Support for this study was provided by the U.S. National Science Foundation (Project #: CHE-1339637) with additional support from the U.S. Environmental Protection Agency. We thank Dr. Jone Corrales, Dr. Lauren Kristofco, Gavin Saari, Samuel Haddad, Bekah Burket and Bridgett Hill for general lab support.

Studies in this protocol generally follow standardized experimental design and recommended statistical analysis guidelines from the US Environmental Protection Agency (EPA no. 2000.0) for fathead minnows and the Organization for Economic Cooperation and Development (OECD no. 236) for zebrafish.&#160;These experimental designs (e.g., increasing replication) can be modified within the current protocol for future studies. Fish culture conditions follow previously published literature17. All experimental procedures and fish culture protocols followed Institutional Animal Care and Use Committee protocols approved at Baylor University.
1. Exposing Fish to Chemical Treatment
<ol>
	<li>Prepare caffeine exposure solutions by dissolving caffeine in reconstituted hard water. Perform appropriate serial dilutions by diluting higher caffeine treatments with hard water to produce lower caffeine treatment levels. 
	NOTE: Table 1 summarizes each of the treatment levels used in this experiment.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Zebrafish</td>
			<td></td>
			<td colspan="2">Fathead Minnow</td>
		</tr>
		<tr>
			<td>Treatment</td>
			<td>Nominal Caffeine Concentration (mg/L)</td>
			<td>Measured Caffeine Concentration (mg/L)</td>
			<td>Treatment</td>
			<td>Nominal Caffeine Concentration (mg/L)</td>
			<td>Measured Caffeine Concentration (mg/L)</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>0</td>
			<td>&#60;LOD</td>
			<td>Control</td>
			<td>0</td>
			<td>&#60;LOD</td>
		</tr>
		<tr>
			<td>75th Centile*</td>
			<td>0.001</td>
			<td>0.001</td>
			<td>75th Centile*</td>
			<td>0.001</td>
			<td>0.001</td>
		</tr>
		<tr>
			<td>95th Centile*</td>
			<td>0.039</td>
			<td>0.013</td>
			<td>95th Centile*</td>
			<td>0.039</td>
			<td>0.009</td>
		</tr>
		<tr>
			<td>99th Centile*</td>
			<td>0.412</td>
			<td>0.361</td>
			<td>99th Centile*</td>
			<td>0.412</td>
			<td>0.310</td>
		</tr>
		<tr>
			<td>THV</td>
			<td>4.07</td>
			<td>3.81</td>
			<td>THV</td>
			<td>4.07</td>
			<td>4.12</td>
		</tr>
		<tr>
			<td>10% LC50</td>
			<td>48.46</td>
			<td>46.66</td>
			<td>10% LC50</td>
			<td>14.1</td>
			<td>14.7</td>
		</tr>
		<tr>
			<td>40% LC50</td>
			<td>193.82</td>
			<td>186.67</td>
			<td>40% LC50</td>
			<td>56.38</td>
			<td>53.91</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental caffeine treatments for zebrafish and fathead minnow experiments. Nominal and measured values of caffeine for each treatment are given. *The caffeine treatments used in this study represent the upper centiles of exposure distributions based on measured environmental values of caffeine16. THV: Therapeutic Hazard Value. LOD: Limit of Detection
<ol start="2">
	<li>Pour the prepared solution in individual exposure champers. Use 100 mL glass beakers filled with 20 mL of exposure solution for zebrafish exposure chambers and 500 mL beakers with 200 mL of exposure solution for fathead minnow exposure chambers.</li>
	<li>Using a transfer pipette, place 10 zebrafish embryos aged 4&#8211;6 h post fertilization (hpf) in each of four replicate exposure chambers per treatment.</li>
	<li>Place 10 fathead minnow larvae aged within 24 h of hatching in each of three replicate exposure chambers per treatment. To accommodate the larger size of fathead minnow larvae, cut the tip of the transfer pipette off prior to transfer.</li>
	<li>Maintain zebrafish experiments at a 16:8 h light:dark photoperiod and a constant temperature of 28 &#177; 1 &#176;C. Use the same photoperiod regime for fathead minnow studies, but at a temperature of 25 &#177; 1 &#176;C.</li>
	<li>After 96 h of chemical exposure, load individual fish in separate wells of 48 (for zebrafish) and 24 (for fathead minnow) well plates.
	<ol>
		<li>To ensure that each well contains an equal volume of solution, transfer zebrafish larvae to 48 well plates using a 5,000 &#181;L autopipette for a 1,000 &#181;L volume per well. Use the autopipette to withdraw and transfer both the zebrafish larvae and exposure solution simultaneously.</li>
		<li>Due to their larger size, transfer fathead minnow larvae using a transfer pipette with the tip cut off. Prior to transferring fathead minnow larvae to individual wells, fill each well to 2,000 &#181;L using an autopipette. When transferring individual fathead larvae to wells, place the tip of the transfer pipette in the well solution and allow the fish to swim from the pipette tip into the well.</li>
	</ol>
	</li>
</ol>
2. Calibration of Video-tracking Parameters
<ol>
	<li>Prior to behavioral measures, set observation and calibration parameters in the video track software (see Table of Materials).
	<ol>
		<li>Place a well plate in the recording chamber with at least 1 larval fish in an individual well. Use the plate and associated fish as representations to set calibration parameters.</li>
		<li>In the video track software, click &#34;File | Generate Protocol&#34;, which will open a &#8220;protocol creation wizard&#8221; dialogue box. In the &#34;Location Count&#34; field, enter the number of individuals wells of the well plate and then click &#34;OK&#34;.</li>
		<li>At the top of the screen, click &#34;View | Full Screen&#34;, which will prompt the system to display an overhead camera view of the well plate.</li>
		<li>Click the &#34;Draw Areas&#34; icon, which appears as three multicolored shapes. To the right of the well plate viewing area, select the circle icon in the field labeled &#34;Areas&#34;.</li>
		<li>Use the cursor to delineate the circular video tracking area in the top left well of the well plate. Select &#34;Top-Right Mark&#34; and then outline the viewing area of the top right well. Then, select &#34;Bottom Mark&#34; to outline the bottom right well. 
		NOTE: After drawing the circular outline, its position will likely need to be adjusted.&#160; To adjust the position of the outline, click &#8220;select&#8221; and then use the cursor to move the outlined area. Also, outlines can be replicated by clicking &#34;Copy&#34; and then &#34;Paste&#34;.&#160;</li>
		<li>After the top left, top right, and bottom right well tracking areas have been defined, click &#34;Build&#34; to prompt the software to automatically delineate the viewing areas of the remaining wells.</li>
		<li>In the area labeled &#34;Calibration&#34;, click &#34;Draw Scale&#34;. Use the cursor to draw a horizontal line across the plate. Once the line is drawn, a dialogue box labeled &#8220;Calibration measurement&#8221; will appear. Enter the well plate length and click &#34;OK&#34;.</li>
		<li>Exit the drawing manager by clicking the &#34;Draw Areas&#34; icon.</li>
		<li>Click the &#34;Tiles&#34; icon.&#160; Using the cursor, highlight all the boxes that appear on the viewing screen so that each box is green. 
		NOTE: The Tiles icon appears as a group of six individual small squares</li>
		<li>&#34;Click View| Full Screen&#34;. &#160;To the right of the plate viewing area, click &#34;Bkg&#34; in the box labeled &#34;Detection Threshold&#34;. Use the threshold adjustment bar to set the pixel detection threshold. Once, the appropriate pixel detection threshold is selected, click &#34;Apply to Group&#34;. 
		NOTE: This protocol sets the detection threshold at 13 in black mode for zebrafish observations and at 110 in transparent mode for fathead minnow observations.</li>
		<li>In the box labeled &#34;Movement Threshold&#34;, enter the desired movement speed tracking parameters. Once speed parameters are set, click &#34;Apply to Group&#34;. 
		NOTE: This protocol sets small/large movements at 20 mm/s and inactive/small movements at 5 mm/s. These selections program the software to track larval fish movement at three different speed levels: inactive (freezing) = &#60;5 mm/s, small (cruising) = 5&#8211;20 mm/s, and large (bursting) = &#62;20 mm/s.</li>
		<li>Click &#34;Parameters | Protocol Parameters&#34; from the drop-down menu. In the dialogue box, select the &#34;Time&#34; tab. Enter the observation time and the integration time. After parameters are entered click &#34;Ok&#34;.</li>
		<li>To set the light/dark photoperiod times and light intensity for each photoperiod open the light driver settings dialogue box by selecting &#34;Light Driving&#34; from the &#34;Parameters&#34; drop down menu. 
		NOTE: See protocol video for setting multiple light-dark photoperiods.</li>
		<li>After the video tracking parameters have been set, save the observation protocol. 
		NOTE: This protocol observes fish behavior over a 50 min period that includes a 10 min acclimation phase followed by 4 altering light/dark phases consisting of two 10 min light periods and two 10 min dark periods. The integration time is set to measure behavior for each minute of the 50 min behavioral trial.</li>
	</ol>
	</li>
</ol>
3. Observation of Larval Fish Locomotor and Photomotor Behavior
<ol>
	<li>Place the well plate containing experimental fish in the behavioral recording chamber.</li>
	<li>In the video tracking software, open the tracking protocol developed in step 3.</li>
	<li>In the video tracking viewer, check to make sure that all larvae are visible on the computer screen, that only one individual larva is present in each well, and that individual wells are aligned within the observation areas that were defined in steps 2.1.5 and 2.1.6.</li>
	<li>Click on &#34;Experiment | Execute&#34;. 
	NOTE: The system will prompt the user to provide a name and location to save the observation data.</li>
	<li>Once the name and save location of the observation data have been specified, click on the &#34;Several Live Images&#34; icon to highlight all the pre-defined viewing areas 
	NOTE: This icon is located at the top of the computer screen and appears as a box divided into four smaller squares. Clicking on this icon will highlight all the pre-defined viewing areas.</li>
	<li>Close the panel of the recording chamber and click &#34;Background | Start&#34; on the computer monitor.</li>
</ol>
4. Analyzing Behavioral Data
<ol>
	<li>To retrieve larval fish activity data, open the spreadsheet, which is automatically compiled by the tracking software and is in the folder specified by the user before initiating behavioral trials (Step 3.4).</li>
	<li>Refer to Figures 1A and 1B for representative measurements of naive locomotor activity of unexposed zebrafish and fathead minnow larvae, respectively. Refer to Figures 1C and 1D for PMR calculations, which effectively examine the magnitude of movement difference between light to dark or dark to light transitions.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57938/57938fig1.jpg" /> 
Figure 1: Example of baseline activity of unexposed zebrafish (A and B) and fathead minnow (C and D). The mean (&#177; SEM) distance swam for zebrafish (A) and fathead minnow (C) is given by dots each representing one-minute intervals of activity. Two dark and two light periods of photomotor responses are measured. The last (a, c, e, and g) and first (b, d, f, and h) minute of each photoperiod are used to calculate PMRs. Photomotor responses of zebrafish (B) and fathead minnow (D) are measured as the change in mean (&#177;SEM) distance traveled between the last minute of an initial photoperiod and the first minute of the following period. <a href="https://www.jove.com/files/ftp_upload/57938/57938fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol><li>Malaj, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organic+chemicals+jeopardize+the+health+of+freshwater+ecosystems+on+the+continental+scale%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Organic chemicals jeopardize the health of freshwater ecosystems on the continental scale.</a> Proceedings of the National Academy of Sciences. 111 (26), 9549-9554 (2014).</li>
<li>Schäfer, R. B., Kühn, B., Malaj, E., König, A., Gergs, R. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/fwb.12811">Contribution of organic toxicants to multiple stress in river ecosystems.</a> Freshwater Biology. 61 (12), 2116-2128 (2016).</li>
<li>Andersen, M. E., Krewski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicity+testing+in+the+21st+century%3A+bringing+the+vision+to+life%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Toxicity testing in the 21st century: bringing the vision to life.</a> Toxicological Sciences. 107 (2), 324-330 (2008).</li>
<li>Rovida, C., Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Re-evaluation+of+animal+numbers+and+costs+for+in+vivo+tests+to+accomplish+REACH+legislation+requirements+for+chemicals-a+report+by+the+transatlantic+think+tank+for+toxicology+%28t+%284%29%29%5BTitle%5D)+AND+%22Altex%22%5BJournal%5D)">Re-evaluation of animal numbers and costs for in vivo tests to accomplish REACH legislation requirements for chemicals-a report by the transatlantic think tank for toxicology (t (4)).</a> Altex. 26 (3), 187-208 (2009).</li>
<li>Council, N. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCouncil%2C+NR+%22Toxicity+testing+in+the+21st+century%3A+a+vision+and+a+strategy%22">Toxicity testing in the 21st century: a vision and a strategy</a>. , National Academies Press. (2007).</li>
<li>Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Opportunities+and+challenges+for+use+of+tumor+spheroids+as+models+to+test+drug+delivery+and+efficacy%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.</a> Journal of Controlled Release. 164 (2), 192-204 (2012).</li>
<li>Scholz, S., Fischer, S., Gündel, U., Küster, E., Luckenbach, T., Voelker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+embryo+model+in+environmental+risk+assessment-applications+beyond+acute+toxicity+testing%5BTitle%5D)+AND+%22Environmental+Science+and+Pollution+Research%22%5BJournal%5D)">The zebrafish embryo model in environmental risk assessment-applications beyond acute toxicity testing.</a> Environmental Science and Pollution Research. 15 (5), 394-404 (2008).</li>
<li>Fraysse, B., Mons, R., Garric, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+zebrafish+4-day+embryo-larval+bioassay+to+assess+toxicity+of+chemicals%5BTitle%5D)+AND+%22Ecotoxicology+and+Environmental+Safety%22%5BJournal%5D)">Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals.</a> Ecotoxicology and Environmental Safety. 63 (2), 253-267 (2006).</li>
<li>Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+morphological-behavioral+test+platform+reveals+neurodevelopmental+defects+in+embryonic+zebrafish+exposed+to+comprehensive+suite+of+halogenated+and+organophosphate+flame+retardants%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Advanced morphological-behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.</a> Toxicological Sciences. 145 (1), 177-195 (2015).</li>
<li>Colón-Cruz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+of+larval+photo-dependent+swimming+responses+%28PDR%29%3A+New+endpoints+for+rapid+and+diagnostic+screening+of+aquatic+contamination%5BTitle%5D)+AND+%22Ecotoxicology+and+Environmental+Safety%22%5BJournal%5D)">Alterations of larval photo-dependent swimming responses (PDR): New endpoints for rapid and diagnostic screening of aquatic contamination.</a> Ecotoxicology and Environmental Safety. 147, 670-680 (2018).</li>
<li>Kristofco, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age+matters%3A+developmental+stage+of+Danio+rerio+larvae+influences+photomotor+response+thresholds+to+diazinion+or+diphenhydramine%5BTitle%5D)+AND+%22Aquatic+Toxicology%22%5BJournal%5D)">Age matters: developmental stage of Danio rerio larvae influences photomotor response thresholds to diazinion or diphenhydramine.</a> Aquatic Toxicology. 170, 344-354 (2016).</li>
<li>Rihel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+behavioral+profiling+links+drugs+to+biological+targets+and+rest%2Fwake+regulation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation.</a> Science. 327 (5963), 348-351 (2010).</li>
<li>Kokel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+behavior-based+identification+of+neuroactive+small+molecules+in+the+zebrafish%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Rapid behavior-based identification of neuroactive small molecules in the zebrafish.</a> Nature Chemical Biology. 6 (3), 231-237 (2010).</li>
<li>Bruton, T., Alboloushi, A., DeL a Garza, B., Kim, B. -O., Halden, R. U. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBruton%2C+T+%22Contaminants+of+Emerging+Concern+in+the+Environment%3A+Ecological+and+Human+Health+Considerations%22">Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations</a>. , ACS Publications. 257-273 (2010).</li>
<li>Woudenberg, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+embryotoxicity+test+for+developmental+%28neuro%29+toxicity%3A+Demo+case+of+an+integrated+screening+approach+system+using+anti-epileptic+drugs%5BTitle%5D)+AND+%22Reproductive+Toxicology%22%5BJournal%5D)">Zebrafish embryotoxicity test for developmental (neuro) toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs.</a> Reproductive Toxicology. 49, 101-116 (2014).</li>
<li>Rodríguez-Gil, J., Cáceres, N., Dafouz, R., Valcárcel, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caffeine+and+paraxanthine+in+aquatic+systems%3A+Global+exposure+distributions+and+probabilistic+risk+assessment%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Caffeine and paraxanthine in aquatic systems: Global exposure distributions and probabilistic risk assessment.</a> Science of the Total Environment. 612, 1058-1071 (2018).</li>
<li>Corrales, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+the+Design+of+Less+Hazardous+Chemicals%3A+Exploring+Comparative+Oxidative+Stress+in+Two+Common+Animal+Models%5BTitle%5D)+AND+%22Chemical+Research+in+Toxicology%22%5BJournal%5D)">Toward the Design of Less Hazardous Chemicals: Exploring Comparative Oxidative Stress in Two Common Animal Models.</a> Chemical Research in Toxicology. 30 (4), 893-904 (2017).</li>
<li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/8589427">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li>
<li>Kristofco, L. A., Brooks, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+scanning+of+antihistamines+in+the+environment%3A+Analysis+of+occurrence+and+hazards+in+aquatic+systems%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Global scanning of antihistamines in the environment: Analysis of occurrence and hazards in aquatic systems.</a> Science of the Total Environment. 592, 477-487 (2017).</li>
<li>Saari, G. N., Scott, W. C., Brooks, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+assessment+of+calcium+channel+blockers+in+the+environment%3A+Review+and+analysis+of+occurrence%2C+ecotoxicology+and+hazards+in+aquatic+systems%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Global assessment of calcium channel blockers in the environment: Review and analysis of occurrence, ecotoxicology and hazards in aquatic systems.</a> Chemosphere. , (2017).</li>
<li>Corrales, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+the+Design+of+Less+Hazardous+Chemicals%3A+Exploring+Comparative+Oxidative+Stress+in+Two+Common+Animal+Models%5BTitle%5D)+AND+%22Chemical+Research+in+Toxicology%22%5BJournal%5D)">Toward the Design of Less Hazardous Chemicals: Exploring Comparative Oxidative Stress in Two Common Animal Models.</a> Chemical Research in Toxicology. 30 (4), 893-904 (2017).</li>
<li>Berninger, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+antihistamine+diphenhydramine+on+selected+aquatic+organisms%5BTitle%5D)+AND+%22Environmental+Toxicology+and+Chemistry%22%5BJournal%5D)">Effects of the antihistamine diphenhydramine on selected aquatic organisms.</a> Environmental Toxicology and Chemistry. 30 (9), 2065-2072 (2011).</li>
<li>Brooks, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fish+on+Prozac+%28and+Zoloft%29%3A+ten+years+later%5BTitle%5D)+AND+%22Aquatic+Toxicology%22%5BJournal%5D)">Fish on Prozac (and Zoloft): ten years later.</a> Aquatic Toxicology. 151, 61-67 (2014).</li>
<li>Huggett, D., Cook, J., Ericson, J., Williams, R. <a target="_blank" href="https://www.tandfonline.com/doi/abs/10.1080/714044797">A theoretical model for utilizing mammalian pharmacology and safety data to prioritize potential impacts of human pharmaceuticals to fish.</a> Human and Ecological Risk Assessment. 9 (7), 1789-1799 (2003).</li>
<li>Fitzsimmons, P. N., Fernandez, J. D., Hoffman, A. D., Butterworth, B. C., Nichols, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Branchial+elimination+of+superhydrophobic+organic+compounds+by+rainbow+trout+%28Oncorhynchus+mykiss%29%5BTitle%5D)+AND+%22Aquatic+Toxicology%22%5BJournal%5D)">Branchial elimination of superhydrophobic organic compounds by rainbow trout (Oncorhynchus mykiss).</a> Aquatic Toxicology. 55 (1-2), 23-34 (2001).</li>
<li>Scheiner, S. M., Gurevitch, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aScheiner%2C+SM+%22Design+and+Analysis+of+Ecological+Experiments%22">Design and Analysis of Ecological Experiments</a>. , Oxford University Press. (2001).</li>
<li>Nakagawa, S. <a target="_blank" href="https://academic.oup.com/beheco/article/15/6/1044/206216">A farewell to Bonferroni: the problems of low statistical power and publication bias.</a> Behavioral Ecology. 15 (6), 1044-1045 (2004).</li>
<li>Bean, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmaceuticals+in+water%2C+fish+and+osprey+nestlings+in+Delaware+River+and+Bay%5BTitle%5D)+AND+%22Environmental+Pollution%22%5BJournal%5D)">Pharmaceuticals in water, fish and osprey nestlings in Delaware River and Bay.</a> Environmental Pollution. 232, 533-545 (2018).</li>
<li>Richendrfer, H., Pelkowski, S., Colwill, R., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+edge%3A+pharmacological+evidence+for+anxiety-related+behavior+in+zebrafish+larvae%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">On the edge: pharmacological evidence for anxiety-related behavior in zebrafish larvae.</a> Behavioural Brain Research. 228 (1), 99-106 (2012).</li>
<li>Padilla, S., Hunter, D., Padnos, B., Frady, S., MacPhail, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+locomotor+activity+in+larval+zebrafish%3A+Influence+of+extrinsic+and+intrinsic+variables%5BTitle%5D)+AND+%22Neurotoxicology+and+Teratology%22%5BJournal%5D)">Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables.</a> Neurotoxicology and Teratology. 33 (6), 624-630 (2011).</li>
<li>Sukardi, H., Chng, H. T., Chan, E. C. Y., Gong, Z., Lam, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+for+drug+toxicity+screening%3A+bridging+the+in+vitro+cell-based+models+and+in+vivo+mammalian+models%5BTitle%5D)+AND+%22Expert+Opinion+on+Drug+Metabolism+%26+Toxicology%22%5BJournal%5D)">Zebrafish for drug toxicity screening: bridging the in vitro cell-based models and in vivo mammalian models.</a> Expert Opinion on Drug Metabolism & Toxicology. 7 (5), 579-589 (2011).</li>
<li>Ankley, G. T., Villeneuve, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fathead+minnow+in+aquatic+toxicology%3A+past%2C+present+and+future%5BTitle%5D)+AND+%22Aquatic+Toxicology%22%5BJournal%5D)">The fathead minnow in aquatic toxicology: past, present and future.</a> Aquatic Toxicology. 78 (1), 91-102 (2006).</li>
<li>Hutson, L. D., Liang, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+an+impact%3A+zebrafish+in+education%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Making an impact: zebrafish in education.</a> Zebrafish. 6, 119(2009).</li>
<li>Hutson, L. D., Liang, J. O., Pickart, M. A., Pierret, C., Tomasciewicz, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+a+difference%3A+education+at+the+10th+international+conference+on+zebrafish+development+and+genetics%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Making a difference: education at the 10th international conference on zebrafish development and genetics.</a> Zebrafish. 9 (4), 151-154 (2012).</li>
<li>Kane, A., Salierno, J., Brewer, S. <a target="_blank" href="https://pdfs.semanticscholar.org/8988/2abeade06cda10e67bb5c88b2c564f74f44a.pdf">Fish models in behavioral toxicology: automated techniques, updates and perspectives.</a> Methods in Aquatic Toxicology. 2, 559-590 (2005).</li>
<li>Rodriguez, A., et al. <a target="_blank" href="https://arxiv.org/abs/1706.02577">ToxTrac: a fast and robust software for tracking organisms.</a> Methods in Ecology and Evolution. 9 (3), 460-464 (2018).</li>
<li>Hamm, J., Wilson, B., Hinton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+uptake+and+bioactivation+with+development+positively+modulate+diazinon+toxicity+in+early+life+stage+medaka+%28Oryzias+latipes%29%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).</a> Toxicological Sciences. 61 (2), 304-313 (2001).</li>
<li>Kristofco, L. A., Haddad, S. P., Chambliss, C. K., Brooks, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+uptake+of+and+sensitivity+to+diphenhydramine+in+embryonic+and+larval+zebrafish%5BTitle%5D)+AND+%22Environmental+Toxicology+and+Chemistry%22%5BJournal%5D)">Differential uptake of and sensitivity to diphenhydramine in embryonic and larval zebrafish.</a> Environmental Toxicology and Chemistry. 37, 1175-1181 (2018).</li>
<li>Steele, W. B., Kristofco, L. A., Corrales, J., Saari, G. N., Haddad, S. P., Gallagher, E. P., Kavanagh, T. J., Kostal, J., Zimmerman, J. B., Voutchkova-Kostal, A., Anastas, P. T., Brooks, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+behavioral+toxicology+of+two+common+larval+fish+models%3A+exploring+relationships+between+modes+of+action+and+locomotor+responses%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Comparative behavioral toxicology of two common larval fish models: exploring relationships between modes of action and locomotor responses.</a> Science of the Total Environment. 460-461, 1587-1600 (2018).</li>
</ol>

The authors have nothing to disclose.

When selecting chemical treatment levels for behavioral toxicology studies, several factors must be considered. Caffeine treatment levels in the present study were selected based on upper centile values for predicted environmental exposure scenarios from wastewater effluent16. When possible, we routinely select treatment levels for aquatic toxicology studies using probabilistic exposure assessments of environmental observations19,20,21. A THV, which is calculable for medicines, was also included as a treatment level in the present study. THV values (Eq. 1)22,23 are defined as predicted water concentrations leading to human therapeutic doses (Cmax) of pharmaceuticals in fish23, are inspired from initial plasma modeling efforts24, and are calculated based on blood:water chemical partitioning coefficients (Eq. 2)25.
THV = Cmax / log PBW&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (Eq. 1)
log PBW = log [(100.73 . log Kow &#183; 0.16)+0.84]&#160;&#160;&#160;&#160; (Eq. 2)
Here, we also select sublethal treatment levels relative to zebrafish and fathead minnow LC50 values. We consider this approach a useful benchmarking procedure for behavioral responses, particularly when comparing thresholds of specific behaviors with a fish model across multiple chemicals. It further facilitates calculations of acute to chronic ratios, which can be diagnostically useful in aquatic toxicology for mechanistic studies and assessments. LC50 values were obtained from preliminary toxicity bioassays following the standardized guidelines given in step 2.1.
In this protocol, we employ common experimental designs and statistical techniques recommended by the US EPA and OECD standardized methods for toxicology studies with fish models. Though we report p values (e.g., &#60;0.01, &#60;0.05, &#60;0.10), significant differences (&#945; = 0.10) in activity levels are identified among treatments using analysis of variance (ANOVA) if normality and equivalence of variance assumptions are met. Dunnett&#39;s or Tukey&#39;s HSD post hoc tests are performed to identify treatment level differences. We select this alpha (&#945; = 0.10) value to reduce type II errors, particularly for early tier assays and when an understanding of biologically important effect size is limited for understudied behavioral endpoints and model organisms26, instead of employing procedures more common in the biomedical sciences for multiple comparisons (e.g., Bonferroni correction for RNA-Seq data)27.&#160;&#160;Future studies are needed to understand variability of these behavioral responses and potentially modify experimental designs (e.g., increase replication) accordingly.
A number of factors can influence behavior of larval fish in addition to chemical exposure. For example, time of day, age, well size, temperature, lighting condition, and volume of exposure solution in each well represent important considerations11,30. For these reasons, precautions should be taken to minimize the effects of external factors that could influence locomotor behavior of the larval fish during experimentation. Behavioral observations should be performed in narrow time windows (3 to 4 h) and across time periods when time of day effects are expected to have minimal influence on larval locomotor behavior11. Additionally, larval fish should be maintained at a consistent temperature (28 &#177; 1 &#176;C for zebrafish and 24 &#177; 1 &#176;C for FHM) and on a defined light/dark cycle in temperature-controlled incubators throughout the exposure period. In addition, the temperature of the laboratory where behaviors are recorded should be maintained to conditions approximating experimental conditions to avoid temperature influences on behaviors. Further, wells used during behavioral observations should be maintained at a consistent volume for each individual fish.
Larval and embryonic zebrafish PMRs have been previously used in the biomedical sciences to identify potential therapeutic targets for novel compounds12,13. This protocol expands on previous behavioral research with zebrafish by utilizing 38 endpoints to investigate chemical bioactivity of environmental contaminants. Although caffeine is a common aquatic contaminant with an understood mechanism of action (MoA), many compounds in commerce lack important mechanistic data. Therefore, this protocol can be employed to gain insight of MoAs for compounds lacking toxicity data, including commercial chemicals39. Furthermore, the protocol provides methods for two of the most commonly used fish models. As noted previously, whereas the zebrafish is a common biomedical fish model that is becoming increasingly popular in ecotoxicology, the fathead minnow is commonly used as an ecological model for environmental assessment applications but has received comparatively less attention in behavioral studies with automated systems compared to the zebrafish.&#160;Though there remains no standardized regulatory methods for fish behavioral toxicology studies, this protocol provides an approach to support future efforts.
Caffeine elicited behavioral responses in each of the fish models at levels that have been detected in the aquatic environment16. Rodriguez-Gil et al. 2018 developed global environmental exposure distributions in aquatic systems based on measured values of caffeine16. Specifically, 95% of predicted wastewater effluent concentrations would fall below the LOECs for the most sensitive behavioral endpoints of zebrafish and fathead minnow in the present study (Table 2). Though several behavioral effects of caffeine were observed in zebrafish (particularly in dark conditions) at environmentally relevant levels, it is unclear whether these behavioral modifications might occur in natural fish populations or result in ecologically important adverse outcomes. Though useful for sensitive, diagnostic screening purposes, larval fish behavioral thresholds may not be representative of other life history stages or of fish in natural populations. Further research is warranted to determine whether similar behavioral response thresholds would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Though fish models are increasingly used for biomedical studies, fish have been routinely employed for ecology and physiology studies, to examine contamination of surface waters, and to understand toxicological thresholds of chemicals. Such efforts are important because chemical contamination can impair aquatic ecosystems and jeopardize the quality of source water supplies1,2. Most of the chemicals in commerce, however, lack even basic toxicology information3.
Animal model assays traditionally used in regulatory toxicity testing are resource intensive and cannot provide the high throughput, early tier screening needed for toxicity testing in the 21st century4. Subsequently, there is a growing impetus to adopt and utilize in vitro models that can more rapidly and efficiently screen compounds for biological activities3,5. Though cell based models present many opportunities, they often lack biological complexity, and thus do not account for many important whole organism processes, including metabolism6.
The zebrafish is a common biomedical animal model that is gaining popularity as an alternative model in aquatic toxicology and ecotoxicology7,8. Given their small size, rapid development, and high fecundity, fish models can be used to rapidly and efficiently screen chemicals for bioactivity and toxicity at the whole organism scale9. With the aid of automated tracking software, larval zebrafish behaviors provide enhanced diagnostic utility in screening contaminants for toxicity10,11. Studies in the pharmaceutical sciences have demonstrated that locomotor endpoints are informative of chemical mechanisms of action, can be used to phenotype behaviors, and then may tentatively identify subcellular targets for novel molecules12,13. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common, ecologically important fish model that is used for ecotoxicology studies and during prospective (e.g., new chemical evaluations) and retrospective (e.g., ambient surface water or wastewater effluent discharge monitoring) environmental assessments. Unfortunately, behavioral responses of larval fathead minnows have received markedly less attention than zebrafish. Our ongoing research with two common larval fish models, the zebrafish and fathead minnow, suggests that larval fish swimming patterns appear unique to anticipated modes or mechanisms of action for diverse chemicals. Thus, behavioral endpoints provide the potential to rapidly and sensitively examine chemicals for toxicity and to identify subcellular targets for industrial chemical and other contaminants, particularly during early tier assessments.
Here, we report a protocol for examining behavioral response profiles in larval fish. We demonstrate these methods using caffeine, a model neurostimulant and a common aquatic contaminant that is introduced to aquatic systems through discharge from wastewater treatments plants following human consumption of foods, beverages, and pharmaceuticals formulated with caffeine14. We examine behavioral responses to caffeine in both larval zebrafish and fathead minnow, including to a sudden change in lighting condition, which is often referred to as a photomotor response (PMR) during pharmaceutical studies with embryonic and larval zebrafish13,15. We further identify effects of caffeine across several locomotor endpoints to develop chemical response profiles for each fish model. Caffeine treatment levels used in this study represent the upper centiles of exposure distributions based on measured environmental values of caffeine16. We also include treatments benchmarked to larval fish LC50 values, and the therapeutic hazard value (THV), a pharmaceutical concentration in water that is anticipated to result in plasma levels in fish consistent with a human therapeutic plasma dose.

<table><tbody><tr><td>ViewPoint Zebrabox</td><td>ViewPoint</td><td></td><td>ZebraLab and ZebraLab platform for automated behavioral observations</td></tr><tr><td>Caffeine</td><td>Sigma-Aldrich</td><td>C0750-100G</td><td>Study chemical</td></tr><tr><td>Incubator</td><td>VWR</td><td>9110589</td><td>Maintains light/dark cycle and temperature for fathead minnow experiments</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td>35824-636</td><td>Maintains light/dark cycle and temperature for zebrafish experiments</td></tr><tr><td>100 mL glass beakers</td><td>VWR</td><td>89000-200</td><td>Zebrafish exposure chambers&amp;nbsp;</td></tr><tr><td>500 mL glass beakers&amp;nbsp;</td><td>Pyrex</td><td>EW-34502-03</td><td>Fathead minnow exposure chambers</td></tr><tr><td>5,000 &amp;micro;L auto-pipette</td><td>Eppendorf</td><td>Research 5000</td><td>Used to fill individual wells in well plates</td></tr><tr><td>Transfer Pippettes</td><td>VWR</td><td>414-004-004</td><td>Used to transfer study organisms&amp;nbsp;</td></tr><tr><td>48-well plates</td><td>Fisher Scientific</td><td>08-772-52</td><td>Larval zebrafish behavioral recording chambers</td></tr><tr><td>24-well plates</td><td>VWR</td><td>10062-896</td><td>Larval fathead minnow behavioral recording chambers</td></tr><tr><td>Calcium sulfate dihydrate</td><td>Sigma-Aldrich</td><td>C3771</td><td>For reconstituted hard water</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td>For reconstituted hard water</td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S5761</td><td>For reconstituted hard water</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td>For reconstituted hard water</td></tr><tr><td>z-mod recirculating system</td><td>Marine Biotech Systems</td><td></td><td>Recirculating system to maintian zebrafish cultures</td></tr><tr><td>Statistical analysis software</td><td>Sigma Plot&amp;nbsp;</td><td>Version 13.0</td><td>Used to analyze beahvioral data and produce figures</td></tr><tr><td>Statistical analysis software</td><td>Graphpad Prism</td><td>Prism 5</td><td>Used to produce figures&amp;nbsp;</td></tr><tr><td>Autosampler/quaternary pumping system</td><td>Agilent Technologies</td><td>Infinity 1260 model&amp;nbsp;</td><td>Analytical verification of caffeine treatment levels</td></tr><tr><td>Jet stream thermal gradient electrospray ionization source</td><td>Agilent Technologies</td><td></td><td>Analytical verification of caffeine treatment levels</td></tr><tr><td>Triple quadrupole mass analyzer&amp;nbsp;</td><td>Agilent Technologies</td><td>Model 6420</td><td>Analytical verification of caffeine treatment levels</td></tr><tr><td>10&amp;nbsp;cm&amp;nbsp;&amp;times;&amp;nbsp;2.1&amp;nbsp;mm Poroshell 120 SB-AQ column (120&amp;Aring;, 2.7)&amp;nbsp;</td><td>Agilent Technologies</td><td>685775-914T</td><td>Caffiene chromatography&amp;nbsp;</td></tr><tr><td>MassHunter Optimizer Software&amp;nbsp;</td><td>Agilent Technologies</td><td></td><td>Determine the ionization mode, monitored transitions, and instrumental parameters for caffeine/caffeine-d9 and paraxanthine/paraxanthine-d6</td></tr></tbody></table>

experimental protocol for examining behavioral response profiles in larval fish: application to the neuro-stimulant caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Caffeine treatment levels did not appreciably vary during the 96 h experiments with zebrafish and fathead minnows. For example, Table 1 presents analytically verified concentrations of each treatment level. This protocol verified water samples for caffeine treatment levels by isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) generally following previously reported methods28. The formation of paraxanthine, the primary metabolite of caffeine, was also quantified. A description of these analytical procedures is provided in the supplemental analytical information. Because of the similarities between nominal and analytical verification of treatments, nominal treatment levels are presented throughout the remainder&#160;of this manuscript. Caffeine significantly altered zebrafish and fathead minnow behaviors. However, zebrafish locomotor responses were consistently more sensitive to caffeine than fathead minnows. The most sensitive behavioral endpoints for zebrafish and fathead minnow larvae were affected by caffeine at a concentration of 0.039 mg/L. Table 2 summarizes lowest observed effect concentrations (LOECs) and no observed effect concentrations (NOECs) for each behavioral endpoint in both fish models.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Zebrafish&#160;&#160;</td>
			<td colspan="3">Fathead minnow&#160;&#160;</td>
		</tr>
		<tr>
			<td>Endpoint&#160;</td>
			<td>LOEC (mg/L)</td>
			<td>NOEC (mg/L)</td>
			<td>Endpoint&#160;</td>
			<td>LOEC (mg/L)</td>
			<td>NOEC (mg/L)</td>
		</tr>
		<tr>
			<td>Total Distance Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Total Distance Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Total Distance Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Total Distance Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Total Counts Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Total Counts Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Total Counts Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Total Counts Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Distance Dark</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Bursting Distance Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Distance Light</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Bursting Distance Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Counts Dark</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Bursting Counts Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Counts Light</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Bursting Counts Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Duration Dark</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Bursting Duration Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Duration Light</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Bursting Duration Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Distance Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Cruising Distance Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Distance Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Distance Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Counts Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Cruising Counts Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Counts Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Counts Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Duration Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Cruising Duration Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Duration Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Duration Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Distance Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Freezing Distance Dark</td>
			<td>0.039</td>
			<td>0.001</td>
		</tr>
		<tr>
			<td>Freezing Distance Light</td>
			<td>0.039</td>
			<td>0.001</td>
			<td>Freezing Distance Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Counts Dark</td>
			<td>0.412</td>
			<td>0.039</td>
			<td>Freezing Counts Dark</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Counts Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Freezing Counts Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Duration Dark</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Freezing Duration Dark</td>
			<td>56.38</td>
			<td>14.10</td>
		</tr>
		<tr>
			<td>Freezing Duration Light</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Freezing Duration Light</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Dark 1 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Dark 1 PMR</td>
			<td>0.039</td>
			<td>0.001</td>
		</tr>
		<tr>
			<td>Light 1 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Light 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Dark 2 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Dark 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Light 2 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Light 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Dark 1 PMR</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Bursting Dark 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Light 1 PMR</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Bursting Light 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Dark 2 PMR</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Bursting Dark 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Bursting Light 2 PMR</td>
			<td>&#8722;</td>
			<td>193.82</td>
			<td>Bursting Light 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Dark 1 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Dark 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Light 1 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Light 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Dark 2 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Cruising Dark 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Cruising Light 2 PMR</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Cruising Light 2 PMR</td>
			<td>56.38</td>
			<td>14.10</td>
		</tr>
		<tr>
			<td>Freezing Dark 1 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Freezing Dark 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Light 1 PMR</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Freezing Light 1 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Dark 2 PMR</td>
			<td>48.46</td>
			<td>4.07</td>
			<td>Freezing Dark 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>Freezing Light 2 PMR</td>
			<td>193.82</td>
			<td>48.46</td>
			<td>Freezing Light 2 PMR</td>
			<td>&#8722;</td>
			<td>56.38</td>
		</tr>
	</tbody>
</table>
Table 2: Zebrafish and fathead minnow behavioral NOECs and LOECs for Caffeine. No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) (mg/L) values for each of the light/dark swimming activity endpoints and photomotor responses for zebrafish and fathead minnows exposed to caffeine. Dashes indicate that no effects were observed at a particular endpoint across all treatment levels.
Figure 2 presents total locomotor activity and PMRs of zebrafish and fathead minnow following 96 h exposure to caffeine. Fathead minnow larvae PMRs were altered by caffeine at lower treatment levels (0.038 mg/L) than zebrafish, but a markedly larger number of photomotor endpoints were affected in zebrafish. The highest treatment level of caffeine (193.82 mg/L) altered PMR in zebrafish, in which these responses were exactly opposite from controls. At this elevated treatment level, however, PMRs decreased in dark and increased in light conditions.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57938/57938fig2.jpg" /> 
Figure 2: Swimming activity and photomotor responses of zebrafish (A and B) and fathead minnow (C and D) after 96 h exposure to caffeine. The mean (&#177; SEM) distance swam for zebrafish (A) and fathead minnow (C) is given by dots each representing 1-min intervals of activity. Photomotor responses of zebrafish (B) and fathead minnow (D) are measured as the change in mean (&#177; SE) total distance traveled between the last minutes of an initial photoperiod and the first minute of the following period. Two dark and two light period photomotor responses were measured. A total of 24 (4 replicates each of 6 larvae) zebrafish and 12 (3 replicates each of 4 larvae) fathead minnows were used for behavioral observation. *p &#60;0.10; ** p &#60;0.05; *** p &#60;0.01. <a href="https://www.jove.com/files/ftp_upload/57938/57938fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition to measuring larval PMRs, light and dark locomotor activity was analyzed across three speed thresholds for distance moved, number of movements, and duration of movements. This data is used to develop behavioral response profiles for caffeine (Figure 3, Supplemental Figure 1). In both of the fish models, caffeine inhibited activity at all significantly affected locomotor endpoints. Both fish models demonstrated increased activity at the bursting speed thresholds following exposure to caffeine, though not significantly. Similar to the results of the PMR observations, caffeine effected a greater number of zebrafish locomotor endpoints. In fact, caffeine significantly altered several locomotor responses under dark conditions at environmentally realistic levels below the THV. However, fathead minnow locomotor activity was not significantly affected under light conditions by any treatment level.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57938/57938fig3.jpg" /> 
Figure 3: Response profiles of larval zebrafish and fathead minnows after 96 h exposure to caffeine. Mean zebrafish dark (A) and light (B) swimming activity compared to mean fathead minnow dark (C) and light (D) activity after 96 h exposure to caffeine. Plotted data represents activity over two 10 min dark photoperiods and two 10 min light photoperiods for each fish model. Data is normalized to control, which is represented at the 0 axis in each figure. Behavioral parameters include distance swam, number of movements (counts), and duration of each movement across 3 speed levels, bursting (&#62;20 mm/s), cruising (5&#8211;20 mm/s), and freezing (&#60;5 mm/s). In addition to movement patterns at each of the speed thresholds, total distance swam, and total number of movements is represented. &#8593; represents a significant increase in activity in comparison to control and &#8595; indicates a significant decrease in activity in comparison to control. A total of 24 (4 replicates each of 6 larvae) zebrafish and 12 (3 replicates each of 4 larvae) fathead minnow where used in behavioral observations for each group. *p &#60;0.10; ** p &#60;0.05; *** p &#60;0.01. <a href="https://www.jove.com/files/ftp_upload/57938/57938fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Photomotor responses of zebrafish (A and B) and fathead minnow (C and D) across three speed thresholds. Zebrafish (A, B, and C) and fathead minnow larvae (D, E, and F) photomotor responses across three speed thresholds (Freezing: 20 mm/s) after 96hr exposure to caffeine. Photomotor responses of zebrafish and fathead minnow are measured as the change in mean (&#177;SE) total distance traveled between the last minutes of an initial photoperiod and the first minute of the following period. Two dark and two light period photomotor responses were measured. A total of 24 (4 replicates each of 6 larvae) zebrafish and 12 (3 replicates of 4 larvae) fathead minnows were used for behavioral observation. *p &#60;0.01 <a href="https://www.jove.com/files/ftp_upload/57938/Steele_et_al_JoVE_Supplemental_Figure_1_01302018.pptx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine at JoVE.com

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

experimental-protocol-for-examining-behavioral-response-profiles

Research

JoVE Journal

Environment

11.1K Views.  Baylor University. Hier presenteren we een protocol te onderzoeken larvale zebravis en fathead minnow motorische activiteiten en photomotor Reacties (PMR) met behulp van een geautomatiseerd tracking-software. Wanneer opgenomen in algemene toxicologie bioassays, geven analyses van deze gedragingen een diagnostisch instrument om te onderzoeken van chemische topicale. Dit protocol wordt beschreven met behulp van cafeïne, een neurostimulant model.

Video: Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

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

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

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

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

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

Neuroscience

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

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

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

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

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

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

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

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

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

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

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

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

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

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

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

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

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

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

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

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

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

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

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://app.jove.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Samenvatting

Meer video's verkennen