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/pubmed?term=((Zebrafish+based+assays+for+the+assessment+of+cardiac%2C+visual%2C+and+gut+function+-+Potential+safety+screens+for+early+drug+discovery%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+gastrointestinal+physiology+using+a+novel+intestinal+transit+assay+in+zebrafish%5BTitle%5D)+AND+%22Neurogastroenterology+and+Motility%22%5BJournal%5D)">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/pubmed?term=((Zebrafish+model+in+drug+safety+assessment%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">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/pubmed?term=((The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/21550442">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/pubmed?term=((The+effects+of+endogenous+and+exogenous+nitric+oxide+on+gut+motility+in+zebrafish+Danio+rerio+embryos+and+larvae%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">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/pubmed?term=((Development+of+enteric+and+vagal+innervation+of+the+zebrafish+%28Danio+rerio%29+gut%5BTitle%5D)+AND+%22The+Journal+of+Comparative+Neurology%22%5BJournal%5D)">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/pubmed?term=((In+vivo+analysis+of+gut+function+and+disease+changes+in+a+zebrafish+larvae+model+of+inflammatory+bowel+disease%3A+A+feasibility+study%5BTitle%5D)+AND+%22Inflammatory+Bowel+Disease%22%5BJournal%5D)">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/pubmed?term=((Lessons+learned+from+the+fate+of+AstraZeneca%27s+drug+pipeline%3A+A+five-dimensional+framework%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+Zebrafish+Book.+A+Guide+for+the+Laboratory+Use+of+Zebrafish+%28Danio+Rerio%29%22">The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio Rerio)</a>. , University of Oregon Press. Eugene, OR, USA. (2000).</li>
<li>Cassar, S., Huang, X., Cole, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+method+for+predicting+drug+effects+on+gut+transit+time+using+larval+zebrafish%5BTitle%5D)+AND+%22Journal+of+Pharmacolical+and+Toxicological+Methods%22%5BJournal%5D)">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/pubmed?term=((Building+a+tiered+approach+to+in+vitro+predictive+toxicity+screening%3A+A+focus+on+assays+with+in+vivo+relevance%5BTitle%5D)+AND+%22Combinatorial+Chemistry+and+High+Throughput+Screening%22%5BJournal%5D)">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/pubmed?term=((Discovering+novel+neuroactive+drugs+through+high-throughput+behavior-based+chemical+screening+in+the+zebrafish%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">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/pubmed?term=((Automated+zebrafish+chorion+removal+and+single+embryo+placement%3A+optimizing+throughput+of+zebrafish+developmental+toxicity+screens%5BTitle%5D)+AND+%22Journal+of+Laboratory+Automation%22%5BJournal%5D)">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><tbody><tr><td>Wild type zebrafish breeding pair</td><td>Various sources - for example ZIRC (Zebrafish International Resource Center)</td><td>ZL-1</td><td>Adult wild type zebrafish of AB lineage</td></tr><tr><td>1.7 L Breeding Tank - Beach Style Design</td><td>Tecniplast</td><td>1.7L SLOPED</td><td>Breeding tank</td></tr><tr><td>Instant Ocean sea salt</td><td>Intant Ocean</td><td>SS15-10</td><td>Dehydrated sea salt</td></tr><tr><td>First Bites larval fish food</td><td>Hikari</td><td>20095</td><td>Powdered larval fish food</td></tr><tr><td>Yellow-green (505/515) Fluospheres</td><td>Invitrogen</td><td>F8827</td><td>Fluorescent label</td></tr><tr><td>V-bottom 96-well polystyrene microplates</td><td>Thermo Fisher Scientific</td><td>249570</td><td>Multiwell microplate with V-shaped bottom in each well</td></tr><tr><td>Atropine</td><td>Sigma Aldrich</td><td>A0132</td><td></td></tr><tr><td>Amitriptyline</td><td>Sigma Aldrich</td><td>A8404</td><td></td></tr><tr><td>Tegaserod</td><td>Sigma Aldrich</td><td>SML1504</td><td></td></tr><tr><td>Metoclopramide</td><td>Sigma Aldrich</td><td>M0763</td><td></td></tr><tr><td>Erythromycin</td><td>Sigma Aldrich</td><td>E5389</td><td></td></tr><tr><td>Spectramax M2e microplate reader</td><td>Molecular Devices</td><td>Spectramax M2e</td><td>A multi-well plate spectrophotometer capable of fluorescent excitation and emission detection.</td></tr><tr><td>SoftMax Pro</td><td>Molecular Devices</td><td>SoftMax Pro</td><td>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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Biology

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

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Developmental Biology

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

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

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

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

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

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

Immunology and Infection

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Bioengineering

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

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

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

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

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Summary

Explore More Videos

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

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

Microgavage of Zebrafish Larvae

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

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