Name: behavioral approaches to studying innate stress in zebrafish
Uploaded: 2019-05-01T14:00:00
Description: Watch this Scientific Journal Video about Behavioral Approaches to Studying Innate Stress in Zebrafish at JoVE.com

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.

The protocol has been approved by the Institutional Animal Care and Use Committeeat Florida Atlantic University .
1. Preparation
<ol>
	<li>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.</li>
	<li>Move the following materials and equipment into the behavioral room: (i) a camera and lens, (ii) an infrared filte...

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<s...

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 studied extensively3,4,5,6. Despite these critical findings, much remains unknown about genetic and neuronal regulation of stress. As such, many stress related disorders suffer from a lack of therapeutics.
Genetically amendable model organisms provide a useful tool in the study of genetic and neuronal control of behavior. Fish models, in particular, are extremely powerful: they are small organisms with short generation times, their use in a laboratory setting is facile, their genomes are easily modified, and, as a vertebrate, they share not only genetic, but also neuroanatomical homology with their mammalian counterparts7,8. Standard assays for measuring stress can be paired with zebrafish lines harboring genetic mutations, or those in which manipulation of precise neuronal subsets is possible, and the effects of single genes or defined neurons can be assessed rapidly and efficiently.
Behaviorally, stress responses can be characterized in fish as periods of hyper-activity or prolonged periods of inactivity (akin to &#39;freezing&#39;)9, reduced exploration10, rapid breathing, reduced food intake11, and a place-preference for the bottom of a tank12. For example, when placed into an unfamiliar tank, adult zebrafish and other small fish models show an initial preference for the bottom half of the tank, yet, over time, the fish begin exploring top and bottom halves with near-equal frequency12. Treatment of adults with drugs known to reduce anxiety cause fish to explore immediately the top half10,13. Conversely, drugs that increase anxiety cause fish to show strong preference for the bottom half of the tank12,14,15. Thus, reduced exploration and preference for the bottom half of the tank are simple and reliable indicators of stress.
Like most vertebrates, stress responses in fish are driven by activation of hypothalamic-pituitary-inter-renal axis (HPI; analogous to the hypothalamic-pituitary-adrenal [HPA] axis in mammals)14,16. Hypothalamic neurons expressing the hormone corticotropin-releasing hormone (CRH) signal to the pituitary, which in turn releases adrenocorticotropic releasing hormone (ACTH). ACTH then signals to the inter-renal gland to produce and secrete cortisol, which has a number of downstream targets16, one of them being negative feedback of the crh-producing hypothalamic neurons3,17,18,19.
Here, we describe a method to assess behavioral measures of innate stress. For the behavior, we detail protocols using the novel tank diving test12,14. We then demonstrate, as an example, that a known anxiolytic drug, buspirone, reduces behavioral measures of stress.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Camera</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Infrared filter</td>
			<td style="width:264px;">Edmund Optics</td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Video Acquisition Program</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Infrared LED lights</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Assay tank</td>
			<td style="width:264px;">Aquaneering</td>
			<td style="width:181px;">Part number ZT180</td>
			<td style="width:380px;">Size: M3 1.8 liter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stand and clamp, or standard tripod for camera</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:217px;">250mL beaker</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Tracking software</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">We use Ethovision XT 13 from Noldus Information Technology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Buspirone chloride</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:181px;">B7148</td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Randomized trial generator</td>
			<td></td>
			<td></td>
			<td>We use the RANDBETWEEN function in Microsoft Excel</td>
		</tr>
	</tbody>
</table>

behavioral approaches to studying innate stress in zebrafish

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.

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...

Watch this Scientific Journal Video about Behavioral Approaches to Studying Innate Stress in Zebrafish at JoVE.com

Behavioral Approaches to Studying Innate Stress in Zebrafish

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 ...

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