We thank members of the UNC Zebrafish Aquaculture facility for maintenance of the zebrafish. We would also like to thank Diagnosys, LLC for assistance with the setup of the ERG apparatus. Additional thanks go to Dr. Portia McCoy and the laboratory of Dr. Ben Philpot for assistance with electrophysiological methods. We also wish to thank Lizzy Griffiths for her illustration of a larval zebrafish. This work was supported by National Institutes of Health awards F32 EY022279 (to J.D.C) and R21 EY019758 (to E.R.W).

Animal upkeep and experimental protocols were approved by the Institutional Animal Care and Use Committees of the University of North Carolina at Chapel Hill, and meet all requirements of the NIH Office of Laboratory Animal Welfare and the Association for Assessment and Accreditation of Laboratory Animal Care International. 
NOTE: To obtain larvae for ERG analysis, published protocols for standard zebrafish husbandry and maintenance were employed18. Larvae are obtained through natural breeding and housed under a 14 hr light/1...

<ol>
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In this protocol a simple procedure for ERG recordings of larval zebrafish is detailed. This procedure allows for a quick and comprehensive assay of visual function.There are several critical steps throughout the procedure that should be kept in mind. The zebrafish larvae should be healthy before the experiment to prevent death during potential drug treatments and ensure prolonged livelihood during the ERG recordings. In addition, it is important that the larvae utilized in experiments are closely age-matched. This is du...

The electroretinogram (ERG) is a noninvasive electrophysiological method that has been used extensively in the clinic for determining the function of the retina in humans. The electrical activity in response to a light stimulus is measured by placing recording electrodes on the outer surface of the cornea. The characteristics of the stimulus paradigm and the response waveform define the retinal neurons contributing to the response. This method has been adapted for use with a number of animal models including mice and zebrafish. The typical vertebrate ERG response has four principal components: the a-wave, which is a cornea-negative potential derived from photoreceptor cell activity; the b-wave, a cornea-positive potential derived from the ON bipolar cells; the d-wave, a cornea-positive potential interpreted as the activity of the OFF bipolar cells; and the c-wave, which occurs several seconds after the b-wave and reflects activity in M&#252;ller glia and the retinal pigment epithelium1-4. Additional references for understanding the history and principles of ERG analysis in humans and model animals are the online textbook, Webvision, from the University of Utah and texts such as the Principles and Practice of Clinical Electrophysiology of Vision4,5.
Daniorerio (zebrafish) has long been favored as a model for vertebrate development, due to its rapid maturation and transparency, which allows for noninvasive morphological analysis of organ systems, behavioral assays and both forward and reverse genetic screens (for review, see Fadool and Dowling6). Zebrafish larvae are highly amenable to genetic and pharmacological manipulation, which, when coupled with their high fecundity, make them an excellent animal model for high-throughput biological analyses. The higher ratio of cones to rods in larval zebrafish &#8211; roughly 1:1 compared to mice (~3% cones) &#8211; make them particularly useful for the study of cone function7-9.
In the vertebrate retina, cones develop before rods10. Interestingly, zebrafish cones are operative as early as 4 dpf, allowing for selective electrophysiological analysis of cones at that stage6,11,12. In contrast, ERG responses in rods appear between 11 and 21 dpf13. Therefore, zebrafish larvae at 4-7 dpf serve functionally as an all-cone retina. However, the native photopic ERG response of 4-7 dpf larvae is dominated by the b-wave. Application of pharmacological agents, such as L-(+)-2-amino-4-phosphono-butyric acid (L-AP4), an agonist for the metabotropic glutamate (mGluR6) receptor expressed by the ON bipolar cells, effectively blocks the generation of the b-wave and reveals the isolated cone mass receptor potential, (the &#8220;a-wave&#8221;)14-17.
Here we describe a simple and reliable method for ERG analysis using commercially available ERG equipment designed for use with mice that have been adapted for use with zebrafish larvae. This system can be utilized on zebrafish larvae of varying genetic backgrounds, as well as those treated with pharmacological agents, to aid researchers in the identification of signaling pathways that contribute to visual sensitivity and light adaptation16. The experimental procedures outlined in this protocol will guide investigators in the use of ERG analysis to answer a variety of biological questions pertaining to vision, and demonstrate the construction of a flexible ERG setup.

<table border="0" cellpadding="0" cellspacing="0" width="2170">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"><a name="RANGE!A1:D30">Name of the Material/Equipment</a></td>
			<td style="width:168px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td style="width:1504px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday cage</td>
			<td>80/20 Inc</td>
			<td>custom</td>
			<td>Custom designed aluminum &#34;Industrial Erector Set&#34; for Cage framework</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PVA sponge</td>
			<td>Amazon</td>
			<td>B000ZOWG1C</td>
			<td>Provides a soft, moist platform for placement of zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 ml Sterile Filter systems</td>
			<td>Corning</td>
			<td>431154</td>
			<td>Filtering solutions to prevent small articulates from blocking micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Espion E2</td>
			<td>Diagnosys, LLC</td>
			<td>contact</td>
			<td>Modular electrophysiology system capable of generating visual stimuli for any stimulator and digital recording and analysis of responses using propietary software, more information at http://www.diagnosysllc.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Colordome</td>
			<td>Diagnosys, LLC</td>
			<td>contact</td>
			<td>Light stimulator with RGB LED and Xenon light sources for Ganzfeld ERG, more information at http://www.diagnosysllc.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Drummond</td>
			<td>3-000-024-R</td>
			<td>Holding and positioning the recording microelectrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic ring stand</td>
			<td>Drummond</td>
			<td>3-000-025-MB</td>
			<td>Holding and positioning of the camera and refrence electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lead extensions</td>
			<td>Grass Technologies</td>
			<td>F-LX</td>
			<td>Spare female to male 1.5 mm lead cables for connecting electrodes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:333px;">Male Pin to Female SAFELEAD Adaptor</td>
			<td>Grass Technologies</td>
			<td>DF-215/10</td>
			<td>Connecting 2 mm pins to 1.5 headboard pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Window screen frame (metal) and spline</td>
			<td>Lowes or Home Depot</td>
			<td>various</td>
			<td>For attaching copper mesh to Faraday cage framework</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Steriflip 50 ml filters</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td>Filtering solutions to prevent small articulates from blocking micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BNC adaptor</td>
			<td>Monoprice</td>
			<td>4127</td>
			<td>Connecting camera to BNC cable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BNC cable</td>
			<td>Monoprice</td>
			<td>626</td>
			<td>Connecting camera to video adaptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera lens</td>
			<td>Navitar</td>
			<td>1582232</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera coupler</td>
			<td>Navitar</td>
			<td>1501149</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luna BNC to VGA + HDMI Converter</td>
			<td>Sewell</td>
			<td>SW-29297-PRO</td>
			<td>BNC to VGA adaptor allowing camera image to project on computer monitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APB</td>
			<td>Sigma</td>
			<td>A1910</td>
			<td>mGluR6 agonist, blocks b-wave allowing analysis of the isolated cone mass receptor potential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosilicate glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td>Fire- polished borosilicate glass (metling temperature = 821&#176;C) with filament and dimensions of 1.5mm x 0.86 mm (outer diameter by inner diameter)&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P97 Flaming/Brown puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td>For pulling glass micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorbothane sheet</td>
			<td>Thorlabs</td>
			<td>SB12A</td>
			<td>Synthetic viscoelastic urethane polymer, placed under Passive Isolation Mounts and ERG platform to absorb shock and prevent slipping, can be cut to size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Breadboard</td>
			<td>Thorlabs</td>
			<td>B2436F</td>
			<td>Vibration isolation platfrom for ERG stimulator and zebrafish specimen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Isolation Mounts</td>
			<td>Thorlabs</td>
			<td>PWA074</td>
			<td>Provides vibration isolation to breadboard</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Copper mesh</td>
			<td>TWP</td>
			<td>022X022C0150W36T</td>
			<td>To line Faraday Cage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette pump</td>
			<td>VWR</td>
			<td>53502-233</td>
			<td>Used with Pasteur pipettes to carefully transfer zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pasteur pipettes</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td>Used with Pipette pump to carefully transfer zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera</td>
			<td>Watec</td>
			<td>WAT-902B</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine (MS-222)</td>
			<td>Western Chemical</td>
			<td>Tricaine-S</td>
			<td>Pharmaceutical-grade anesthetic,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-fil</td>
			<td>WPI</td>
			<td>MF28G-5</td>
			<td>Filling microelectrode holder and microelectrode glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microelectrode holder</td>
			<td>WPI</td>
			<td>MEH2SW15</td>
			<td>Holds glass microelectrode, connects to ERG equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reference Electrode</td>
			<td>WPI</td>
			<td>DRIREF-5SH</td>
			<td>Carefully break off last centimeter of casing to drain electrolyte and expose sintered Ag/AgCl pellet electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reference Electrode (alternative)</td>
			<td>WPI</td>
			<td>EP1</td>
			<td>Alternative to DRIREF-5SH. Ag/AgCl electrode that must be wired/soldered to connecting lead</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low-noise cable for Microelectrode holder</td>
			<td>WPI</td>
			<td>13620</td>
			<td>Connecting recording microelctrode holder to adaptor/headboard</td>
		</tr>
	</tbody>
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electroretinogram analysis of the visual response in zebrafish larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

Typically, ERGs are recorded from zebrafish larvae at 5 dpf, since a number of studies have published ERG recordings at this stage9,16,20. Larval responses were measured under dark-adapted conditions with no background illumination using a 20 msec stimulus of white LED light. We utilized a commercially available ERG system consisting of a Ganzfeld light stimulator and computer controller/recorder. The stimulator uses a tightly controlled proprietary pulse width modulation (PWM) system to control the...

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Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

electroretinogram-analysis-of-the-visual-response-in-zebrafish-larvae

Research

JoVE Journal

Neuroscience

15.5K visualizações.  University of North Carolina at Chapel Hill. We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.