulating Anxiety, Circadian Rhythm Locomotor Activity, and Short-Term Memory

“…These behavior tests were started with a three-dimensional (3D) locomotor activity test that tracked the detailed trajectories. This assay was reported as a highly sensitive method to evaluate the potential chemical toxicity in fish behavior [38,39]. After a 15-min acclimation in the experiment tank, the lpar3 KO fish showed similar behavior with the control fish in terms of their locomotor activity as indicated by similar average speed, freezing time movement ratio, and rapid movement ratio (Figure 2A,E,F).…”

“…The behavior tests were as follows: novel tank, shoaling, mirror biting, social interaction, predator avoidance, color preference, and three-dimensional (3D) locomotion tests. Three-dimensional locomotor activity was measured by using an acrylic tank (20 cm × 20 cm × 20 cm) with~6 L water according to a previously described protocol [39]. The novel tank, mirror biting, predator avoidance, social interaction, and shoaling tests were performed by a designed zebrafish tower consisting of ten trapezoid test tanks (28 cm × 5 cm × 15 cm) with~1.25 L water in each according to our previously published protocols [40].…”