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 filter which can be attached to the lens, (iii) a camera stand, (iv) a computer with camera acquisition software, (v) a firm and stable table to perform the assay on, (vi) infrared lights (IR lights; 850 nm or 940 nm), (vii) a white acrylic diffuser, which is longer than the length of the recording tank (viii) 1.8 L trapezoidal plastic assay tank (referred to as the &#39;novel tank&#39;; the one used here is 12 x 18 inches), and (ix) a bucket of fish system water. 
	NOTE: For the novel tank, our lab uses commercially available plastic vessels, which are trapezoidal in shape. The dimensions of the tank are roughly 6 in x 9 in (detailed dimensions are provided in Figure 1A). The diffuser board we use is slightly larger than the novel tank (12 in x 18 in). Novel tank experiments have been performed with tanks having differing shapes, such as those which are rectangular or those with different trapezoidal dimensions20,21. Typically, fish behavior is similar in all tanks regardless of their dimensions: for all containers, fish initially prefer the bottom half, yet over time begin exploring top half with greater frequency.</li>
	<li>Attach the infrared filter to the camera lens. The wavelengths of the infrared light strips typically range from 850 nm to 940 nm. The filter is a long pass filter that restricts light of wavelengths less than 720 nm from transmitting through to the camera.</li>
	<li>Select suitable parameters for the camera acquisition software. For most recordings, set the camera acquisition to a rate of 30 frames-per-second, and recording duration to 10-min. 
	NOTE: These parameters may differ, depending on the experiment. For example, to study habituation in a novel tank22,23, longer recordings may be required.</li>
</ol>
2. Setup
NOTE: The steps in this section describe setting up the novel tank assay. A diagram of the end product is given in Figure 1B.
<ol>
	<li>Place the novel tank in the middle of the table.</li>
	<li>Position the infrared lights behind the tank and place the white acrylic sheet or diffuser screen in between the tank and LED light source.
	<ol>
		<li>Place the diffuser so that it maximally spreads the light coming from the LEDs, and the intensity of the light is enough to illuminate the novel tank. The closer the board is to the light source, the brighter the lights will be, yet the less it will diffuse. By contrast, placing the diffuser board away from the light source will reduce light intensity, but spread the light better.</li>
	</ol>
	</li>
	<li>Fill approximately three quarters of the novel tank with fish system water. 
	NOTE: System water is generated using reverse osmosis of tap water, followed by dosing such that conductivity equals 900 &#177; 100 &#181;S, that pH is neutral (7.2), and that the temperature is 27 &#177; 1 &#176;C.</li>
	<li>Attach the camera to the camera stand and connect the camera to the computer. Open up the video acquisition software and adjust the camera to face the front of the tank and ensure the entire novel tank can be seen and that there are no obscured areas in the video. Adjust the tank and the infrared lights such that there is sufficient and even illumination throughout the tank when observed through the camera. 
	NOTE: Before proceeding to experiments, it can often be useful to perform a trial run, in which video of a fish is captured and tracking is performed. This will ensure that the setup is sufficient for experimentation of behavior.</li>
</ol>
3. Novel tank test setup
<ol>
	<li>Prepare a 250 mL beaker pre-filled with fish system water, and at least two holding tanks.</li>
	<li>On the morning of the test, transfer at least 10 test adult zebrafish to be used for each experimental condition (controls and experimental adults) from fish facility into a holding tank, transfer them to the behavior room, and allow them to acclimate for at least one hour. 
	NOTE: A power analysis should be performed before experimentation, yet in our hands, an n = 10 is usually sufficient to detect statistical significance. Moreover, the holding tank should contain no more than five individuals per liter of water. An acclimation of one hour is sufficient as zebrafish adults have been shown to habituate within 30 minutes of a new tank22. Also, behavioral rhythms are affected by circadian processes, and thus experimental replicates done on different days should be performed within the same hours. We typically perform all experiments between the hours of 11:00 am and 6:00 pm.</li>
	<li>Label the tanks such that the condition or genotype of the animals is blind to the experimenter. 
	NOTE: Experiments can easily be blinded to the experimenter by labeling tanks using a letter or number system (i.e., one tank is labeled &#39;A&#39;, another &#39;B&#39;, etc.). A party not involved in the experiments labels the tanks with such a system, and masks the identities from the experimenter until after post-analysis is complete.</li>
	<li>Using a net, gently place a single adult in the pre-filled beaker from step 3.1. Allow the adult fish to acclimate in the beaker for 10 minutes. 
	NOTE: Record the sex of the adult, as it might be important post-analysis to look for sex-specific differences.</li>
	<li>After acclimation in the beaker, introduce the fish into the novel tank (set up in section 1) by gently pouring out the water and adult from the beaker.</li>
	<li>After introducing the adult into the novel tank, start the camera recording, and move away from the setup to prevent additional distress to the fish.</li>
	<li>After the recording has finished, remove the individual from the novel tank and place into a new holding tank. 
	NOTE: A different holding tank from the one in step 3.2 should be used to prevent repeated testing on the same individuals.</li>
	<li>Repeat steps 3.4 to 3.7 for each adult until all animals have been tested. 
	NOTE: In addition to blinding conditions or genotypes, randomize trials. Use a random number generator or any tool that allows one to randomize between the trials. This should be done before experimentation so that each trial is determined before the experiments begin.</li>
	<li>At the end of all tests, return the fish back to the fish facility.</li>
</ol>
4. Pretreatment with drug
NOTE: The aim of the following steps is to compare the behavior of an individual before and after the use of drugs. This comparison is achieved by first performing a novel tank test as in step 3.4 to 3.6, followed by drug treatment, and then a second novel tank test (Figure 3A).
<ol>
	<li>Prepare a stock solution of the drug, including positive and negative controls. 
	NOTE: If the drug has previously been used in the literature, find an appropriate working dose and use this. For example, for buspirone in the representative results, we make a 100x stock solution and use 0.05 mg/mL as the final concentration, as described in the literature13,20. If suggested dose is unknown, a dose response curve should be performed by examining several concentrations. Set up more beakers with serial dilutions of drug. If the drug is not dissolvable in water, use dimethyl sulfoxide (DMSO) as a solvent.</li>
	<li>Dilute drugs to working concentration in 250 mL beakers with system water. For example, if a 100x solution was made, dilute 1:100 in system water. Set up a beaker with only system water as a control. 
	NOTE: If DMSO was used as a solvent in step 3.1, use an equal volume of DMSO in the control beaker.</li>
	<li>With the help of another researcher, mask the identities of the drug and control beakers to ensure that the tester is blind to the treatment conditions until post-analysis. 
	NOTE: A number or letter system may be used.</li>
	<li>Perform a novel tank test by following steps 3.1 to 3.6 to obtain a baseline behavioral stress response.</li>
	<li>After the baseline recording, use a net to immediately remove the adult fish from the novel tank and place it in the beaker dosed with drug or vehicle, as described in step 4.2. Allow the adult to stay in the beaker for 10 min. 
	NOTE: Ensure that the net does not touch the water in the beakers to prevent cross-contamination of drugs. Ensure proper dosage and administration time depending on the drug used. A 10 min treatment time might not work for all drugs.</li>
	<li>After treatment, use a net to remove the adult from the beaker in step 4.5 and place it in another beaker filled with fresh system water only. This is the washout period to minimize further dosing during the second novel tank test. Allow the adult to stay in the wash out beaker for an additional 10 min. 
	NOTE: Separate nets should be used for each drug condition to prevent any unwanted cross treatment with drug. The washout period may be skipped if the experimenter wishes.</li>
	<li>Perform the novel tank diving test a second time by removing that adult from the beaker in previous step, place it in a new novel tank, and follow steps 3.5 to 3.6.</li>
	<li>After the second novel tank test, remove the individual into a separate holding tank. Pour away the system water in the second novel tank and fill it with fresh system water for the next test. This step prevents cross-contamination of any drug. 
	NOTE: Depending on the half-life of the drugs, fresh beakers containing drugs should be made every 3 hours. For buspirone, make fresh solutions every 3 hours. In addition, following the note in step 3.8, the trials should be randomized between controls and drug treatments.</li>
	<li>At the end of all trials, return individuals back into the fish facility. 
	NOTE: Depending on type of drug used, the effects of these treatments on individuals can be long lasting. Therefore, do not use these individuals in other experiments.</li>
</ol>
5. Video analysis
<ol>
	<li>After all trials, load video files into tracking software of choice. 
	NOTE: We typically use commercially available tracking software, yet several freely available software packages can be used. The steps to achieve tracking may differ according to the software package used.</li>
	<li>Using a still frame from the video, define imaginary boundaries around (i) the entire novel tank arena that is filled with water, and boundaries around (ii) the top third, (iii) middle third, and (iv) bottom third of the tank. Use these to establish time that the fish spent in each portion of the novel tank.</li>
	<li>Calculate x-y displacement per frame for each arena defined in step 5.2.</li>
	<li>Define top, middle, and bottom areas of the tank. Each region should be similar in size. A brief method for determining these regions is to calculate the length of the tank in the y-direction, and divide this by 3. 
	NOTE: Variations to the general protocol do exist. For example, some labs use halves instead of thirds14.</li>
	<li>Determine the time spent in each arena, the distance, and the velocity. 
	NOTE: Most tracking packages will automatically calculate these for the user. However, if the tracking software does not, these can be calculated easily from the x-y displacement values. For example, distance can be determined using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/59092/59092eq1.jpg" /> 
	and velocity can be determined following the formula: 
	<img alt="Equation 2" src="/files/ftp_upload/59092/59092eq2.jpg" /></li>
	<li>Repeat steps 5.2 to 5.4 to acquire tracks and measurements for all trials. 
	NOTE: Variations to this general protocol</li>
</ol>
6. Testing for normality
<ol>
	<li>Perform statistics before proceeding to calculate statistical differences. Check to see if the data is normally distributed using a Shapiro-Wilk test.</li>
	<li>If the null hypothesis that the data is normally distributed is rejected (i.e., the data does not follow a Gaussian distribution), perform all tests using non-parametric tests. Conversely, if the data is found to follow a normal distribution, proceed to use parametric tests. 
	NOTE: We use commercially available software to perform statistics; however, the R programming language can also be used. A Shapiro-Wilk analysis can be performed using R&#39;s shapiro.test function.</li>
</ol>

The authors declare that they have no competing or financial interests.

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 fish12,14,21,35,36, and zebrafish has been shown to be a powerful model to examine the pharmacological effects of anxiety-related drugs. Similar to humans, these studies have demonstrated that drugs such as buspirone, nicotine, fluoxetine, and scopolamine have anxiolytic effects in zebrafish12,13,14,37. Moreover, drugs such as scopolamine that are typically not used to treat anxiety in humans can have additional anxiolytic effects37. Drug screens demonstrating these anxiolytic effects in zebrafish can facilitate the study of side effects and their pharmacological mechanisms. Further, the zebrafish has a comparable stress response pathway to humans, hence pairing the assay with quantification of the release of cortisol after a stressor or drug treatment can be used to validate the behavioral responses14. Finally, we wish to point out that this assay is not specific to zebrafish, and has also been extended to other fish species such as the Mexican blind cavefish, Astyanax mexicanus21. It is likely that the assay can be extended to cichlids38, mosquitofish39, killifish40, and other piscine systems.
An important advantage of the novel tank test is its ecological relevance; as the assay measures innate preference for the bottom half of the tank, the response is likely one that would occur in the wild. In addition to the novel tank test, other behavioral assays that have been used in other model organisms can be used to further validate the behavioral stress response, such as an open field test or a light/dark assay41,42. These assays are based on the tendency for an animal to follow the sides of the arena (thigmotaxis), and preference for exploration in the dark (scototaxis) after being exposed to a stressful cue42,43. In addition, electric shock has been used to measure either innate or conditioned fear responses9,44,45, though the ecological relevance of this approach is unclear.
When one is considering an assay for his/her study, it is important to take into account innate bias within strains or species. In addition to maintaining and reducing environmental fluctuations in behavioral assays, keeping the genetic background of the test adults consistent will be vital since research has shown variability within and between individuals of the same genotype41,46. A comprehensive review of the advantages, disadvantages, validity of each common behavioral assay used to study anxiety, and also variations in behavior within common wildtype lines can be found elsewhere41.
Zebrafish as a model for examining stress 
Zebrafish are becoming a popular model for examining genetic and neuronal pathways that modulate precise behaviors47,48, and recently developed brain atlases allow for mapping neurons regulating behavior with precision49,50,51,52,53. The approach we describe here to measuring innate anxiety is able to harness powerful genetic and neural circuits tools in zebrafish. Two foreseeable approaches rely on the large collection of mutant lines and transgenic driver lines. Mutant lines, for example, will facilitate investigators to examine the role that precise genes have in modulating stress. Additionally, transgenic Gal4/UAS and QF/QUAS system have been extensively applied to zebrafish54,55, and when crossed to UAS or QUAS effector lines, the function of precise neuronal circuits can be manipulated and behavior assessed. These approaches provide a complement to genetic mutant lines, and permit investigation of how precise neural circuits contribute to stress.
Novel techniques for examining neural activity can be fully integrated with this assay. Quantification of c-fos mRNA or protein are widely used to examine neuronal activity56. This gene is an immediate early gene, whose transcription is activated by neuronal activity. Newer approaches based on similar methodology have been developed. For example, the extracellular-signal-regulated kinase (ERK) was recently developed for examining neuronal activity in zebrafish50. The ERK protein exists in nearly all cells of the central nervous system. Upon neuronal activation, the ERK peptide become phosphorylated. Moreover, reliable antibodies for both un-phosphorylated ERK (total ERK, tERK) and phosphorylated ERK (pERK) have been developed and work well in zebrafish. Thus, by co-labeling with antibodies specific to tERK and pERK, neuronal activity can be reliably measured. Using this approach, adults that significantly display more bottom dwelling in the novel tank test can be removed after recording, stained for either c-fos or tERK/pERK, and resulting brain sections imaged.
Taken together, these approaches should facilitate a facile approach for dissecting the genetic and neuronal mechanisms underlying stress in zebrafish. Moreover, due to the high conservation of genetic and neuronal pathways in zebrafish and mammals, we expect these methods to reveal conserved mechanisms underlying stress behavior.

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.

<table><tbody><tr><td>Camera</td><td></td><td></td><td>We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics.</td></tr><tr><td>Infrared filter</td><td>Edmund Optics</td><td></td><td></td></tr><tr><td>Video Acquisition Program</td><td></td><td></td><td>Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set.</td></tr><tr><td>Infrared LED lights</td><td></td><td></td><td></td></tr><tr><td>Assay tank</td><td>Aquaneering</td><td>Part number ZT180</td><td>Size: M3 1.8 liter</td></tr><tr><td>Stand and clamp, or standard tripod for camera</td><td></td><td></td><td></td></tr><tr><td>250mL beaker</td><td></td><td></td><td></td></tr><tr><td>Tracking software</td><td></td><td></td><td>We use Ethovision XT 13 from Noldus Information Technology</td></tr><tr><td>Buspirone chloride</td><td>Sigma-Aldrich</td><td>B7148</td><td></td></tr><tr><td>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 activity was recorded for 10-min, and tracking was performed offline using commercially available software. Comparison of locomotor activity between the first and last minute showed dramatic differences (Figure 2A,B). When first introduced into the tank, fish spent the majority of the time in the bottom (Figure 2B), yet over time, adults had a gradual increase in the amount of time spent in the top (Figure 2B,C). Total duration spent in the top in the first minute compared to the last minute revealed significant differences (6.29 &#177; 8.21 s vs. 23.23 &#177; 9.02 s; paired t- test, p &#60; 0.05) (Figure 2C). By contrast, total distance traveled between first and last minute revealed no significant differences (440.4 &#177; 110.3 cm vs. 405.5 &#177; 49.71 cm; paired t- test, p = 0.375) (Figure 2D). Because innate preference was different between the first and last minute, and not distance traveled, we believe the change in behavior represents a stress response, and not merely a change in locomotor activity. These results demonstrate that zebrafish exhibit an easily measurable innate stress response. This approach also establishes a foundation to compare stress differences between different groups of animals, and assess the differences in stress between them.
Effects of anxiolytic drugs on stress behavior in zebrafish 
Zebrafish are a powerful system for screening drugs, since application of drug can be applied in non-invasive ways by simply adding to the water25,26,27. To validate that bottom dwelling in zebrafish represents an innate stress response, we compared behavior in groups of adult zebrafish tested before and after exposure to an anxiolytic drug; as a control, we handled a separate of adults similarly, yet applied only vehicle (system water) instead of drug. We used the 5HT1A receptor agonist buspirone, which is not a controlled substance and is prescribed to human patients suffering from generalized anxiety disorder28. Buspirone has been validated to cause reduction in behavioral stress responses in various fish and mammalian models10,13,21,29,30,31,32,33,34 . As described in the protocol, zebrafish were first recorded in the novel tank test, then retrieved and placed in a beaker of drug or vehicle for 10 min. Fish were then given a &#39;wash-out&#39; period, where they were placed in a new beaker for 10 min, and then re-recorded in the novel tank test (Figure 3A).
Analysis of locomotor paths revealed little difference before and after treatment for groups of adults exposed to vehicle alone (Figure 3B). By contrast, adults exposed to buspirone spent a large amount of time in the top compared to the locomotor paths of the same fish before drug exposure (Figure 3B,C). Quantification of duration of time spent in the top revealed no significant differences between pre- and post-treatment in control animals (183.9 &#177; 90.46 s before vs. 113.8 &#177; 81.88 s after; one-way ANOVA followed by Sidak&#39;s multiple comparisons test, p = 0.4254), yet animals exposed to buspirone spent significantly more time in the top relative to pre-treatment, and control individuals after treatment (Buspirone: 201.4 &#177; 49.95 s before vs. 552.2 &#177; 42.97 s after; one-way ANOVA followed by Sidak&#39;s multiple comparisons test, p &#60; 0.0001; Control vs. Buspirone post-treatment: p &#60; 0.0001.) (Figure 3C). To examine whether the differences were due to less locomotion in general, we measured total distance traveled. These data revealed no significant differences for any of the groups (4134 &#177; 601.9 cm before vs. 3471 &#177; 766 cm after for control; Kruskal-Wallis test, p &#62; 0.05; 3904 &#177; 301.3 cm before vs. 3644 &#177; 566.3 cm after for buspirone; Kruskal-Wallis test, p &#62; 0.05) (Figure 3D). Taken together, these data demonstrate that bottom dwelling in adult zebrafish is a measure of innate stress, and establish a foundation for further pharmacological experiments in adult zebrafish.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59092/59092fig1.jpg" /> 
Figure 1. Diagram of the novel tank setup. (A) Dimensions of the 1.8 L trapezoidal novel tank as seen from the recording side of the tank. (B) Diagram of the setup including positions of the infrared lights, camera, and barriers used to minimize human interference. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59092/59092fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59092/59092fig2.jpg" /> 
Figure 2. Examining innate stress responses in wild-type zebrafish. (A) Representative swim paths of an individual adult in the novel tank test in the first minute (left) and last minute (right) of a 10-min recording. Imaginary lines defining the top, middle, and bottom zones of the tank are indicated with dotted lines. (B) Quantification of total time spend in the top zone for each minute of the 10-min recording. (C &#38; D) Comparisons of total duration spent in the top zone (C) and total distance traveled (D) in the first and last minute of all trials. Paired t - tests were used for analysis since the data passed the Shapiro-Wilk test for normality. n = 5; *: p = 0.028. Error bars indicate standard error of the mean. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59092/59092fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59092/59092fig3.jpg" /> 
Figure 3. Examining the effects of anxiolytic drugs on stress behavior. (A) Schematic of experimental flow. (B) Representative swim paths pre- and post- treatment of an individual from a control individual treated with system water only, and another individual treated with buspirone in the novel tank test. Dotted lines define separation of top, middle, and bottom zones of the tank. Grey tracks represent the control individual, and blue tracks represent the buspirone-treated individual. (C &#38; D) Comparisons of total duration spent in the top zone (C) and total distance traveled (D) between control (Ctrl) and buspirone-treated (Busp) trials. A test for normality using the Shapiro-Wilk test was first done. Where the test for normality failed, Kruskal-Wallis test followed by Dunn&#39;s multiple comparisons test was used (C); and if the data passed normality, one-way ANOVA followed by Sidak&#39;s multiple comparisons test was used for analysis (D). n= 5 for each condition; ***: p = 0.001. Error bars indicate standard error of the mean. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59092/59092fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

behavioral-approaches-to-studying-innate-stress-in-zebrafish

Nanyang Technological University

Research

JoVE Journal

Behavior

8.9K Views.  Florida Atlantic University. 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.

Video: Behavioral Approaches to Studying Innate Stress in Zebrafish

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth&#8217;s magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

We describe a behavioral protocol designed to assess how zebrafish&#8217;s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed.

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Medicine

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPI&#160;assay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

Here we describe a protocol that utilizes commercially available automated systems to pharmacologically validate the prepulse inhibition (PPI) assay in larval zebrafish.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Biology

Light-Dark Preference Test: A Method to Study Anxiety-Related Behavior in Zebrafish

Source: Holcombe, A., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/51851/" target="_blank">A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal</a>. &#160;J. Vis. Exp.&#160;(2014)
The video describes the light-dark preference assay to study the anxiety-related behavior in zebrafish following the exposure to ethanol.

Source: Holcombe, A., et al. A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal. &#160;J. Vis. ...