Name: high throughput danio rerio energy expenditure assay
Uploaded: 2016-01-27T17:00:00
Description: Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
<ol>
	<li>Prepare E3 embryo medium8. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g&#160;KCl, 5.8g CaCl2&#9679;2H2O, and 9.78 g MgCl2&#9679;6H2O in 1.95 L ddiH2O....

<ol>
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Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 ...

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through &#946;-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 - 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P&#60;0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are ...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

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