Zebrafish (Danio rerio) has been used as a New Alternative Model (NAM) for the last 40 years. This organism conserves approximately 70% of gene homology with humans, in addition to having the benefit of possessing fast development, high fecundity, feasible maintenance, and ease of manipulation. This NAM complies with the 3Rs principle (Replacement, Reduction and Refinement), since it is possible to perform multiple assays on embryos. The embryos present functionality in most organs at 5 dpf (day post-fertilization).
This makes zebrafish an ideal and cost and time effective model in areas such as biological development, neurophysiology, biomedicine, behavioral studies, toxicology and Early Drug Discovery.
Behavioral studies have been used with zebrafish in complex paradigms such as in scenarios related to memory, anxiety, stress, addiction, and the reinforcing properties of drug abuse. Among the various behavioral assays employed, the novel tank diving test (NTT) stands out as a valuable tool for investigating anxiety-related behaviors in zebrafish.
The Novel Tank Diving Test
The NTT is a behavioral assay designed to evaluate anxiety-like behaviors in zebrafish. Conceptually, it is similar to the open-field and elevated plus maze tasks, often used to measure exploration and anxiety-related behaviors in rodents. Thus, the NTT is based on zebrafish’s instinctive behavior to seek protection in novel environments. The test involves placing an individual in an unfamiliar, transparent tank and recording its behavior in vertical distribution over a specified period.
The tank is typically divided into zones, such as top, middle, and bottom, to facilitate behavioral analysis. When exposed to a novel environment, zebrafish initially dive down to the bottom and then gradually start exploring, first the bottom of the tank, then the top half. This gradual increase in exploration is interpreted as a reduction in anxiety. These typical behaviors that are correlated with an augmentation in the levels of anxiety include inhibited exploration, speed of locomotion, an increase in freezing behavior, and erratic movements. By observing such parameters zebrafish anxiety levels can be studied.
The NTT is quick to implement (5-10 minutes) and sensitive to pharmacological, genetic, and environmental manipulations. The automation of the assay allows a High-Content Screening (HCS) of compound candidates. The procedure typically involves the following steps:
- Habituation: Before the test, zebrafish are acclimated to the experimental room for a specified period. This minimizes stress due to sudden changes in the environment.
- Transfer to Novel Tank: The zebrafish is gently netted and transferred to the novel tank.
- Recording: A camera is used to record the fish's behavior for a predetermined duration, usually ranging from 5 to 10 minutes.
- Behavioral Analysis: The recorded video is then analyzed using specialized software to quantify various behavioral parameters, including time spent in different zones, locomotor activity, and freezing behavior.
Interpreting the Results
The NTT provides valuable information about zebrafish response to a novel environment, which is linked to anxiety-like behaviors. The zebrafish feels anxious when spending long periods of time at the bottom of the tank, and this behavior allows us to determine if a compound has an anxiolytic effect when the bottom-dwelling time is decreased. Common variables measured in a novel tank diving assay include:
- The latency to enter the top zone: It is the main measure of anxiety level and refers to the time that it takes the fish to enter the top zone for the first time. The longer the fish takes to enter the top zone, the higher anxiety level is interpreted.
- Time spent in each zone: Total time spent in each zone. A longer duration at the top of the tank indicates lower anxiety levels.
- The number of transitions: It reflects the number of crossings between the zones. More top entries indicate lower anxiety levels.
- Distance traveled in the top zone: More distance traveled in the bottom of the tank and less in the top indicates higher anxiety levels.
- Total distance traveled: The total distance the fish has traveled throughout the entire novel tank. It can reflect general motor/neurological phenotypes.
- Velocity: It reflects the activity of the fish and is expressed as the mean and the maximum speed of swimming.
- Mobility: Sometimes called activity, refers to the time that zebrafish spends swimming or is immobile (frozen).
- Erratic movements: Sharp changes in swimming direction.
These parameters can be interpreted to determine anxiety-like behavior. The time spent in different zones is used as a parameter because anxious fish tend to spend more time near the bottom, while less anxious fish explore the entire tank, including the upper regions. Freezing behavior such as cessation of movement is often associated with increased anxiety. On the other hand, highly anxious fish may exhibit erratic and unpredictable swimming patterns. It has been demonstrated that anxiolytic drugs or other anxiety-reducing manipulations decrease the latency to explore the top part of the aquarium. Thus, the fish show scarcer erratic movements, fewer and shorter freezing bouts, and they spend more time in the top part and less in the bottom.
Applications in Research
The NTT has found applications in a wide range of research areas, including:
- Drug Screening: It is used to evaluate the anxiolytic or anxiogenic effects of various compounds, providing insights into the Early Drug Discovery Phase of potential therapeutic interventions for anxiety-related disorders.
- Genetic Studies: Researchers employ this assay to investigate the genetic basis of anxiety-related behaviors by comparing wild-type zebrafish with genetically modified strains.
- Neurobiology: It offers a window into the neural circuits and molecular pathways underlying anxiety, aiding in the understanding of the brain's role in this behavior.
- Neuroimmunology: Investigating the interactions between the nervous and immune systems in zebrafish behavior can provide novel insights into the role of neuroinflammation in anxiety-related disorders.
- Toxicology: The test can be used to assess the potential neurotoxic effect of any chemical compound and the impact of environmental contaminants on anxiety levels in zebrafish, serving as a bioindicator for aquatic ecosystems.
The (NTT) in zebrafish has revolutionized the study of anxiety-related behaviors and has become an indispensable and reliable tool in behavioral research with applications in a wide range of research areas such as drug screening, genetic studies, neurobiology, neuroimmunology or toxicology.
The automation of the test allows a High-Content Screening of candidates. Its ability to reliably assess anxiety-like behaviors provides researchers with a valuable platform for studying a range of phenomena, from genetics to neurobiology with a steadfast commitment to ethical practices.
By understanding the biological underpinnings of anxiety-like behaviors in zebrafish, researchers are not only contributing to the fundamental knowledge of neuroscience but also paving the way for potential clinical applications and toxicology studies. These applications could include the development of novel therapeutics, personalized medicine approaches, or improved diagnostic tools for anxiety disorders. In relation to toxicology, the determination of the neurotoxicity of the many novel chemicals under development and a deeper knowledge of the neurotoxicity profile of already existing ones is a priority to establish their safety for industrial or consumption use.