The measurement of multiple behavior endpoints in zebrafish can provide informative clues within neurobehavioral field. However, multiple behavior evaluations usually require complicated and costly instrumental settings. Here, we reported a versatile setting that applied ten acrylic tanks arranging into five vertical layers and two horizontal columns to perform multiple behavior assays simultaneously, such as the novel tank diving test, mirror-biting test, social interaction, shoaling, and predator escape assay. In total, ten behavioral performance were collected in a single video, and the XY coordination of fish locomotion can be tracked by using open source software of idTracker and ImageJ. We validated our setting by examining zebrafish behavioral changes after exposure to low dose ethanol (EtOH) for 96 h. Fish were observed staying longer time at bottom of the tank, less mirror biting interest, higher freezing time, less fear in predator test, and tight shoaling behaviors which indicated the anxiogenic effect was induced by low dosage exposure of EtOH in zebrafish. In conclusion, the setting in this study provided a simple, versatile and cost-effective way to assess multiple behavioral endpoints in zebrafish with high reliability and reproducibility for the first time.

Zebrafish has an advantage of optical clarity within the body during its early developmental stage. Therefore, organs, including the heart, can be observed and analyzed in a non-invasive manner. Previously, heart rate measurement was analyzed by a complicated system, programming language, fluorescence transgenic zebrafish, or expensive setup that was not feasible and practical. In this study, we established a simple dissecting microscope with Charge-coupled Device (CCD) to capture the signal of heartbeat, and used ImageJ software as an open source platform to analyze cardiac rhythm for the atrium and the ventricle. We subjected astemizole to zebrafish embryos, and applied our apparatus to precisely detect heart arrhythmia (Atrium-Ventricle block) induction. In other words, we provided a simple, economical, and reliable method to measure cardiac rhythm in zebrafish embryos, which is able to evaluate whether cardiac rhythm is affected by chemical compounds.

In this study, we evaluated the acute (24, 48, 72, and 96 h) and chronic (21 days) adverse effects induced by low doses (0.1, 0.5, 1, and 1.5 mg/L) of zinc chloride (ZnCl2) exposure in adult zebrafish by using behavioral endpoints like three-dimensional (3D) locomotion, passive avoidance, aggression, circadian rhythm, and predator avoidance tests. Also, brain tissues were dissected and subjected to analysis of multiple parameters related to oxidative stress, antioxidant responses, superoxide dismutase (SOD), neurotoxicity, and neurotransmitters. The results showed that ZnCl2-exposed fishes displayed decreased locomotor behavior and impaired short-term memory, which caused an Alzheimer’s Disease (AD)-like syndrome. In addition, low concentrations of ZnCl2 induced amyloid beta (amyloid β) and phosphorylated Tau (p-Tau) protein levels in brains. In addition, significant induction in oxidative stress indices (reactive oxygen species (ROS) and malondialdehyde (MDA)), reduction in antioxidant defense system (glutathione (GSH), GSH peroxidase (GSH-Px) and SOD) and changes in neurotransmitters were observed at low concentrations of ZnCl2. Neurotoxic effects of ZnCl2 were observed with significant inhibition of acetylcholine (ACh) activity when the exposure dose was higher than 1 ppm. Furthermore, we found that zinc, metallothionein (MT), and cortisol levels in brain were elevated compared to the control group. A significantly negative correlation was observed between memory and acetylcholinesterase (AChE) activity. In summary, these findings revealed that exposure to ZnCl2 affected the behavior profile of zebrafish, and induced neurotoxicity which may be associated with damaged brain areas related to memory. Moreover, our ZnCl2-induced zebrafish model may have potential for AD-associated research in the future.

Leptin, a hormone secreted by peripheral adipose tissues, regulates the appetite in animals. Recently, evidence has shown that leptin also plays roles in behavioral response in addition to controlling appetite. In this study, we examined the potential function of leptin on non-appetite behaviors in zebrafish model. By using genome editing tool of Transcription activator-like effector nuclease (TALEN), we successfully knocked out leptin a (lepa) gene by deleting 4 bp within coding region to create a premature-translation stop. Morphological and appetite analysis showed the lepa KO fish display a phenotype with obese, good appetite and elevation of Agouti-related peptide (AgRP) and Ghrelin hormones, consistent with the canonical function of leptin in controlling food intake. By multiple behavior endpoint analyses, including novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm, and color preference assay, we found the lepa KO fish display an anxiogenic phenotype showing hyperactivity with rapid swimming, less freezing time, less fear to predator, loose shoaling area forming, and circadian rhythm and color preference dysregulations. Using biochemical assays, melatonin, norepinephrine, acetylcholine and serotonin levels in the brain were found to be significantly reduced in lepa KO fish, while the levels of dopamine, glycine and cortisol in the brain were significantly elevated. In addition, the brain ROS level was elevated, and the anti-oxidative enzyme catalase level was reduced. Taken together, by performing loss-of-function multiple behavior endpoint testing and biochemical analysis, we provide strong evidence for a critical role of lepa gene in modulating anxiety, aggression, fear, and circadian rhythm behaviors in zebrafish for the first time.

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,