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This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.
Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.
Stress responses are altered behavioral and physiological states resulting from potentially harmful or aversive stimuli. Stress responses are conserved throughout the animal kingdom, and are critical for the survival of an organism1. Decades of research have greatly expanded our knowledge of some of the genetic and neuronal mechanisms underlying stress states. Today, areas of the brain such as the amygdala and the striatum2, and genetic factors such as corticotropin releasing hormone (crh), and the glucocorticoid (gr) and mineralocorticoid receptors (mr) have been stu....
The protocol has been approved by the Institutional Animal Care and Use Committeeat Florida Atlantic University .
1. Preparation
Examining stress in zebrafish
To examine stress behavior over time in wild-type zebrafish, we tested adult fish from the AB strain24 in the novel tank test. AB adults were subjected to the protocol as described above. Briefly, fish were given a 1-h acclimation period in a tank in the behavior room. An individual was placed in a beaker for 10-min, and then placed gently in an unfamiliar tank (novel tank) filled with fresh system water. Locomotor activ.......
Zebrafish exhibit a robust stress response in a novel tank
Here, we describe a simple behavioral approach for examining stress responses in adult zebrafish, and validate the approach as a simple measure of stress using pharmacology.
The novel tank test is a widely used test for examining innate stress in zebrafish and other species of fish12,14,21,35
This work was supported by funding from the Jupiter Life Science Initiative at Florida Atlantic University to ERD and ACK. This work was also supported by grants R21NS105071 (awarded to ACK and ERD) and R15MH118625 (awarded to ERD) from the National Institutes of Health.
....Name | Company | Catalog Number | Comments |
Camera | We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics. | ||
Infrared filter | Edmund Optics | ||
Video Acquisition Program | Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set. | ||
Infrared LED lights | |||
Assay tank | Aquaneering | Part number ZT180 | Size: M3 1.8 liter |
Stand and clamp, or standard tripod for camera | |||
250mL beaker | |||
Tracking software | We use Ethovision XT 13 from Noldus Information Technology | ||
Buspirone chloride | Sigma-Aldrich | B7148 | |
Randomized trial generator | We use the RANDBETWEEN function in Microsoft Excel |
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