Name: high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake
Uploaded: 2016-10-27T15:00:00
Description: Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

The authors thank Meng-Chieh Shen for images, Jennifer Anderson for providing helpful comments on the manuscript, and members of the Farber laboratory for their contributions in developing these techniques. This study was funded by NIDDK-NIH award RO1DK093399 (S.A.F.), RO1GM63904 (The Zebrafish Functional Genomics Consortium: PI Stephen Ekker and Co-PI S.A.F), and F32DK096786 (J.P.O.). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Additional support was provided by the G. Harold and Leila Y. Mathers Charitable Foundation to the laboratory of S.A.F and the Carnegie Institution for Science endowment.

These protocols have been approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee (protocol no. 139).
1. Animal Preparation
<ol>
	<li>Maintain adults and larvae at 28 &#176;C on a 14 hr:10 hr light:dark cycle. Feed adults twice daily with shell free Artemia (decapsulated, non-hatching, starting at 14 dpf) and commercial micropellets.</li>
	<li>These protocols are optimized for the use of 6-7 dpf larvae collected by natural spawning of the AB background. Protocols can...

<ol>
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C., Amsterdam, A., Soroka, C., Boyer, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+zebrafish+identifies+the+mutants+vps18,+nf2+and+foie+gras+as+models+of+liver+disease.">A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.</a> Development. 132 (15), 3561-3572 (2005).</li><li>Matthews, R. 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Y., 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=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), 36474 (2012).</li><li>Song, Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21 (9), 2042-2049 (2007).</li><li>Watts, S. A., Powell, M., D'Abramo, L. 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The techniques described here allow researchers to treat larval zebrafish with a lipid-rich feed, visualize dietary lipid processing in live larvae, and quantify larval food intake. To ensure success, special attention should be given to several critical steps. Commercial chicken eggs vary; to minimize potential variability we perform all assays on organic eggs from cage-free chickens that have not been enriched for omega-3 fatty acids. Lower feeding rates may be observed in fish younger than 6 dpf with remaining endogen...

The mechanisms by which the intestine regulates dietary lipid processing, the liver controls complex lipid synthesis and lipoprotein metabolism, and how these organs work with the central nervous system to control food intake are incompletely understood. It is of biomedical interest to elucidate this biology in light of the current epidemics of obesity, cardiovascular disease, diabetes, and non-alcoholic fatty liver disease. Studies in cell culture and mice have provided the majority of our understanding of the mechanistic relationships between dietary lipids and disease, and zebrafish (Danio rerio) are emerging as an ideal model to complement this work.
Zebrafish have similar gastrointestinal (GI) organs, lipid metabolism, and lipoprotein transport to higher vertebrates 1,2, develop rapidly, and are genetically tractable. The optical clarity of the larval zebrafish facilitates in vivo studies, a particular advantage for study of the GI system as its extracellular milieu (i.e., bile, microbiota, endocrine signaling) is virtually impossible to model ex vivo. In accordance, a body of research combining the genetic tractability and conduciveness to live imaging of zebrafish larvae with a variety of dietary manipulations (high-fat3,4, -cholesterol5, and -carbohydrate diets6,7), and models of cardiovascular disease8, diabetes9,10, hepatic steatosis11-13, and obesity14-16, are emerging to provide a host of metabolic insights.
An essential aspect of transitioning the larval zebrafish into metabolic research is the optimization of techniques developed in other model animals to the zebrafish and the development of novel assays that exploit the unique strengths of the zebrafish. This protocol presents techniques developed and optimized to feed larval zebrafish a lipid-rich meal, visualize dietary lipid processing from whole body to subcellular resolution, and measure food intake. Chicken egg yolk was chosen to compose the lipid-rich meal as it contains high levels of fats and cholesterol (lipids compose ~58% of chicken egg yolk, of which ~5% is cholesterol, 60% are triglycerides, and 35% are phospholipids). Chicken egg yolk provides more fat than typical commercial zebrafish micropellet foods (~15% lipids) and the advantage that it is a standardized feed with known percentages of specific fatty acids species, as zebrafish diets and feeding regiments have not been standardized across labs17. Moreover, fluorescent lipid analogs provided in the egg yolk visualize transport and accumulation of dietary lipids18, image cellular components including lipid droplets by acting both as vital dyes3 and through covalent incorporation into complex lipids, investigate metabolism through thin layer chromatography (TLC)19 and high performance liquid chromatography (HPLC) (S.A.F. unpublished data), and provide a quantitative assay for total food intake20.

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:127px;">A5040-25G</td>
			<td style="width:335px;">Anesthesia for larval zebrafish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Chicken eggs</td>
			<td style="width:143px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Organic, cage-free eggs, not enriched for omege-3 fatty acids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Ultrasonic processor 3000 sonicator</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">S-3000</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Sonabox acoustic enclosure</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">432B</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">1/8&#8221; tapered microtip</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">419</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Amber vials (4 ml, glass)</td>
			<td style="width:143px;">National Scientific</td>
			<td style="width:127px;">13-425</td>
			<td>Lipid storage; includes vials, open-top caps, and cap septa</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Incu-Shaker Mini&#160;</td>
			<td style="width:143px;">Benchmark</td>
			<td style="width:127px;">1222U12</td>
			<td>Incubated shaker for feeds</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C16&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3821</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C12&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3822</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3834</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D2183</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">TopFluor cholesterol&#160;</td>
			<td style="width:143px;">Avanti Polar Lipids Inc.</td>
			<td style="width:127px;">810255</td>
			<td>Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fatty acid-free BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">A0281-1G</td>
			<td>For TopFluor cholesterol solubilization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">M0387</td>
			<td>Mounting media for live larval imaging; 75 x 25 x 1 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Low melt agarose</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">BP165-25</td>
			<td>Mounting media for live larval imaging; 22 x 30</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">VWR microscope slides&#160;</td>
			<td style="width:143px;">VWR&#160;</td>
			<td style="width:127px;">16004-422</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Coverslips&#160;</td>
			<td style="width:143px;">Cover Glass</td>
			<td style="width:127px;">12-544A</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Super glue</td>
			<td style="width:143px;">Loctite</td>
			<td style="width:127px;">LOC01-30379</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">FluoroDish (glass bottom dish)</td>
			<td style="width:143px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:127px;">FD35-100</td>
			<td>Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Confocal microscope</td>
			<td>Leica Microsytems</td>
			<td style="width:127px;">SP-2, SP-5</td>
			<td>Microscope for high magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Stereoscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:127px;">SM21500</td>
			<td>Microscope for low magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Glass culture tubes&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">73500-13100</td>
			<td>Lipid extraction; (13 x 100 mm; 13 ml)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Savant SpeedVac Plus&#160;</td>
			<td style="width:143px;">ThermoQuest</td>
			<td style="width:127px;">SC210A</td>
			<td>Lipid extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Whatman Scientific</td>
			<td style="width:127px;">WC4855-821</td>
			<td>Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Analtech, Inc.</td>
			<td style="width:127px;">66911</td>
			<td>Food intake assay; Direct replacement for Whatman Scientific TLC plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9410 Variable Mode Imager</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:127px;">9410</td>
			<td>Fluorescent plate reader for food intake assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">ImageQuant software</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>29000605</td>
			<td>Analysis of food intake assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">5 3/4&#8217; Wide bore, borosilicate disposable pasteur pipets&#160;&#160;&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">63A53WT</td>
			<td>Transfering larvae</td>
		</tr>
	</tbody>
</table>

high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

When fed on a rocker at 29-31 &#176;C, the majority of healthy larvae (&#8805;95%) will eat within 1 hr. Upon consuming the egg yolk emulsion, the larval intestine darkens in color. Very dark intestines can be observed at 2 hr (Figure 1). If larvae are unfed or fail to feed, the intestine remains clear. Larvae fed egg white exhibit a distended intestinal lumen that does not darken in color.
<img alt="Figure 1" src="/files/ftp_upload/54735/54735...

Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

The overall goal of this experimental protocol is to feed larval Zebrafish a lipid rich meal that can be spiked with a fluorescent lipid analog to mount the larvae to visual lipid uptake dynamics and to quantify dietary lipid intake. This method can help answer key questions in the fields of metabolism and gastrointestinal biology, such as how dietary lipids are metabolized and how food intake is regulated. The main advantage of this technique is that fluorescent lipid analogs can efficiently be delivered in dietary liposomes.The optical clarity of the larval Zebrafish enables microscopy to reveal subcellular lipid processing. To begin, suspend one milligram of powdered fluorescent lipid analog in two milliliters of 100%chloroform for a final concentration of 0.5 micrograms per microliter. Aliquot the analog into amber glass tubes with screw caps compatible with the solvent that contain a Teflon gasket.Store these stocks in the freezer. Add 10.4 microliters of the 0.5 microgram per microliter fluorescent lipid analog stock to a 1.5 milliliter tube and dry under a stream of nitrogen, taking care not to blow the lipid out of the tube. Immediately resuspend the dried lipid in five microliters of 100%ethanol by pipetting a stream down the sides of the tube.Then, add 95 microliters of embryo medium and mix thoroughly by pipetting up and down until the mixture appears homogeneous. Protect the tube from light and store it on ice until needed. To make a fluorescent cholesterol liposome solution, add enough fluorescent cholesterol analog stock into a 1.5 milliliter tube to make a final concentration of 2.5 micrograms per milliliter when it is added to the egg yolk emulsion.Dry the cholesterol stock under a stream of nitrogen, as before. Immediately resuspend the fluorescent cholesterol analog in 15 microliters of room temperature 100%ethanol by pipetting a stream down the side of the tube. And mix with 85 microliters of 1%fatty acid free BSA and water.It is essential to completely solubilize the fluorescent lipid analog in ethanol and embryo media prior to adding it to the egg yolk emulsion to ensure homogeneous distribution of the lipid in the feed and even availability to all larvae. Protect the tube from light with foil and store it on ice. To make the feeding solution, add 19 milliliters of embryo medium to a 50 milliliter conical tube, and then add a one milliliter egg yolk aliquot.Vortex the mixture for one to two minutes to make a homogeneous emulsion. Pour the mix through a fine strainer to remove protein conglomerates and collect it into a fresh 50 milliliter conical tube. Next, pulse sonicate the egg mix five times, one second on, one second off, with a 1/4 inch tapered microtip at six watts.Repeat the sonication program four additional times. This creates the liposomes, which should now be continuously rocked until use to maintain homogeneity. Add the appropriate amount of fluorescent lipid analogs to five milliliters of the liposome mixture immediately after sonication.Vortex at high speed for 30 seconds to incorporate the fluorescent lipid analogs into the liposomes. Protect the mixture from light by wrapping the tube in foil, and return it to the rocker. Add fifty larvae to a feeding dish, remove the embryo medium, and add 5 milliliters of feed.To encourage food intake, gently rock the larvae in an incubated rocker and shield from light if fluorescent lipid analogs are in use. At the end of the feeding period, wash the larvae in five milliliters of embryo medium three times. To immobilize the larvae, place a metal block on ice, overlay a tissue to prevent excessive cooling, and then place the larvae in the dish of embryo medium on top to cool.Observe the larvae at 10x under a dissecting microscope, and determine which of them have consumed the liposome feed. Larvae that have consumed liposomes will have a darkened intestine. It is critical to determine whether a larvae have fed by checking for a darkened intestine.Typically, one to five percent of the larvae will not eat, and could confound the experimental results if not identified and removed. For long-term, high magnification imaging with an inverted objective, first cool the larvae on a metal block on ice. Transfer a larva to a glass bottom dish, and remove the embryo medium by wicking with a tissue.Place a drop of 42 degree Celsius low melt 1.2%agarose on top of the larva, and immediately position with a poker. Remove the dish from the cold block and allow the agarose to sit for two to three minutes. Finally, add a sufficient volume of fresh embryo medium to cover the agarose and prevent desiccation.Specimens are now ready for imaging. At the end of a liposome feed, pool a minimum of 10 washed larvae in a 1.5 milliliter tube. Remove the embryo medium and then snap freeze the larvae.Repeat in a separate tube with 10 unfed larvae as a control. Keeping the sample on ice, add 100 microliters of homogenization buffer to each tube, and then homogenize with a microtip sonicator. Transfer the tissue to a 13 milliliter culture tube.Add 375 microliters of one to two chloroform to methanol to each homogenization buffer slurry. Vortex for 30 to 60 seconds, and then incubate for 10 minutes at room temperature. Add a further 125 microliters of chloroform, and vortex for 30 seconds.Next, add 125 microliters of 200 millimolar TRIS pH 7.5, vortex for 30 seconds, and then centrifuge at 2000 times g for five minutes protected from light. Remove samples from the centrifuge carefully to avoid disturbing the separate phases. Using a clean glass pipette, collect the lower organic phase and transfer it into a clean 13 milliliter glass tube.Carefully avoid transferring any of the upper aqueous or middle larval debris phases and discard them. Dry the organic phase under vacuum at 0.12 atmospheric pressure, while protecting it from light, taking care to stop once the liquid has evaporated. Resuspend a single sample in 10 microliters of two to one chloroform to methanol and spot the entire sample volume onto a channeled, thin-layer chromatography plate.Repeat with the remaining samples, spotting in evenly spaced intervals. Finally, scan the plate with a fluorescent plate reader. Examination of larvae fed on a rocker under experimental conditions shows darkened intestines, typical of fish that have consumed the egg yolk emulsion.Unfed larvae usually have clear intestines, so those that have not consumed the diet formula during the feeding period are easily distinguished. A larva fed with a fluorescent lipid analog visualizes the transport and accumulation of this dietary lipid. Here, the fluorescent signal is seen throughout the digestive organs after eight hours of feeding.Various lipid analogs are incorporated in, and thus visualize, unique sets of cellular structures since they are differentially metabolized into complex lipids. Here, Fluorescent-C16 and C12 analogs label lipid droplets. Fluorescent-C5 labels hepatic and pancreatic ducts, lipid droplets, cellular membranes, and arterial networks.And Fluorescent-C2 analog labels hepatic and pancreatic ducts and cellular membranes. Larval food intake assays were used to study the feeding of wild-type versus transgenic larvae overexpressing Apolipoprotein A4B1, and showed that over four hour feedings the transgenic larvae ate less than the wild-type. Breedings were normalized used paired, unfed control larvae.Quantification of the fluorescence of wild-type and transgenic larvae, which overexpress Apo A4 following feeding, show significantly reduced ingestion by the transgenic larvae when compared to the wild-type. Once mastered, the egg yolk fluorescent lipid analog feed can be prepared in about 15 minutes. Food intake can be quantified from 10 to 12 pooled samples in approximately four hours.While attempting this procedure, it's important to remember to screen larvae for developmental defects that may impair food intake, such as a malformed jaw, an underdeveloped intestine, or decreased mobility. Methods like pharmaceutical treatments or genome engineering can be incorporated into this experimental protocol in order to answer additional questions, like how specific proteins and cell signaling pathways regulate food intake and lipid metabolism. After it's development, this technique paved the way for researchers in the fields of gastrointestinal physiology and cell biology to visualize dietary lipid transport and accumulation in larval Zebrafish.After watching this video, you should have a good understanding of how to prepare a lipid rich larval Zebrafish feed, screen larvae for food intake, mount larvae for short and long-term imaging, and quantify food intake. Don't forget that working with a sonicator can be hazardous. Ear protection should be worn while performing this procedure.

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