Funding for this project was provided by a grant from the Melbourne Neuroscience Institute (to PTG, PRJ &#38; BVB).

All electroretinogram (ERG) procedures were performed according to the provisions of the Australian National Health and Medical Research Council code of practice for the care and use of animals and were approved by the institutional animal ethics committee at the University of Melbourne.
1. Buffer Preparation
<ol>
	<li>Prepare the 10x goldfish Ringer&#8217;s buffer (1.25 M NaCl, 26 mM KCl, 25 mm CaCl2, 10 mM MgCl2, 100 mM glucose, 100 mM HEPES) using reverse osmosis (RO...

<ol>
	<li>Roper, C., Tanguay, R. L., Slikker, W., Paule, M. G., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Developmental Neurotoxicology (Second Edition). , 143-151 (2018).</li><li>Nguyen, C. T., 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=Simultaneous+Recording+of+Electroretinography+and+Visual+Evoked+Potentials+in+Anesthetized+Rats.">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a> Journal of visualized experiments: JoVE. , e54158 (2016).</li><li>Chrispell, J. D., Rebrik, T. I., Weiss, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroretinogram+analysis+of+the+visual+response+in+zebrafish+larvae.">Electroretinogram analysis of the visual response in zebrafish larvae.</a> Journal of visualized expriment: JoVE. (97), (2015).</li><li>Seeliger, M. W., Rilk, A., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganzfeld+ERG+in+zebrafish+larvae.">Ganzfeld ERG in zebrafish larvae.</a> Documenta Ophthalmologica. 104 (1), 57-68 (2002).</li><li>Fleisch, V. C., Jametti, T., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroretinogram+(ERG)+Measurements+in+Larval+Zebrafish.">Electroretinogram (ERG) Measurements in Larval Zebrafish.</a> Cold Spring Harbor Protocols. 2008, (2008).</li><li>Biehlmaier, O., Neuhauss, S. C., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+and+functionality+at+the+cone+terminal+of+the+developing+zebrafish+retina.">Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina.</a> Developmental Neurobiololgy. 56 (3), 222-236 (2003).</li><li>Gestri, G., Link, B. A., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visual+system+of+zebrafish+and+its+use+to+model+human+ocular+diseases.">The visual system of zebrafish and its use to model human ocular diseases.</a> Developmental Neurobiololgy. 72 (3), 302-327 (2012).</li><li>Saszik, S., Bilotta, J., Givin, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERG+assessment+of+zebrafish+retinal+development.">ERG assessment of zebrafish retinal development.</a> Visual Neuroscience. 16 (5), 881-888 (1999).</li><li>Niklaus, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+accumulation+in+zebrafish+eyes+leads+to+augmented+amplitudes+in+the+electroretinogram.">Cocaine accumulation in zebrafish eyes leads to augmented amplitudes in the electroretinogram.</a> Matters. 3 (6), e201703000003 (2017).</li><li>Tanvir, Z., Nelson, R. F., DeCicco-Skinner, K., Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+month+of+hyperglycemia+alters+spectral+responses+of+the+zebrafish+photopic+ERG.">One month of hyperglycemia alters spectral responses of the zebrafish photopic ERG.</a> Disease models &#38; mechanisms. , (2018).</li><li>Kakiuchi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+electroretinogram+as+an+early+marker+of+visual+abnormalities+in+vitamin+A+deficiency.">Oscillatory potentials in electroretinogram as an early marker of visual abnormalities in vitamin A deficiency.</a> Molecular medicine reports. 11 (2), 995-1003 (2015).</li><li>Emran, F., Rihel, J., Adolph, A. R., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+larvae+lose+vision+at+night.">Zebrafish larvae lose vision at night.</a> Proceedings of the National Academy of Sciences. , (2010).</li><li>Makhankov, Y. V., Rinner, O., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inexpensive+device+for+non-invasive+electroretinography+in+small+aquatic+vertebrates.">An inexpensive device for non-invasive electroretinography in small aquatic vertebrates.</a> Journal of Neuroscience Methods. 135 (1-2), 205-210 (2004).</li><li>Bilotta, J., Saszik, S., Sutherland, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+contributions+to+the+electroretinogram+of+the+dark-adapted+developing+zebrafish.">Rod contributions to the electroretinogram of the dark-adapted developing zebrafish.</a> Developmental Dynamics. 222 (4), 564-570 (2001).</li><li>Cameron, M. A., Barnard, A. R., Lucas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electroretinogram+as+a+method+for+studying+circadian+rhythms+in+the+mammalian+retina.">The electroretinogram as a method for studying circadian rhythms in the mammalian retina.</a> Journal of genetics. 87 (5), 459-466 (2008).</li><li>Bui, B. V., Armitage, J. A., Vingrys, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+modelling+of+oscillatory+potentials.">Extraction and modelling of oscillatory potentials.</a> Documenta Ophthalmologica. 104 (1), 17-36 (2002).</li><li>Bui, B. V., Fortune, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+contributions+to+the+rat+full-field+electroretinogram.">Ganglion cell contributions to the rat full-field electroretinogram.</a> The Journal of Physiology. 555 (1), 153-173 (2004).</li></ol>

The authors have no disclosures relevant to this work.

Functional readouts such as the ERG have become increasingly important in the suite of tools used to study larval zebrafish8,9,12,14. Due to the small size of the larval zebrafish eye, glass micropipettes have been adapted as recording electrodes in most published protocols3,4,5,<sup class="...

The zebrafish (Danio rerio) has become a widely used genetic vertebrate model, including studies of the visual neurosciences. The increasing popularity of this species can be attributed to advantages including ease of genetic manipulation, the highly conserved vertebrate visual system (neuron types, anatomical morphology and organization, and underlying genetics), high fecundity and lower cost of husbandry compared to mammalian models1. The non-invasive electroretinogram (ERG) has long been used clinically to assess human visual function, and in the laboratory setting to quantify vision in a range of large and small species including rodents and larval zebrafish2,3,4,5. The most commonly analyzed ERG components are the a-wave and b-wave, originating from the light-sensing photoreceptors and bipolar interneurons, respectively. In larval zebrafish, distinct layers in the retina are established by 3 days post-fertilization (dpf) and the morphology of the photoreceptor cone terminal synapses mature before 4 dpf6,7. Outer retinal function of larval zebrafish is thus established before 4 dpf, meaning that the ERG is measurable from this early age onwards. Because of the short experimental cycle and the high-throughput properties of the model, the ERG has been applied to larval zebrafish for functional assessment of disease models, analyzing color vision and retinal development, studying visual circadian rhythms and testing drugs8,9,10,11,12.
However, current approaches for larval zebrafish ERG has some complexities that may make it harder to adopt. Published larval zebrafish ERG protocols commonly use a glass micropipette filled with conductive liquid as the recording electrode3,4,5,13, which requires a high quality micropipette tip3. Specialized equipment, such as a micropipette puller and in some cases a microforge, are required for their manufacture. This can be a challenge for laboratories with limited resources and leads to extra costs even when adapting available small animal ERG systems for measurement of larval zebrafish visual function. Even when smoothed, the sharp micropipette tip can damage the surface of the larval eye. Additionally, commercial micropipette holders for electrophysiology are constructed with a fixed silver wire. These fixed wires become passivated after repetitive use, requiring the purchase of new holders leading to increased maintenance costs.
Here we describe an ERG method using a cone-shaped sponge-tip recording electrode, that is particularly useful for adapting established small-animal ERG setups for larval zebrafish ERG measurements. The electrode is easily made using common polyvinyl acetate (PVA) sponge and fine silver wire without any other specialized equipment. Our data show that this novel electrode is sensitive and reliable enough to demonstrate the functional development of retinal neural circuits in larval zebrafish between 4 and 7 dpf. This economical and practical sponge-tip electrode may be useful to researchers establishing new ERG systems or modifying existing small-animal systems, for zebrafish studies.

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millex GP</td>
			<td>SLGP033RS</td>
			<td>Filters the 10&#215; goldfish ringer&#39;s buffer for sterilizatio</td>
		</tr>
		<tr>
			<td>1-mL syringe</td>
			<td>Terumo</td>
			<td>DVR-5175</td>
			<td>With a 30G &#215; &#189;&#34; needle to add drops of saline to the electrode sponge tip to prevent drying and increased noisein the ERG signals.</td>
		</tr>
		<tr>
			<td>30G &#215; &#189;&#34; needle</td>
			<td>Terumo</td>
			<td>NN*3013R</td>
			<td>For adding saline toteh sopnge tip electrode.</td>
		</tr>
		<tr>
			<td>Bioamplifier</td>
			<td>ADInstruments</td>
			<td>ML135</td>
			<td>For amplifying ERG signals.</td>
		</tr>
		<tr>
			<td>Bleach solution&#160;</td>
			<td>King White</td>
			<td>9333441000973</td>
			<td>For an alternative method of sliver electrode chlorination. Active ingredient: 42 g/L sodium hypochlorite.</td>
		</tr>
		<tr>
			<td>Circulation water bath</td>
			<td>Lauda-Ko&#776;nigshoffen</td>
			<td>MGW Lauda</td>
			<td>Used to make the water-heated platfrom.</td>
		</tr>
		<tr>
			<td>Electrode lead</td>
			<td>Grass Telefactor</td>
			<td>F-E2-30</td>
			<td>Platinum cables for connecting silver wire electrodes to the amplifier.</td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Photometric Solution International&#160;</td>
			<td></td>
			<td>For maintianing dark adaptation and enclosing the Ganzfeld setup to improve signal-to-noise ratio.</td>
		</tr>
		<tr>
			<td>Ganzfeld Bowl</td>
			<td>Photometric Solution International&#160;</td>
			<td></td>
			<td>Custom designed light stimulator: 36 mm diameter, 13 cm aperture size.</td>
		</tr>
		<tr>
			<td>Luxeon LEDs</td>
			<td>Phillips Light Co.</td>
			<td></td>
			<td>For light stimulation twenty 5W and one 1W LEDs.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Harvard Apparatus</td>
			<td>BS4 50-2625</td>
			<td>Holds the recording electrode during experiments.</td>
		</tr>
		<tr>
			<td>Microsoft Office Excel</td>
			<td>Microsoft</td>
			<td>version 2010</td>
			<td>Spreadsheet software for data analysis.</td>
		</tr>
		<tr>
			<td>Moisturizing eye gel</td>
			<td>GenTeal Gel</td>
			<td>9319099315560</td>
			<td>Used to cover zebrafish larvae during recordings to avoiding dehydration. Active ingredient: 0.3 % Hypromellose and 0.22 % carbomer 980.</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Copan</td>
			<td>200C</td>
			<td>Used to caredully transfer larval zebrafish.</td>
		</tr>
		<tr>
			<td>Powerlab data acquisition system</td>
			<td>ADInstruments</td>
			<td>ML785</td>
			<td>Controls the LEDs to generate stimuli.</td>
		</tr>
		<tr>
			<td>PVA sponge</td>
			<td>MeiCheLe</td>
			<td>R-1675</td>
			<td>For the placement of larval zebrafish and making the cone-shaped electrode ti</td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Aaxis Pacific</td>
			<td>13317002</td>
			<td>For electroplating silver wire electrode.</td>
		</tr>
		<tr>
			<td>Scope Software</td>
			<td>ADInstruments</td>
			<td>version 3.7.6</td>
			<td>Simultaneously triggers the stimulus through the Powerlab system and collects data</td>
		</tr>
		<tr>
			<td>Silver (fine round wire)</td>
			<td>A&#38;E metal</td>
			<td>0.3 mm</td>
			<td>Used to make recording and reference ERG electrodes.</td>
		</tr>
		<tr>
			<td>Stereo microscope&#160;</td>
			<td>Leica</td>
			<td>M80</td>
			<td>Used to shape and measure the cone-shaped sponge apex (with scale bar on eyepiece). Positioned in the Faraday cage for electrode placement.</td>
		</tr>
		<tr>
			<td>Tricaine&#160;</td>
			<td>Sigma-aldrich</td>
			<td>E10521-50G</td>
			<td>For anaethetizing larval zebrafish.</td>
		</tr>
		<tr>
			<td>Water-heated platform</td>
			<td>custom-made</td>
			<td></td>
			<td>For maintianing the temperature of the sponge platform and the larval body during ERG recordings</td>
		</tr>
	</tbody>
</table>

electroretinogram recording in larval zebrafish using a novel cone-shaped sponge-tip electrode

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

This section provides representative results for ERG measurements taken daily from 4 to 7 dpf. From 4 dpf, ERG responses show robust a- and b-wave components, which arise from photoreceptors and bipolar cells, respectively. At each age tested, the amplitude of the b-wave increased with light intensity (Figure 2; Figure 3). Notably, the sensitivity of the larval zebrafish retina to dimmer flashes increased with age. The a- and b-wave were not ...

Watch this Scientific Journal Video about Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode at JoVE.com

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...

This protocol helps simplify the measurements of electroretinograms or ERGs in larval zebrafish which provides an important functional readout of visual development and genetic disease models. The soft sponge tips of recording electrodes used in this protocol is easy to manufacture using economical and readily available materials and avoids potential damage to the larval eyes. The softer sponge-tipped electrode can facilitate repeated ERG measurements of the same eye.Individuals new to this procedure may find it difficult to work under the dim red lighting that's used throughout the procedure. But it's important that dark adaptation is maintained. To prepare the cone-shaped sponge recording electrode, cut the end from a platinum electrode lead extension and use a scalpel to remove 10 millimeters of the outer polytetrafluoroethylene insulation coating from the new end of the extension.Cut a 40 millimeter piece of a 0.3 millimeter diameter silver wire and entwine the silver wire with the exposed inner wire to securely attach the wire to the electrode lead. Encase the joint with insulating tape leaving an approximately 15 millimeter length of silver wire exposed. Connect another wire to the negative terminal of the battery and immerse the other end of the wire into the saline.Immerse the exposed silver wire in normal saline and connect the other end to the positive terminal of a nine volt direct current source. After 60 seconds, place the wire on absorbent tissue to dry. In the meantime, cut a 20 by 20 millimeter square of polyvinyl acetate sponge into a cone shape and saturate the sponge in one times Ringer's buffer.Under a light microscope with a scale bar on the eyepiece, use a scalpel blade to shape the apex of the cone to an approximately 40 micrometer diameter. Air-dry the cone-shaped sponge on absorbent tissue paper until it is solid. Before inserting the silver wire into the dried solid cone-shaped polyvinyl acetate sponge through the base of the cone, using mask tape to insulate any excess exposed metal to reduce photovoltaic artifacts.On the day of the experiment, immerse the sponge-tipped electrode into Ringer's buffer for 15 minutes to fully saturate the sponge. At least eight hours before the recordings, place no more than 20 zebrafish larvae in a single 15 milliliter tube without a lid and wrapped in aluminum foil in a dark incubator. At the end of the incubation, pour the dark-adapted zebrafish into a petri dish under dim red illumination from a light-emitting diode.To prepare the sponge platform, cut a piece of polyvinyl acetate sponge roughly equal in thickness to the depth of a 35 millimeter petri dish so that the sponge will fit snugly within the dish. Make a small cut vertically through one end of the sponge to accommodate the silver wire of the reference electrode and soak the sponge in goldfish Ringer's buffer until the sponge is saturated. Then place the sponge into a clean 35 millimeter petri dish and use a paper towel to absorb the extra liquid until no solution exudes from the sponge in response to a light finger press.To position the animal for the experiment, use a three millimeter Pasteur pipette to transfer an anesthetized larva onto a three by three centimeter square of paper towel and use forceps to place the square onto the moistened sponge. Using a fine brush soaked in Ringer's buffer, adjust the position of the larva with one eye facing upward and away from any liquid on the towel under the larva. Glaze the larval body with moisturizing eye gel to keep the animal hydrated throughout the electroretinogram recording.And place the dish on a small, water-heated platform in front of the Ganzfeld bowl light stimulus situated inside a Faraday cage. Insert the reference electrode into the cut made in the platform sponge. And connect a commercially-obtained ground electrode to the Faraday cage.Attach the recording electrode to an electrode holder and secure the holder to the stereotaxic arm of a micromanipulator. Using a three millimeter Pasteur pipette, resaturate the sponge tip of the electrode with one drop of Ringer's solution and position the microscope in the Faraday cage over the electroretinogram platform. Use the tip of an absorbent tissue to remove any excess liquid from the electrode sponge tip and under the microscope, position the active electrode so that the sponge tip gently touches the central corneal surface of the larval zebrafish eye.Then move the Ganzfeld bowl towards the sponge platform taking care that the larva is covered by the bowl and close the cage to reduce extraneous electromagnetic noise. The sensitivity of the larval zebrafish retina to dimmer flashes increases with age. As the a-and b-waves are not recognizable at intensities lower than minus 1.61 log candela second per meter squared at four days post fertilization while clear signals are detectable at these intensities in the older larvae.The b-wave amplitude increases markedly between four and five post fertilization. At seven days, the signal at 2.48 log candela second per meter squared is greater compared to days five and six post fertilization. A-and b-wave implicit times become significantly faster after five days post fertilization.With overall, these results demonstrating a maturation in zebrafish retinal function between four to seven days post-fertilization. To successfully perform this protocol it is critical that sponges are fully saturated with fluid which helps maintain the conductivity of the recording electrode and lowers the noise levels. If the a-wave is of particular interest, pharmacological treatments can be applied to block the b-wave component thus exposing the full a-wave.

electroretinogram-recording-larval-zebrafish-using-novel-cone-shaped

Research

JoVE Journal

Developmental Biology

6.3K visualizações.  University of Melbourne. Este protocolo ajuda a simplificar as medidas de eletroretinogramas ou ERGs em zebrafish larval que fornece uma leitura funcional importante do desenvolvimento visual e modelos de doenças genéticas. As pontas de esponja macia de gravação de eletrodos usados neste protocolo são fáceis de fabricar usando materiais econômicos e prontamente disponíveis e evita danos potenciais aos olhos larvais. O eletrodo mais macio com ponta de esponja pode facilitar repetidas medições de ERG do mesmo...