Behavioral Approaches to Studying Innate Stress in Zebrafish

Summary

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.

Abstract

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.

Introduction

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....

Protocol

The protocol has been approved by the Institutional Animal Care and Use Committeeat Florida Atlantic University .

1. Preparation

1. Designate an isolated room for performing behavioral studies, or close off a section of a room so that it is isolated.
   NOTE: The room should be undisturbed and have low traffic to avoid disrupting normal behavior of the fish.
2. Move the following materials and equipment into the behavioral room: (i) a camera and lens, (ii) an infrared filte.......

Discussion

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 fish^12,14,21,35

Materials

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