Simon Cassar of carpetbones.com designed and created the animated figure used to demonstrate the in-life procedure of the GI transit assay.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of AbbVie.Abbvie operates under the National Institutes of Health Guide for Care and Use of Laboratory Animals in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). No animal health concerns were observed in these studies.
1. Breed Adult Zebrafish and Collect Embryos
<ol>
	<li>House and breed adult zebrafish using general husbandry and breeding practices. For example, see Westerfield10.</li>
	<li>Prepare embryo medium by dissolving dehydrated sea salt (see Table of Materials) in deionized water at a concentration of 60 mg/L.</li>
	<li>Collect fertilized eggs from the adult breeding chamber, rinse well with embryo medium and house embryos in about 50 mL of embryo medium in 10 cm Petri dishes (50 embryos per dish) at 28 &#177; 1 &#176;C on a 14:10 h light:dark cycle.</li>
	<li>Remove non-surviving embryos after 24 h and supplement with surviving embryos so that each dish contains 50 embryos per dish.</li>
</ol>
2. Train Larvae to Feed Using Non-dyed Food
<ol>
	<li>On the 4th day post-fertilization (4 dpf), feed the larvae in each Petri dish 2 mg of powdered larval fish food (see Table of Materials) by sprinkling the food on top of the water.</li>
	<li>Allow the larvae 3&#8211;4 h to feed and then transfer them to a clean (no food) Petri dish containing about 50 mL fresh embryo medium.
	<ol>
		<li>To aid in transferring as little food as possible, rinse each larva in a Petri dish containing about 50 mL fresh embryo medium before the final transfer to the new dish.</li>
	</ol>
	</li>
	<li>Repeat feeding and rinsing of the larvae on 5 dpf and 6 dpf.</li>
</ol>
3. Prepare Fluorescent Food on the 6 dpf and Feed to Larvae on the 7 dpf
<ol>
	<li>Prepare food containing a fluorescent label (hereafter referred to as fluorescent food, according to methods of Field et al.3). Briefly, mix 300 &#181;L of fluorescent label (see Table of Materials), 100 &#181;L of deionized water, and 200 mg of powdered larval food on a 10 cm watch glass.</li>
	<li>Spread the resulting paste into a thin layer on the watch glass. Allow the paste to dry at room temperature in the dark for &#62;8 h.</li>
	<li>Scrape the dried mixture off the watch glass, crush to powder, and store at room temperature in the dark. The fluorescent food is now ready to be fed to the larvae.</li>
	<li>On the 7 dpf, feed the fluorescent food to larvae in the same manner as done for previous feedings, that is provide 2 mg fluorescent food per dish (see steps 2.1&#8211;2.2.1). 
	NOTE: Ensure that the food is finely ground to a powder. Rubbing fluorescent food that is wrapped in weighing paper between thumb and forefinger is a useful method for ensuring finely ground food.</li>
</ol>
4. Prepare Concentrated Dosing Solutions of Test Compounds
<ol>
	<li>Dissolve each test compound in embryo medium to a concentration that is 2x the target dose. If testing of a dose range is desired, prepare multiple concentrated dosing solutions of the appropriate concentrations for the desired doses.</li>
	<li>Prepare enough of each concentrated dosing solution so that 24 larvae can be treated. Each larva will require 100 &#181;L dosing solution (the final volume per well is 200 &#181;L), so at the very least, 2.4 mL dose solution is needed for each treatment group; 2.5 mL would be an appropriate volume.</li>
	<li>If a solvent (i.e., dimethyl sulfoxide) was used for initial solubilizing of test compound, prepare the appropriate vehicle control dose (i.e., same amount of solvent as the compound treatment but without the compound). And, as was done for each compound-treated group, prepare enough vehicle solution to treat 24 larvae.</li>
</ol>
5. Transfer Larvae to a 96-well plate and Apply Treatments
<ol>
	<li>After larvae have been allowed to feed on fluorescent food for 2 h, use a transfer pipette to move them to a rinsing dish, as was done after feeding on prior days.</li>
	<li>After each larva is rinsed, withdraw it along with 100 &#181;L embryo medium, and move it into a well of a 96-well polystyrene conical-bottom multi-well plate (see Table of Materials), dispensing the full 100 &#181;L embryo medium into the well with the larva.</li>
	<li>Once the required numbers of larvae have been transferred to the 96-well-plate, add 100 &#181;L of the 2&#215; concentrated dosing solutions to each well. 
	NOTE: The use of a 12-channel multichannel pipette facilitates rapid dosing of the larvae (12-at-a-time). To avoid accidentally adding the wrong treatment, keep close track of which larvae have been dosed with which treatment and be sure to change pipette tips between treatments.</li>
</ol>
6. Measure Initial and Subsequent Fluorescence from Each Well
<ol>
	<li>After adding the dose solutions, place the 96-well-plate into a plate spectrophotometer capable of exciting and detecting emissions from the fluorescent label. 
	NOTE: For the yellow-green label used (see the Table of Materials), the appropriate wavelengths of light are exciting at 505 nm and detecting at 515 nm.</li>
	<li>Read the fluorescence of the 96-well-plate from the bottom 5 times in immediate succession without shaking the plate. Use the minimum value of the 5 readings from each well as the initial fluorescence from the well. 
	NOTE: The reason that 5 readings are taken whenever the plate is read is that larvae sometimes swim in the path of the excitation light and the food in their gut emits a very large signal which is unrepresentative of the released feces. Taking the minimum of the 5 reads can help to avoid using artificially high measurements from un-transited food.</li>
	<li>Read the fluorescence of the 96-well-plate (as was done for the initial reading) every 20 min for the first 2 h post-dosing, every 30 min for hours 3 and 4 post dosing, and then once every hour for hours 5, 6, 7, and 8 post-dosing.
	<ol>
		<li>Take caution to not disturb (by shaking the 96-well-plate) settled fecal matter between reads and incubate the larvae at 28 &#177; 1 &#176;C between reads.</li>
	</ol>
	</li>
	<li>Incubate the larvae over night at 28 &#177; 1 &#176;C and read the fluorescence from the 96-well-plate the following morning around the same time that the larvae were dosed the day before. Use this measurement as the 24 h post-dose fluorescence.</li>
</ol>
7. Analyze the Data
NOTE: Here we calculate accumulation per well and group averages for each time point.
<ol>
	<li>To calculate accumulation per well, using only minimum values from each of the 5 reads, subtract the initial value from each time point&#39;s value. Perform this calculation for the initial value as well (this results in an initial accumulation of zero for each well).
	<ol>
		<li>If the accumulation in a well at the 24 h time point is less than 150 relative fluorescent units, exclude that well from further analysis; these low values are most likely due to low or no intake of the fluorescent food by the larvae during feeding, and thus those larvae are not good subjects for measuring transit time.</li>
	</ol>
	</li>
	<li>For each time point, calculate the average accumulation for wells containing co-treated larvae, as well as standard error of those averages.</li>
	<li>Plot the average accumulation on the Y-axis versus time on the X-axis for each treatment group and compare the areas under those curves (AUCs). 
	NOTE: Treatments that significantly slow or accelerate transit time will have AUCs significantly smaller or larger, respectively, than the vehicle-treated group.</li>
</ol>

<ol>
	<li>Berghmans, 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=Zebrafish+based+assays+for+the+assessment+of+cardiac,+visual,+and+gut+function+-+Potential+safety+screens+for+early+drug+discovery.">Zebrafish based assays for the assessment of cardiac, visual, and gut function - Potential safety screens for early drug discovery.</a> Journal of Pharmacological and Toxicological Methods. 58 (1), 59-68 (2008).</li><li>Field, H. A., Kelley, K. A., Martell, L., Goldstein, A. M., Serluca, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gastrointestinal+physiology+using+a+novel+intestinal+transit+assay+in+zebrafish.">Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish.</a> Neurogastroenterology and Motility. 21, 304-312 (2009).</li><li>Kanungo, J., Cuevas, E., Ali, S., Paule, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+in+drug+safety+assessment.">Zebrafish model in drug safety assessment.</a> Current Pharmaceutical Design. 20 (34), 5416-5429 (2014).</li><li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496, 498-503 (2013).</li><li>Shepherd, I., Eisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+zebrafish+enteric+nervous+system.">Development of the zebrafish enteric nervous system.</a> Methods in Cell Biology. 101, 143-160 (2011).</li><li>Holmberg, A., Olsson, C., Holmgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+endogenous+and+exogenous+nitric+oxide+on+gut+motility+in+zebrafish+Danio+rerio+embryos+and+larvae.">The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae.</a> The Journal of Experimental Biology. 209, 2472-2479 (2006).</li><li>Olsson, C., Holmber, A., Holmgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+enteric+and+vagal+innervation+of+the+zebrafish+(Danio+rerio)+gut.">Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut.</a> The Journal of Comparative Neurology. 508, 756-770 (2008).</li><li>Fleming, A., Jankowski, J., Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+gut+function+and+disease+changes+in+a+zebrafish+larvae+model+of+inflammatory+bowel+disease:+A+feasibility+study.">In vivo analysis of gut function and disease changes in a zebrafish larvae model of inflammatory bowel disease: A feasibility study.</a> Inflammatory Bowel Disease. 16 (7), 1162-1172 (2010).</li><li>Cook, D., 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=Lessons+learned+from+the+fate+of+AstraZeneca's+drug+pipeline:+A+five-dimensional+framework.">Lessons learned from the fate of AstraZeneca's drug pipeline: A five-dimensional framework.</a> Nature Reviews Drug Discovery. 13 (6), 419-431 (2014).</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). , (2000).</li><li>Cassar, S., Huang, X., Cole, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+predicting+drug+effects+on+gut+transit+time+using+larval+zebrafish.">A high-throughput method for predicting drug effects on gut transit time using larval zebrafish.</a> Journal of Pharmacolical and Toxicological Methods. 76, 72-75 (2015).</li><li>McKim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+tiered+approach+to+in+vitro+predictive+toxicity+screening:+A+focus+on+assays+with+in+vivo+relevance.">Building a tiered approach to in vitro predictive toxicity screening: A focus on assays with in vivo relevance.</a> Combinatorial Chemistry and High Throughput Screening. 13 (2), 188-206 (2010).</li><li>Bruni, G., Lakhani, P., Kokel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+novel+neuroactive+drugs+through+high-throughput+behavior-based+chemical+screening+in+the+zebrafish.">Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish.</a> Frontiers in Pharmacology. 5, 153 (2014).</li><li>Mandrell, D., 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=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17, 66-74 (2012).</li></ol>

The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the manuscript. S. Cassar, X. Huang, and T. Cole are employees of AbbVie and have no additional conflicts of interest to disclose.

The novel spectrophotometry method for measuring zebrafish larvae GI transit time, presented here, is an efficient assay that can predict treatment effects on mammalian GI-function. Although the assay has low sensitivity, it has high positive predictivity, which is acceptable for first tier assays employed for paring down the number of candidate treatments based on toxicity12. This method is easier to execute, has higher throughput, and uses less animal handling steps than fluorescent microscopy.
There are technical challenges inherent in this method. Catching individual larvae after feeding the fluorescent food and transferring them into individual wells is challenging at first. However, with practice, a skilled technician can fill a 96-well plate in less than 15 min. If, at a given time point, the plate is accidentally shaken and the fecal matter unsettled from the bottom of the wells before reading, the accumulation will appear to decrease. This can be avoided by moving the plate(s) carefully, without shaking. In our experience, normal movement, including that of the plate spectrophotometer motorized drawer, did not disturb the assay. Plate readers equipped with a heater (i.e., the plate does not have to be returned to incubator between measurements), and located near the assay lab bench could optimize for lower chance of disturbance, but this was not necessary in our experience.
Early attempts at the method did not include feeding on the days before the assay. Without those &#39;practice&#39; feedings, lower numbers of larvae consumed sufficient fluorescent food during the time allowed before treatment application. In those attempts, variation within treatment groups was greater, and more data was unusable due to low/no signal accumulation over time. Even with practice feeding, some larvae do not consume sufficient amounts of the fluorescent food, excluding data from those larvae decreases variation within groups and increases the ability to identify treatment effects. Starting with larger group sizes (i.e., 24 versus 12) allows for sufficient numbers of larvae contributing useful data to the analysis.
The fluorescent microscopy reveals that food transit was nearly complete by 24 h in atropine-treated larvae (not shown), however the microplate data from the final time point reflect lower final signal in the atropine group, compared to vehicle group. Conversely, higher final microplate fluorescence was associated with treatments that accelerated transit time, even though the vehicle-treated larvae have apparently voided their GI tract before the final time point. Based on the random assignment to treatment of larvae fed fluorescent food, we assume equivalent consumption of the fluorescent food, on average, among treatment groups. Given this, and based on the patterns described above, fluorescence from fecal matter declines with time spent in the larval GI tract or faster transit adds fluorescence to the feces somehow. The actual cause is unknown and has not been interrogated, however the assay still is capable of measuring and comparing transit time among groups.
The employment of this method in detecting toxic GI effects from small molecule compounds is one application. Other possible applications include disease modeling (e.g., irritable bowel syndrome) and novel therapy discovery for such diseases, or for discovering pro-kinetic compounds. Furthermore, coupled with transgenic models, this method could be used to interrogate the role of genes in normal GI transit, as well as transit disorders, including enteric neuron deficiency. The zebrafish larva offers a whole organism platform on a scale that approximates that of cell culturing, but since there are multiple tissues and myriad cell types functioning together in the zebrafish, systems biology questions can be asked and answered using this model.
With advancements in technology, and new applications thereof, efficiency in the conduct of zebrafish toxicity tests will continue to improve. Larval zebrafish handling methods and assays are continuing to improve in terms of higher throughput13,14. The novel method presented here is one example of an improvement that may make zebrafish studies more impactful.

Zebrafish (Danio rerio) are used to model vertebrate biology and predict drug toxicity and/or efficacy; new applications in these fields emerge each year. The advantages of zebrafish over mammalian models include their fecundity, small size, and transparency through organogenesis. Zebrafish are used to predict drug candidate acute toxicity, as well as to assess compound impact on organ function, e.g., cardiac, ocular, gastrointestinal (GI)1,2. Zebrafish development and physiology are similar to those of mammals in many ways3 and 80% of genes that are associated with human disease have a zebrafish homolog4.
The GI tract of zebrafish has similar physiology to the mammalian GI tract but has a simpler architecture5. Zebrafish have no stomach; the anterior intestinal bulb acts as a food depot. Gene expression in zebrafish intestine has many similarities to that of the mammal5. Like mammals, the enteric nervous system of zebrafish controls gut motility, and intestinal innervation mirrors that of other vertebrates6,7. Based on these similarities, functional disorders of the human intestine have been studied in zebrafish, using methods derived from mammalian models8.
GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. A review of programs in a major pharmaceutical company during 2005&#8211;2010 revealed GI safety as the principal cause for 9% of clinical study terminations9. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, compound, and labor. A predictive high-throughput assay for GI transit can provide flexibility to compound toxicity testing, and deliver 3Rs impact. A novel method offering such an assay is presented by the protocol described herein. This high-throughput assay could be employed early in drug development to prioritize candidates, and contribute to the reduction of GI safety testing in larger species.

<table border="0" cellpadding="0" cellspacing="0" style="width:1031px;" width="1030">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Wild type zebrafish breeding pair</td>
			<td style="width:260px;">Various sources - for example ZIRC (Zebrafish International Resource Center)</td>
			<td style="width:131px;">ZL-1</td>
			<td style="width:424px;">Adult wild type zebrafish of AB lineage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.7-liter Breeding Tank - Beach Style Design</td>
			<td style="width:260px;">Tecniplast</td>
			<td style="width:131px;">1.7L SLOPED</td>
			<td style="width:424px;">Breeding tank</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean sea salt</td>
			<td style="width:260px;">Intant Ocean</td>
			<td style="width:131px;">SS15-10</td>
			<td style="width:424px;">Dehydrated sea salt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">First Bites larval fish food</td>
			<td style="width:260px;">Hikari</td>
			<td style="width:131px;">20095</td>
			<td style="width:424px;">Powdered larval fish food</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yellow-green (505/515) Fluospheres</td>
			<td style="width:260px;">Invitrogen</td>
			<td style="width:131px;">F8827</td>
			<td style="width:424px;">Fluorescent label</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V-bottom 96-well polystyrene microplates</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">249570</td>
			<td style="width:424px;">Multiwell microplate with V-shaped bottom in each well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atropine</td>
			<td style="width:260px;">Sigma Aldrich</td>
			<td style="width:131px;">A0132</td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amitriptyline</td>
			<td style="width:260px;">Sigma Aldrich</td>
			<td style="width:131px;">A8404</td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegaserod</td>
			<td style="width:260px;">Sigma Aldrich</td>
			<td style="width:131px;">SML1504</td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metoclopramide</td>
			<td style="width:260px;">Sigma Aldrich</td>
			<td style="width:131px;">M0763</td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Erythromycin</td>
			<td style="width:260px;">Sigma Aldrich</td>
			<td style="width:131px;">E5389</td>
			<td style="width:424px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectramax M2e microplate reader</td>
			<td style="width:260px;">Molecular Devices</td>
			<td style="width:131px;">Spectramax M2e</td>
			<td style="width:424px;">A multi-well plate spectrophotometer capable of fluorescent excitation and emission detection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SoftMax Pro</td>
			<td style="width:260px;">Molecular Devices</td>
			<td style="width:131px;">SoftMax Pro</td>
			<td style="width:424px;">Software for spectrophotometer data acquisition</td>
		</tr>
	</tbody>
</table>

high-throughput measurement of gut transit time using larval zebrafish

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

This method, which uses plate-based spectrophotometry to assess GI transit, can be used as a high-throughput replacement of fluorescent microscopy, which is a lower throughput method for assessing the same function (Figure 1). To generate the data in Figure 1, identically treated fish were analyzed for GI transit using either fluorescence microscopy (representative images shown) or spectrophotometry at 4 time points, 0, 4, 8, and 24 h post-dosing; comparison of the data from those experiments gave highly correlated results (linear regression of data r2 = 0.95). The linear regression has a negative slope because microscopy measures the retained fluorescence signal and spectrophotometry measures the transited signal.
The effects of compounds of disparate mechanisms, with well-established GI activity in humans, can be detected in zebrafish using the spectrophotometry assay (Figure 2). Compared to vehicle-treated controls, atropine (4.2 &#181;M) and amitriptyline (5 &#181;M) slowed GI transit, while tegaserod (3.3 &#181;M) and metoclopramide (33 &#181;M) accelerated transit time. Erythromycin (14 &#181;M), expected to accelerate transit time had no effect as measured by this method. Treatment group sizes were 24 before removing data from larvae with no or very low signal. The AUC for the average signal per time point was compared between compound-treated and vehicle-treated groups using Tukey&#39;s Honestly Significant Difference for type-1-error control. Effects were considered significant only when p &#8804; 0.05. The concentrations used for the above treatments were the maximum tolerated doses, determined in a prior experiment and defined as the highest dose with no observable adverse effect by gross observation.
The spectrophotometry assay can measure dose-dependent effects of compounds. Figure 3 provides data demonstrating that atropine slows GI transit dose-dependently in zebrafish larvae. The lowest dose of atropine tested, 0.042 &#181;M, had no significant effect, while the two higher concentrations each had significant impact, 0.42 &#181;M having less of an effect than 4.2 &#181;M.
A new assay can be assessed by testing positive and negative controls, that is, compounds known to be active and inactive respectively in the target system (in this case the target system is the mammalian GI transit). For the spectrophotometry assay, 18 active (positive) controls and 6 inactive (negative) controls were tested. Based on these experiments, the spectrophotometry assay has high positive predictive value (90.9%), but low sensitivity (55.6%) and negative predictive value (38.5%). These values are derived from the data presented in Table 1. They reflect, in practice, that if the zebrafish transit time is impacted by a treatment, mammalian transit is likely to be impacted. However, if there is no effect on zebrafish transit time, this is not predictive of mammalian effect.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58497/58497fig1.jpg" /> 
Figure 1: Fluorescent food transit is detected as a loss of signal from microscopic imaging and a corresponding gain in signal by plate-based spectrophotometry. (A) Representative microscopic images from analysis of atropine (4.2 &#181;M) effect on GI transit time. (B) Average signal quantified from microscopic images is highly (negatively) correlated with signal from voided fecal matter (spectrophotometry) from identically treated fish. Data from atropine-treated fish are in red. This figure copied with permission from Cassar et al.11. <a href="https://www.jove.com/files/ftp_upload/58497/58497fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58497/58497fig2.jpg" /> 
Figure 2: Analyzing fluorescent signal accumulation over time from a multi-well plate allows identification of treatments that change GI transit rate. Asterisk (*) indicates significantly different AUC than vehicle-treated fish. Error bars represent the standard error of the mean signal for larvae in the treatment group per time point. This figure has been reused with permission from Cassar et al.11. <a href="https://www.jove.com/files/ftp_upload/58497/58497fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58497/58497fig3.jpg" /> 
Figure 3: Atropine dose-dependently slows zebrafish gut transit time as reflected by fluorescent spectrophotometry of fecal accumulation over time. Asterisk (*) indicates significantly different AUC than vehicle-treated fish. Error bars represent the standard error of the mean signal for larvae in the treatment group per time point. This figure is reused with permission from Cassar et al.11. <a href="https://www.jove.com/files/ftp_upload/58497/58497fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58497/58497table1.jpg" /> 
Table 1: GI activity of 24 compounds in mammals and fish. This table is reused with permission from Cassar et al.11.

Watch this Scientific Journal Video about High-throughput Measurement of Gut Transit Time Using Larval Zebrafish at JoVE.com

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

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

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

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7.4K Views.  AbbVie. The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Video: High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...