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>

<ol>
	<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. 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=Zebrafish+Behavioral+Profiling+Links+Drugs+to+Biological+Targets+and+Rest/Wake+Regulation.">Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation.</a> Science. 327, 348-351 (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=Measuring+behavioral+and+endocrine+responses+to+novelty+stress+in+adult+zebrafish.">Measuring behavioral and endocrine responses to novelty stress in adult zebrafish.</a> Nat. 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. L., 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=Understanding+behavioral+and+physiological+phenotypes+of+stress+and+anxiety+in+zebrafish.">Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.</a> Behav. Brain Res. 205, 38-44 (2009).</li><li>Colwill, R., Creton, R., Kalluef, A., Stewart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish protocols for neurobehavioral research. 66, (2012).</li></ol>

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 border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</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></td>
			<td></td>
			<td></td>
			<td>Equipment</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/m2.</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></td>
			<td></td>
			<td></td>
			<td>Software</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><a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a></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.remotexs.ntu.edu.sg/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 ...

automated-high-throughput-behavioral-analyses-in-zebrafish-larvae

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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 ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...