The authors have nothing to disclose.

All procedures and behavioral testing were approved by MacEwan University&#8217;s Animal Research Ethics Board under protocol number 06-11-12, which is in compliance with the Canadian Council for Animal Care&#8217;s guidelines for the care and use of experimental animals.
1. Prepare Dosing Tanks, Solutions, and Administration Schedule
<ol>
	<li>Prepare an administration schedule such that the animals are dosed in the same environment and during the same time of day to avoid any confounds of time or biasing to visual stimuli.</li>
	<li>Obtain as many identical, 1.5 L, clear polypropylene spawning tanks as necessary for the number of group sizes. Use groups of 8 fish per tank, which allows for 2 groups of fish to be tested per day later on in the procedure (see step 3). Use one holding tank and one dosing tank per group (2x total number of groups).
	<ol>
		<li>Place 400 &#181;m spawning inserts in all holding tanks. Fill the tanks with habitat water or reverse osmosis water at the correct temperature (for zebrafish, 25-28 &#176;C) that is consistent with the temperature fish are normally housed at. 
		NOTE: There can be undesired chemical interactions between some drugs and the chemical components of buffered habitat water. In this situation, use reverse osmosis water buffered with minimal or no aquarium salts for drug administration, as well as for the control groups.</li>
		<li>Ensure that the tanks are in a neutral environment to avoid conditioning fish to external visual stimuli during dosing.</li>
	</ol>
	</li>
	<li>Prepare the drug solution. Mix the appropriate amount of the drug with habitat water in the spawning tanks. Prepare the 0.2% ethanol solution by combining 3 ml of a high grade ethanol (95% non-denatured ethanol) with 1,497 ml of water. Prepare the 1.4% ethanol solution by combining 21 ml of ethanol with 1,479 ml of water.</li>
</ol>
2. Netting Fish and Ethanol Administration Procedure
<ol>
	<li>Carefully net fish from their habitat tanks and transfer into the appropriate holding tank containing the spawning insert. Ideally, house the fish in the spawning insert to eliminate netting altogether.</li>
	<li>With all fish in their respective holding tanks, gently lift the spawning insert out of the holding tank and place it into the appropriate drug solution tank (Figure 1A).
	<ol>
		<li>Record the dosing time as required. Use 30 min in the ethanol solution for the procedure described here.</li>
		<li>If possible, have assistants help with the transfer of all experimental groups to the drug solution simultaneously to ensure precise dosing time. Alternatively, transfer one group at a time and keep track of individual groups&#8217; dosing times (Figure 1A).</li>
	</ol>
	</li>
	<li>At the end of the requisite dosing period, remove the fish from the ethanol solution by carefully lifting the spawning insert out of the drug solution and placing gently back into the holding tank.</li>
	<li>Gently net the fish in the holding tanks and place them back into their habitats until the next scheduled dosing time, or place the spawning insert back into the holding tank to eliminate netting.</li>
	<li>As mentioned previously, if possible within the parameters of animal housing equipment, house the animals in the same tank and spawning insert that is used as the holding tank. This will eliminate netting altogether during the administration procedure.</li>
</ol>
3. Behavioral Testing
<ol>
	<li>Obtain a light/dark arena 9.5 cm wide by 55 cm long and 9.5 cm deep with a white waterproof floor (Figure 1B). Affix white and black waterproof non-reflective paper to the inside walls of the arena using velcro, with half of the arena covered in white and half covered in black. Fill the arena to a depth of 5 cm with habitat water at a temperature of 25-28 &#176;C. Maintain this temperature throughout testing.</li>
	<li>Minimalize external visual stimuli by constructing a white three-sided enclosure for the arena to be situated in. Ensure the testing area has diffuse overhead lighting that does not cause reflections on the water surface, yet is sufficiently bright for the movement tracking software, or post-hoc manual quantification from video images.</li>
	<li>Place the arena in the enclosure and set the recording and movement analysis parameters of the behavior tracking software. Set the trial duration to 5-15 min, depending on the research question. 
	NOTE: Here, we used 5 min.</li>
	<li>Transport the group of fish to be tested to the research area in the habitat tank and place them outside of the arena enclosure. Acclimate the fish for 10 min.</li>
	<li>Gently net a fish from the appropriate group and place in the center of the light/dark arena, being sure to release the fish when it is positioned parallel to the long axis of the arena to avoid biasing the fish to the light or dark zone.</li>
	<li>Begin recording behavior immediately after the animal is released. Watch for any software problems with tracking the fish or for fish jumping or freezing. Rotate the arena 180&#176; after half of the subjects have been tested to prevent any confounds due to biases resulting from which end of the arena is oriented toward the open end of the enclosure.</li>
	<li>After the trial has ended, gently net and remove the fish from the arena to a holding tank or habitat tank.</li>
</ol>
4. Analysis
<ol>
	<li>Examine time spent in light versus dark zones. For each group and each fish, obtain the relative time spent in the light and dark zones and analyze using a one sample t-test (or Wilcoxon signed rank test for nonparametric data; difference from (half of the total trial time) 150 sec) to determine if groups significantly prefer one area over the other.</li>
	<li>To compare preferences, calculate a preference index by subtracting the time spent in the light zone from the time spent in the dark zone and compare differences between groups. t-tests can be used to compare two groups. Compare multiple groups with a one-way analysis of variance utilizing Tukey&#8217;s HSD post hoc test where necessary (or Kruskal-Wallis test with Dunn&#8217;s multiple comparison post hoc test for nonparametric data).</li>
	<li>Compare velocity, number of zone transitions, meandering, and immobility across groups. Use one-way analysis of variance utilizing Tukey&#8217;s HSD post hoc test where necessary (or Kruskal-Wallis test with Dunn&#8217;s multiple comparison post hoc test for nonparametric data).</li>
</ol>

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The authors acknowledge Joshua Gallup for use of his photography equipment used for Figure 1. This work was supported by a Natural Sciences and Engineering Research Council (NSERC) Canada Discovery grant (to T.J.H.).

Previous studies involving drug administration in zebrafish have simply relied on netting fish to transport them from their home tank into the drug solution12,16. Netting is not always consistent and often takes longer than expected due to the escape response of the zebrafish, which has significant individual variability. Traditional transfer methods, while useful, can be improved upon by decreasing the amount of total time the fish spend outside of water, as well as decreasing the amount of variability in transfer time between animals. In addition to improving practicality, this method gives researchers the ability to dose large groups of fish simultaneously in a solution for a precise amount of time. Previous methods require either dosing a single fish, or attempting to net whole groups of fish simultaneously. The former is slow and decreases throughput, whereas the latter is an awkward and difficult task. Furthermore, motion-tracking systems that can assess the behavior of multiple fish at once can benefit from the precision of this method. Fish could be transferred directly from the spawning insert into the shoaling arena, which would ensure near simultaneous exposure to the arena for all fish.
Utilizing this new method produced significant results when implemented on three groups of zebrafish over a 21 day period. Control fish received no alcohol, daily-moderate fish received a small amount of alcohol once per day, and weekly-binge fish received a large dose of alcohol once per week over the 21 day period. In the light/dark task, the dosing procedure did not change the normal preference of the zebrafish for the dark zone under control conditions. However, after 2 days of ethanol withdrawal, zebrafish in the daily-moderate group preferred the light zone of the arena, a reversal of preference. Statistical analysis confirms a significant change in zone preference. It is possible that the dosing procedure resulted in ethanol seeking behavior due to a conditioned place preference (CPP), with the fish seeking ethanol (an appetitive stimulus), and preferring the white zone due to its relative similarity to the bright dosing area. Recent research reveals that CPP can occur in zebrafish and in areas where zebrafish are dosed with ethanol17,18. Weekly binge-fish showed no preference 2 days post ethanol, and though evidence about neural mechanisms accounting for this finding is lacking, it is possible that infrequent but large doses of alcohol may damage the brain, possibly leaving fish unable to discriminate between zones.
Another advantage that this dosing procedure confers on researchers is its generalizability. As described, the procedure produces clear and useful results in zebrafish. However, the procedure is not limited solely to zebrafish. The constraints on the procedure are related to the size and limits of the dosing tanks, animals and testing apparatus, as well as ability to analyze data. With the use of motion tracking software, recent studies have investigated many additional variables throughout a trial. White avoidance habituation, squares crossed, locomotion habituation, latency to the white zone, erratic swimming, thigmotaxis (time spent near the arena walls), and risk assessment can also be quantified14. It is conceivable to use this procedure in other model organisms, such as goldfish, which have been used previously to investigate the role of tolerance and withdrawal19. Researchers hoping to investigate the effects of other pharmacological agents or environmental toxins, the use of other behavioral assays, or other marine or freshwater animals, could also utilize this basic methodology.
Using this protocol is simple and effective. However, if results are not as expected, consider troubleshooting the motion-tracking system to ensure proper data collection. Tracking should be smooth and accurate across the trials. The tracking system should be recording the movement of the body-center of the fish at all times. Any jumping or erratic movement of the tracking dot due to visual artifacts, such as light reflections on water ripples, or improper tracking settings may confound results. Some discrepancies in results may arise from small differences in arena design or dosing environments. To be sure of replication, the arena should be as described, particularly with respect to using a white floor to maximize tracking efficiency in zebrafish. With other fish that are lighter in color, a dark floor should be used to aid in motion tracking. Similarly, dosing the fish in an environment that is free from any visual stimuli that could possibly bias the fish is extremely important. Any distinct color, pattern or hue that is perceivable to the fish could confound results. A neutral, light colored background similar to that in the housing system is recommended.
While it is feasible to modify this method as necessary for other species of fish, results may vary in terms of innate light/dark preference20-21. It is also important to note even the procedure described here still causes some stress. Although netting is significantly reduced, the procedure still involves a very short period of time where the fish must be out of water, possibly inducing a stress response. Ramsay and colleagues22 found that netted zebrafish had at least double the levels of cortisol in their body compared to those that were not netted. Since cortisol is a hormone linked to stress11 this may alter the behavioral response that is measured. While our method reduces netting, it does not eliminate it. However, it gives researchers the opportunity to eliminate the incidence of netting if they desire by housing zebrafish in the spawning inserts. Future studies should investigate whether the use of the spawning insert for transfer of fish can decrease the stress response relative to netting. Additionally, it is crucially important to maintain precise dosing times and transfer times between groups. Changes in dosing times or large differences in time spent out of water could alter results. To reduce possible circadian effects, it is also critical to dose the fish during similar times of the day. In the current experiment fish were dosed and tested between 10 AM and 2 PM. Accurate and reliable replication of this procedure relies on repeating the protocol with precision.

The zebrafish (Danio rerio) is a small teleost species originating in India that is a useful model organism for behavioral1 and medical research2,3. Zebrafish are also commonly used in the testing of various pharmacological substances in order to characterize their impact on behavior. Various dosages and schedules of drug administration have been used to investigate the behavior of the zebrafish after the administration of compounds such as stimulants4, anxiolytics5 and ethanol6-8.
Our lab has investigated the effects of differing schedules of ethanol administration on zebrafish anxiety and locomotion in the well-validated light/dark assay9,20, also commonly referred to as the scototaxic assay. A new method of ethanol administration was developed to increase efficiency for repetitive, daily administration over a long period of time (21 days)6. Previously used methods were practical, however, we sought to develop a method that reduced netting, with its associated time costs, and allowed simultaneous, precisely timed administration of the drug of interest to large numbers of fish. In traditional research using ethanol, zebrafish are netted and transferred from one tank to another containing the appropriate mixture of ethanol and water10-12. While this method is widely accepted, netting zebrafish may increase the variability in the time taken to introduce and remove the fish from the drug solution. Therefore, the exact exposure to the compound of interest may vary over the course of an experiment involving repeated dosing. A method that reduces sources of error stemming from variability in transport times is thus desirable. With our method we are able to move all fish simultaneously, resulting in identical dosing time in each fish. Following ethanol exposure (described here), zebrafish can be tested in any number of behavioral assays, including those that assess anxiety. Dosing groups of fish using the new method has practical uses beyond the ability to accurately replicate and standardize dosing between subjects and across groups of fish. The advent of new software that allows for the tracking of multiple fish at once may see researchers utilize our methods to ensure replicability and accuracy in their experiments. Considering the widespread use of zebrafish as a model organism for behavioral neuroscience, this method will increase efficiency and practicality in future pharmacological studies.
In the present paradigm, a repeated dosing schedule was employed that approximately mirrors human drinking schedules. Fish were randomly assigned to one of three groups: control, daily-moderate, or weekly-binge. The dosing schedule was 21 days in duration, chosen because it significantly exceeded exposure times in previous studies7. Control fish received zero alcohol, daily-moderate fish received 0.2% alcohol once per day, and weekly-binge fish received 1.4% alcohol once per week. The light/dark task was used to assess anxiety after 2 days of withdrawal. This is a relatively simple test to administer which uses a rectangular arena in which the walls on one side are white and on the other side are dark9. Adult zebrafish robustly prefer the dark side of the arena under control conditions6,9,13. Increased anxiety is operationally defined as significantly more time spent in the dark zone, and decreased anxiety can be assumed when the fish spends relatively more time spent in the light zone. With motion-tracking software, other informative variables can also be quantified, including average velocity, immobility, meandering, and zone transitions14.
The dosing method developed in our laboratory can apply to any research in which water-soluble compounds are administered to one or more zebrafish. Many other pharmacological agents which may benefit from this methodology are currently being tested in zebrafish. Commonly tested compounds include nicotine, chlordiazepoxide, buspirone, and scopolamine, which are dissolved in a similar manner to ethanol; by mixing the appropriate amount of the chemical into water. Therefore, the general scope of this procedure is much wider and not limited to ethanol. Furthermore, after dosing with drugs for multiple days, the light/dark task is only one of many behavioral tests that could be employed. After drug administration or during withdrawal, other popular assays that can be utilized include the novel tank diving test15 and tests of social behavior such as shoaling16. The following procedure will outline an efficient method of repeatedly transferring groups of fish or individual fish into solutions containing a pharmacological compound of interest. Additionally, the process of testing anxiety with the light/dark test in groups of fish who are in withdrawal after being exposed to long schedules of alcohol administration will be described.

<table><tbody><tr><td>Three shelf benchtop housing system</td><td><a href="http://aquatichabitats.com/research-systems/z-hab-system/" target="_blank"><img src="/img/new_window.png" /> Aquatic Habitats</a></td><td>N/A</td><td></td></tr><tr><td>1.5 L Spawning tank w/400 &amp;mu;m baffle</td><td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/" target="_blank"><img src="/img/new_window.png" /> Aquatic Habitats</a></td><td>N/A</td><td></td></tr><tr><td>Pure Grain Ethanol</td><td><a href="http://www.luxco.com/brands/vodka-neutral-spirits-neutral-spirits-everclear" target="_blank"><img src="/img/new_window.png" /> Luxco, INC</a></td><td>N/A</td><td></td></tr><tr><td>Ethovision XT Motion tracking software</td><td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt" target="_blank"><img src="/img/new_window.png" /> Noldus Information Technology</a></td><td></td><td></td></tr><tr><td>Pipette</td><td><a href="http://eshop.eppendorf.ca/categories/1" target="_blank"><img src="/img/new_window.png" /> Eppendorf Canada</a></td><td></td><td></td></tr><tr><td>Light/Dark Arena</td><td>Custom</td><td></td><td>Construct as per procedure description. 9.5 cm wide, 9.5 cm deep, 55 cm long.</td></tr></tbody></table>

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.

To maintain accuracy and control in pharmacological studies with zebrafish it is important to time the duration of ethanol administration consistently and accurately as described above. Our procedure can increase the ease and throughput of the dosing procedure. The administration of ethanol on either a weekly-binge or daily-moderate schedule resulted in altered anxiety levels, measured with the light/dark test, compared to controls. When tested two days after the last dosing, zebrafish in the control group (who received no alcohol, but were still moved to dosing tanks) displayed the expected pattern of behavior, with control fish spending significantly more time on the dark side of the arena (Figure 2A) similar to naive zebrafish in other studies6,9,13. Fish in the weekly binge group showed no preference for either the light or dark zones in the task when tested two days after their last ethanol administration (Figure 2B). Interestingly, fish in the daily-moderate group significantly preferred the light side of the arena (Figure 2C); in contrast to the control group. The preference index indicated a significant difference between control and daily-moderate groups (Figure 2D). There were no significant differences in swimming speed, or immobility across groups (Table 1), and therefore, this effect was not due to a motor deficit in the fish.
<img alt="Figure 1" src="/files/ftp_upload/51851/51851fig1highres.jpg" /> 
Figure 1: Transfer Procedure and the Light/Dark arena. These photos illustrate the transfer procedure. (A) First, the fish are situated in the transfer tank (left). Dosing tank containing ethanol is pictured (right). (B-C) Moving the fish from the transfer tank to the ethanol solution requires the researcher to lift the spawning insert out of the transfer tank and into the ethanol solution tank. (D) The light/dark arena as illustrated measured 9.5 cm wide by 9.5 cm deep and 55 cm long. White flooring is used, along with black walls on half of the arena (left) and white walls on the other half, creating a light and dark zone.
<img alt="Figure 2" src="/files/ftp_upload/51851/51851fig2display.jpg" /> 
Figure 2: Representative results and trackplots of ethanol dosing procedure on three groups of zebrafish after 2 days withdrawal. (A) A representative trackplot of a single control zebrafish movement path throughout the 5 min light/dark trial. Below is the same zebrafish trackplot represented as a heatmap, which is a colored representation of zebrafish movement throughout the trial, based on the time the fish spent in the location represented by each pixel. (B) A representative trackplot of a single zebrafish from the weekly-binge group throughout the 5 min light/dark task. Below is the heatmap from the same zebrafish. (C) A representative trackplot of a single daily-moderate zebrafish movement throughout the 5 min light/dark trial. Below is the heatmap from the same zebrafish. (D) The preference index was calculated for all groups by subtracting time spent in the light zone from time spent in the dark zone. Negative numbers indicate preference for the dark zone. Positive numbers indicate preference for the light zone. Results indicate a significant difference in preference between control and daily-moderate groups at 2 days withdrawal *p &#60;0.05 (one-way ANOVA). Note there is also a significant preference for dark in the control group, p &#60;0.05 (one sample t-test, difference from 0), and a significant preference for light in the daily-moderate group, p &#60;0.05 (one sample t-test, difference from 0). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51851/51851fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="43" rowspan="2" style="height:42px;width:125px;"></td>
			<td style="width:89px;">Velocity</td>
			<td style="width:100px;">Immobility</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:89px;">(cm/sec)</td>
			<td style="width:100px;">(sec)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:125px;"></td>
			<td style="width:89px;">2-days Wd</td>
			<td style="width:100px;">2-days Wd</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:125px;">Control (n = 13)</td>
			<td style="width:89px;">9.1 &#177; 0.6</td>
			<td style="width:100px;">1.9 &#177; 0.6</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:125px;">Binge (n = 14)</td>
			<td style="width:89px;">9.8 &#177; 0.5</td>
			<td style="width:100px;">0.8 &#177; 0.2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:125px;">Chronic (n = 15)</td>
			<td style="width:89px;">10.3 &#177; 0.5</td>
			<td style="width:100px;">2.5 &#177; 1.0</td>
		</tr>
	</tbody>
</table>
Table 1: Average velocity and immobility during the light/dark test. The average swim velocity (cm/sec) and immobility (sec) of a representative group of zebrafish after 2 days of withdrawal (mean &#177; S.E.M). Here, no significant differences in either velocity or immobility were found. Used with Permission from Holcombe et al., (2013).

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.1K Views.  MacEwan University. 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.

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

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

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.

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 ...

Behavior

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 ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

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Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Immunology and Infection

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Biology

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

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Medicine

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

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Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

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

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