We thank Sean Pelkowski for assistance in optimization of the behavioral assay. This work was supported by the National Institute of Child Health and Human Development, R01 HD060647 and the National Institute of Environmental Health Sciences, F32 ES021342 and R03 ES017755.

1. Collection of Zebrafish Embryos and Raising Larvae
<ol>
	<li>Glass Pyrex dishes with "fake" grass (made from green yarn) (Figure 1) should be inserted into the tanks at dawn and left in for two hours in order to collect zebrafish embryos. The glass dishes containing the embryos should be poured over a handheld strainer and rinsed with deionized water. The embryos should then be grown in egg water. The egg water contains 60 mg/L of Instant Ocean in deionized water and 0.25 mg/L of methylene blue, which is used as a mold inhibitor.</li>
	<li>Depending upon the hypothesis of the individual experiment, embryos can be treated immediately or during specific stages of development using toxicants or pharmaceuticals. The toxicants and pharmaceuticals are usually dissolved in DMSO (at a 1,000X concentration) and should be further diluted directly in the egg water medium.</li>
	<li>During embryonic and larval treatment with pharmaceuticals or toxicants, the larvae and embryos can be housed in deep Petri dishes at a density of about 50-60 larvae per 50 ml until the behavioral analyses at 7 dpf ( egg water solution should be changed at least every other day to avoid fungal/bacterial growth from dead embryos).</li>
</ol>
2. Preparing Molds for Behavioral Analyses
<ol>
	<li>Specially developed plastic molds which measure 11.7 cm x 7.6 cm x 5 mm were custom-built in-house. The molds are needed to create lanes using agarose which is poured into single well plastic plates from Thermo Scientific. The single well plates measure 12.4 cm x 8.1 cm x 1.2 cm.</li>
	<li>The molds contain five lanes in which the sides are angled at 60&deg;. The lanes in the molds are 3.5 mm high with a base of 18 mm at the top which is the widest whereas the bottom width is 14 mm. There is a 4 mm gap between lanes in the mold (Figure 2).</li>
	<li>To prepare the lanes pour 50 ml of melted agarose (0.8% agarose in deionized water with 60 mg/L Instant Ocean) in a single well plate. The mold should then be placed very slowly on top of the liquid agarose to eliminate any bubble formation and can be removed when the agarose has cooled (which takes roughly 45 min).</li>
	<li>The agarose lanes should be made no earlier than one day before the behavioral experiment is to be run (to avoid the agarose drying out) The lanes can be stored at room temperature with the lids on the dishes for up to roughly 36 hr. The agarose lanes should only be used for one experiment and should then be discarded.</li>
</ol>
3. Image Capture
<ol>
	<li>Up to 20 larvae can be placed in each lane of the plates. Typically 5 larvae per lane are used to facilitate the most accurate tracking of swim speed and to reduce the number of larvae that are needed per experiment. The lanes can be filled with egg water with or without pharmaceuticals or toxicants depending upon the experiment. However, lanes should not be filled up all the way until they are placed in the imaging cabinets; this will prevent overflow. For consistency, the larvae should have an acclimatization period of ten minutes after they are placed in the agarose molds and positioned on top of the laptop screen. Efficiently moving the larvae from the Petri dish to the agarose lane will help to reduce larvae stress. This is easiest when the larvae are housed in shallow tanks or Petri dishes.</li>
	<li>The imaging cabinets include a Canon digital camera used for time-lapse photography and a laptop. The camera should be placed at the top of the cabinet and aimed towards to the bottom of the cabinet where a 15.6 inch screen laptop should be placed with screen facing up (Figure 3). Four plates should be positioned by hand directly on top of the laptop screen. At this time the lanes can be topped off with egg water or chemical treatment so that it is level with the top of the lane (to eliminate shadows on the edges of the lanes in the images).</li>
	<li>A PowerPoint presentation is used as an aversive stimulus for the larvae. In the past moving red balls were shown to zebrafish larvae in 6 or 12 multi-well plates 15,17,18. The current PowerPoint starts with a blank white background for 15 min, followed by 15 min of a moving red bar on the top half of the plate (Figure 4). In order to eliminate overheating the larvae, it is best to purchase a laptop with a screen temperature that does not go above 28 &deg;C. To avoid evaporation of the liquid within the agarose lanes, the maximal imaging time should be kept to below one hour.</li>
	<li>The digital camera should be programmed for time-lapse photography, taking pictures every 6 sec for a total of 300 images per experiment. However, the frequency and length can be adjusted depending on the experiment and behavioral quantification. In the past extended imaging times were employed using longer intervals between each image. The camera can be set at a lower resolution for imaging at video speed (30 frames per second). While the lower resolution limits the recordings to a single multiwell plate, the video recordings are appropriate for imaging rapid swimming events 15.</li>
</ol>
4. Image Analysis
<ol>
	<li>The images should be opened with ImageJ and used with a macro that was specifically written in-house. The macro automatically splits the color channels so that the red color can be removed, subtracts the background, applies a threshold, and identifies the larvae by particle analysis. The most current macro for five lane larval analysis is Zebrafish_macro25k. Use the prompts in the macro to set number of images, color to be subtracted, image threshold, etc.</li>
	<li>After all of the images are run through the ImageJ macro, a results file will be displayed and will contain x,y coordinates of the individual larvae for each image along with the image number and the lane number.</li>
	<li>The results file should be saved in an Excel format and sorted based upon blank background vs. moving bar background and then the well number. An Excel template should be used that has equations built in that automatically determines placement of larvae in the wells, distance between larvae, speed of movement, and amount of rest. The most current Excel template is 25ib created in the Creton lab, which is available upon request. Graphs showing various treatment groups should be built into the Excel sheet along with t-tests for comparison between treatment groups and controls. Further statistical analysis can be performed using SPSS.</li>
</ol>

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	<li>Gerlai, R., Lahav, M., Guo, S., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drinks+like+a+fish:+zebra+fish+(Danio+rerio)+as+a+behavior+genetic+model+to+study+alcohol+effects.">Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects.</a> Pharmacol. Biochem. Behav. 67, 773-782 (2000).</li><li>Selderslaghs, I. W. T., Hooyberghs, J., De Coen, W., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+activity+in+zebrafish+embryos:+A+new+method+to+assess+developmental+neurotoxicity.">Locomotor activity in zebrafish embryos: A new method to assess developmental neurotoxicity.</a> Neurotoxicol. Teratol. 32, 460-471 (2010).</li><li>Norton, W., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+as+a+model+organism+for+behavioural+genetics.">Adult zebrafish as a model organism for behavioural genetics.</a> BMC Neuroscience. 11, 90 (2010).</li><li>Levin, E. D., Cerutti, D., Buccafusco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+15.">Ch. 15.</a> Methods of behavioral analysis in neuroscience. , (2009).</li><li>Kimmel, C., Ballard, W., Kimmel, S., Ullmann, B., Schilling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. , 203-253 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Kokel, D., Bryan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+behavior-based+identification+of+neuroactive+small+molecules+in+the+zebrafish.">Rapid behavior-based identification of neuroactive small molecules in the zebrafish.</a> Nat. Chem. Biol. 6, 231-237 (2010).</li><li>Rihel, J., Prober, D. 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Protoc. 5, 1786-1799 (2010).</li><li>Cachat, J., Stewart, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Neurophenotyping+of+Adult+Zebrafish+Behavior.">Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior.</a> PLoS ONE. 6, e17597 (2011).</li><li>Sledge, D., Yen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+duration+of+exposure+for+developmental+chlorpyrifos-induced+neurobehavioral+toxicity.">Critical duration of exposure for developmental chlorpyrifos-induced neurobehavioral toxicity.</a> Neurotoxicol. Teratol. 33, 742-751 (2011).</li><li>Stewart, A., Wu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+modulation+of+anxiety-like+phenotypes+in+adult+zebrafish+behavioral+models.">Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 35, 1421-1431 (2011).</li><li>Eddins, D., Cerutti, D., Williams, P., Linney, E., Levin, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+provide+a+sensitive+model+of+persisting+neurobehavioral+effects+of+developmental+chlorpyrifos+exposure:+comparison+with+nicotine+and+pilocarpine+effects+and+relationship+to+dopamine+deficits.">Zebrafish provide a sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: comparison with nicotine and pilocarpine effects and relationship to dopamine deficits.</a> Neurotoxicol. Teratol. 32, 99-108 (2010).</li><li>Emran, F., Rihel, J., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+assay+to+measure+responsiveness+of+zebrafish+to+changes+in+light+intensities.">A behavioral assay to measure responsiveness of zebrafish to changes in light intensities.</a> J. Vis. Exp. (20), e923 (2008).</li><li>Pelkowski, S., Kapoor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+imaging+system+for+automated+analyses+of+avoidance+behavior+in+zebrafish+larvae.">A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.</a> Behav. Brain Res. 223, 135-144 (2011).</li><li>Richendrfer, H., Pelkowski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+developmental+toxicity+by+automated+analyses+of+behavior+in+zebrafish+larvae.">Assessment of developmental toxicity by automated analyses of behavior in zebrafish larvae.</a> Unpublished observations. , (2012).</li><li>Richendrfer, H., Pelkowski, S., Colwill, R. M., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+edge:+pharmacological+evidence+for+anxiety-related+behavior+in+zebrafish+larvae.">On the edge: pharmacological evidence for anxiety-related behavior in zebrafish larvae.</a> Behav. Brain Res. 228, 99-106 (2012).</li><li>Richendrfer, H. A., Pelkowski, S., Colwill, R., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+sub-chronic+exposure+to+chlorpyrifos+reduces+anxiety-related+behavior+in+zebrafish+larvae.">Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.</a> Neurotoxicol. Teratol. , (2012).</li><li>Egan, R. J., Bergner, C. 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The authors declare that they have no competing financial interests.

While we are continually improving our novel behavioral assay, it has always been useful for the detection of avoidance and thigmotaxis behavior in zebrafish larvae 15. Many trials have been performed to optimize the results of the assay, such as color of stimulus used, ideal number of larvae per lane, and length of behavioral assay. Previously, we used multi-well plates (with 6 or 12 wells) 15,17,18. However, recently we have created the novel lane mold to create a larger swimming space for the larvae allowing us to gather a larger number of behavioral measures in a single assay 16 (Figure 5). Other modifications include variations of the PowerPoint shown (altered movement or length of assay) and the size of the lanes used (we also have molds for more narrow lanes).
Currently, this high-throughput automated system is unique in its ability to measure a large range of behaviors in zebrafish larvae at the same time such as speed, avoidance, proximity to other larvae, and thigmotaxis in multi-lane plates. Results can be obtained quickly and a large number of larvae can be analyzed at the time of imaging. The system is both inexpensive to build and quick and easy to set up. A limitation of this system is that 3-D movements cannot be assessed in the zebrafish larvae. Automated systems that track adult zebrafish have the 3-D capability and can identify a wider range of behaviors such as movement up or down within the water column 10,19. Another limitation is that our imaging system is currently not optimized for high-throughput analyses at video speed. Video speed imaging is possible when setting the camera to a lower resolution 15, but this restricts the analysis to a single plate.
In using the newly created "lane" method, several parts of the assay needed to be executed in a precise manner. When placing the larvae in the lanes, it is critical to make sure the level of liquid is very shallow until the plates are positioned on top of the laptop screen. If the lanes are too full of liquid, the larvae will escape into the periphery of the plate. In addition, when inserting the mold into the agarose, care must be taken to lower the mold very slowly. If the mold is inserted too quickly, bubbles will form in the agarose and will be identified by the Image J macro as additional larvae. It is advised that if the agarose lanes have even a few bubbles, it is best to make new ones.
In the future, we would like to optimize our behavioral assay to analyze other complex behaviors such as learning in zebrafish larvae and examine how learning may be affected by exposure to toxicants and pharmaceuticals in early development. We are currently working on assays that may be useful for analyzing learning behavior in which the behavioral results may facilitate determining which brain areas are affected by certain toxicants or pharmaceuticals during development. Automated assays have been developed for measuring learning behaviors in zebrafish larvae 20 and these assays may be amendable for high-throughput screening by using the robust avoidance response in multi-lane plates.
We propose that this behavioral assay could be used in future studies for testing the developmental effects of a large number of pharmaceuticals and toxicants. Such studies would provide a wealth of information on specific risk factors and contribute to setting better health and safety guidelines for pregnant women and children.

Zebrafish are becoming a popular model for genetic, developmental and behavioral sciences 1-4. They hatch from their chorions by 2-3 days post fertilization (dpf), develop fully functioning organs by 4-5 dpf, and exhibit a large number of behaviors by 7 dpf 5,6. Zebrafish larvae are ideally suited for high-throughput analyses because of their small size 7,8. Software is commercially available for automated analyses of behavior in larval and adult zebrafish 9-14. However, this software can be expensive and has limited options for measuring complex behaviors of zebrafish larvae in multi-well plates.
We created a novel high-throughput imaging system that is inexpensive to set up and can quantify a number of different behaviors in 7 dpf zebrafish larvae 15,16. The system allows us to quickly and efficiently test subtle behavioral abnormalities after embryonic exposure to a number of pharmaceuticals and environmental toxicants 16-18.
The system was built using wooden cabinets, which house a digital camera at the top of the cabinet. The camera faces downward to the bottom of the cabinet where a laptop is placed with the screen facing up 15. Time lapse imaging is used to capture the placement of the larvae in the lanes. Larvae can be housed in up to four multi-well or multi-lane plates which are positioned on top of the laptop screen. We use a PowerPoint presentation as an aversive stimulus to which the larvae respond by moving away (avoidance) and swimming towards the edge (thigmotaxis) 15,17. The images are imported into ImageJ in which an automated macro is used to split the color channels, subtract the background, and apply a threshold to identify individual larvae. Coordinates are listed for each larva in each image and can be inserted into an Excel file which we use to quantify avoidance and thigmotaxis behavior, fish to fish distance, swim speed, and amount of rest 16.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Instant Ocean</td><td>That Pet Place</td><td>198262</td><td></td></tr><tr><td>Agarose</td><td>Fisher</td><td>BP1356-100</td><td></td></tr><tr><td>Methylene Blue</td><td>That Pet Place</td><td>214325</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>One well plates</td><td>Fisher</td><td>12-565-493</td><td></td></tr><tr><td>Digital camera</td><td>Canon</td><td>EOS Rebel T1i</td><td></td></tr><tr><td>Imaging Cabinets</td><td>WoodCraft Towers</td><td></td><td></td></tr><tr><td>Laptops</td><td>Acer Aspire</td><td>Any is good as long as it has a 15.6 in. LCD screen with 1366 x 768 pixel resolution and a brightness of 220 cd/m&lt;sup&gt;2&lt;/sup&gt;.</td><td></td></tr><tr><td>Camera Lens</td><td>Canon</td><td>EF-S 55 - 250 mm f/4.0 - 5.6 IS zoom lens</td><td></td></tr><tr><td>Plastic diffuser</td><td>Pendaflex</td><td>52345</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>PowerPoint 2010</td><td>Microsoft</td><td></td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/&quot; target=&quot;_blank&quot;&gt;http://rsb.info.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td>Excel 2010</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Statistical software</td><td>SPSS 20</td><td></td><td></td></tr></tbody></table>

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.

In our earlier assays, using the bouncing ball aversive stimulus, wild type larvae that are untreated respond to the moving ball by swimming down in the well (avoidance behavior) and towards the edges of the well (thigmotaxis behavior) 15. We later confirmed that thigmotaxis behavior in this assay is a measure of anxiety-related behavior in zebrafish larvae 17. There were significant differences in the larvae movement away from the ball and preference for the edge when compared to the blank white background. These behaviors have also been confirmed in our new assay using the moving red bar and are even more robust 16. Moreover, we can now sample a larger number of behaviors in a single assay including swim speed, rest, preference for the end or side of the well, and distance between fish (Figure 5). Control larvae grown in egg water show an increased preference to be down in the dish and on the edge of the lane after they are presented with an aversive stimulus (moving red bar). Similar results are obtained when larvae are grown in egg water containing 1 &mu;g/ml DMSO, a solvent that is commonly used to dissolve various pharmaceuticals and toxicants as 1,000X stock solutions.
Representative results are shown in Figure 5 in larvae treated with egg water and DMSO (as controls) and varying concentrations of an organophosphate pesticide commonly found in non-organic foods. The results shown are a sampling from one experiment. However, when repeated, the results indicate that swim speed and thigmotaxis behavior is altered by low concentrations of organophosphate pesticides, which mimic levels in human food consumption 18.
<img alt="Figure 1" src="/files/ftp_upload/50622/50622fig1.jpg" /> 
 Figure 1. Collection Trays. Glass Pyrex dishes are used to collect embryos from the adult fish tanks. Lids from the Pyrex dishes were cut and inserted with plastic grids and green yarn was sewn onto the grids in the plastic. This creates a breeding atmosphere for the adult zebrafish by mimicking the natural environment.
<img alt="Figure 2" fo:content-width="/files/ftp_upload/50622/50622fig2highres.jpg" fo:width="6in" src="/files/ftp_upload/50622/50622fig2.jpg" /> 
 Figure 2. Plastic mold and agarose lanes. A) The mold is shown on the left. 0.8% agarose is poured into a one-well plate; the mold is slowly inserted and then removed when the agarose has cooled. B) The plate on the right shows the lanes created in agarose by the plastic mold.
<img alt="Figure 3" src="/files/ftp_upload/50622/50622fig3.jpg" /> 
 Figure 3. Imaging Cabinets. Imaging cabinets were specially built in our laboratory and used for high-throughput behavioral analyses. A 15 megapixel digital camera was attached to the top of the cabinet facing downwards in order to gather time lapse images of the larvae in multilane plates placed on top of the screen of a laptop. Between the plates and the screen there is a plastic diffuser that is used to prevent moir&eacute; patterns in the images collected.
<img alt="Figure 4" fo:content-width="/files/ftp_upload/50622/50622fig4highres.jpg" fo:width="4.5in" src="/files/ftp_upload/50622/50622fig4.jpg" /> 
 Figure 4. Blank background and PowerPoint aversive stimulus. This is the current PowerPoint that is used to evoke behavioral changes in zebrafish larvae. It provides robust behavioral differences between A) the blank background and B) the moving red bar.
<img alt="Figure 5" fo:content-width="/files/ftp_upload/50622/50622fig5highres.jpg" fo:width="5.5in" src="/files/ftp_upload/50622/50622fig5.jpg" /> 
 Figure 5. Behaviors Quantified in the high-throughput assay. Example of the behaviors that are quantified from our behavioral assay within the Excel sheet that we use for x,y coordinates of the larvae. The white bars show data from larvae exposed to a blank background and the red bars show data from the larvae exposed to the red moving bar in the PowerPoint. The graphs indicate the measurements that can be obtained from behavioral analysis A) Percentage of larvae down in the lane, B) Percentage of larvae on the end of the lane, C) Percentage of larvae on the edge of the lane, D) Distance between fish (mm), E) Swim speed of the larvae (mm/min), F) Percentage rest of the larvae. In the graphs shown, data is from treatment of larvae with DMSO control and several concentrations of a pesticide ranging from 0.001 to 0.1 &mu;M (levels commonly found in the human diet). <a href="https://www.jove.com/files/ftp_upload/50622/50622fig5large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Automated High-throughput Behavioral Analyses in Zebrafish Larvae at JoVE.com

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

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 ...

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15.2K Views.  Brown University. 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.

Video: Automated High-throughput Behavioral Analyses in Zebrafish Larvae

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Environment

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biology

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 ...

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

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

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.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

Source: Steele, W. B., et al. <a href="https://www.jove.com/v/57938/" target="_blank">Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine</a>.&#160;J. Vis. Exp.&#160;(2018).
This video describes the photomotor response assay of zebrafish and fathead minnow larvae using an automated tracking software. The protocol measures the behavioral response of the larval fish following a sudden change in lighting condition.

Source: Steele, W. B., et al. Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Larva

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

Source: Richendrfer, H., et al. <a href="https://www.jove.com/v/50622/" target="_blank">Automated High-throughput Behavioral Analyses in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2013)
The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

Source: Richendrfer, H., et al. Automated High-throughput Behavioral Analyses in Zebrafish Larvae. &#160;J. Vis. Exp.&#160;(2013)
The video describes the ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Summary

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Chapters in this video

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Behavioral Approaches to Studying Innate Stress in Zebrafish

Development of automated imaging and analysis for zebrafish chemical screens.

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus