Name: an assay for lateral line regeneration in adult zebrafish
Uploaded: 2014-04-08T19:00:00
Description: Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

The authors have nothing to disclose.

All procedures are performed following the guidelines described in "Principles of Laboratory Animal Care" (National Institutes of Health publication no. 85-23, revised 1985) and the approved Rosalind Franklin University Institutional Animal Care and Use Committee animal protocol 08-19.
1. Gentamicin-induction of Hair Cell Necrosis
<ol>
	<li>Prepare gentamicin sulfate in normal saline at a final concentration of 0.004% (4.32 mM).</li>
	<li>Place adult fish (D. rerio, 4-6 months of ag...

<ol>
	<li>Dambly-Chaudire, C., Sapde, D., Soubiran, F., Decorde, K., Gompel, N., Ghysen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lateral+Line+of+Zebrafish:+a+Model+System+for+the+Analysis+of+Morphogenesis+and+Neural+Development+in+Vertebrates.">The Lateral Line of Zebrafish: a Model System for the Analysis of Morphogenesis and Neural Development in Vertebrates.</a> Biol. Cell. 95 (9), 579-587 (2003).</li><li>Montgomery, J., Carton, G., Voigt, R., Baker, C., Diebel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+Processing+of+Water+Currents+by+Fishes.">Sensory Processing of Water Currents by Fishes.</a> Phil. Trans. Royal Soc. London B Biol. Sci. 355 (1401), 1325-1327 (2000).</li><li>Buck, L. M., Winter, M. J., Redfern, W., Whitfield, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ototoxin-Induced+Cellular+Damage+in+Neuromasts+Disrupts+Lateral+Line+Function+in+Larval+Zebrafish.">Ototoxin-Induced Cellular Damage in Neuromasts Disrupts Lateral Line Function in Larval Zebrafish.</a> Hearing Res. 284 (1-2), 1-2 (2012).</li><li>Engelmann, J., Hanke, W., Mogdans, J., Bleckmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Stimuli+and+the+Fish+Lateral+Line.">Hydrodynamic Stimuli and the Fish Lateral Line.</a> Nature. 408 (6808), 51-52 (2000).</li><li>Olszewski, J., Haehnel, M., Taguch, M., Liao, J. C. <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+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line.">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line.</a> PloS One. 7 (5), e36661 (2012).</li><li>Raible, D. W., Kruse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Lateral+Line+System+in+Embryonic+Zebrafish.">Organization of the Lateral Line System in Embryonic Zebrafish.</a> J. Comp. Neurol. 421 (2), 189-198 (2000).</li><li>Coffin, A. B., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extracellular Divalent Cations Modulate Aminoglycoside-Induced Hair Cell Death in the Zebrafish Lateral. 253 (1-2), 1-2 (2009).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Ma, E. Y., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Regulates+the+Extent+of+Hair+Cell+Regeneration+in+the+Zebrafish+Lateral+Line).">Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line).</a> J. Neurosci. 28 (9), 2261-2273 (2008).</li><li>Brignull, H. R., Raible, D. W., Stone, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feathers+and+Fins:+Non-Mammalian+Models+for+Hair+Cell+Regeneration.">Feathers and Fins: Non-Mammalian Models for Hair Cell Regeneration.</a> Brain Res. 1277, 12-23 (2009).</li><li>Bibliowicz, J., Tittle, R. K., Gross, J. 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Mol. Biol. 98 (2-3), 2-3 (2008).</li><li>Van Trump, W. J., Coombs, S., Duncan, K., McHenry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+Is+Ototoxic+to+All+Hair+Cells+in+the+Fish+Lateral+Line+System.">Gentamicin Is Ototoxic to All Hair Cells in the Fish Lateral Line System.</a> Hear. Res. 261 (1-2), 1-2 (2010).</li><li>Littleton, R. M., Hove, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+Nontraditional+Model+of+Traditional+Medicine.">Zebrafish: a Nontraditional Model of Traditional Medicine.</a> J. Ethnopharmacol. 145 (3), 677-685 (2013).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Olszewski, J., Haehnel, M., Taguchi, M., Liao, J. C. <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+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line).">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line).</a> PLoS One. 7 (5), 36661-36 (2012).</li><li>Liang, J., Wang, D., Renaud, G., Wolfsberg, T. G., Wilson, A. F., Burgess, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stat3/Socs3a+Pathway+Is+a+Key+Regulator+of+Hair+Cell+Regeneration+in+Zebrafish+[Corrected.">The Stat3/Socs3a Pathway Is a Key Regulator of Hair Cell Regeneration in Zebrafish [Corrected.</a> J. Neurosci. 32 (31), 10662-10673 (2012).</li><li>Nakae, M., Asaoka, R., Wada, H., Sasaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Dye+Staining+of+Neuromasts+in+Live+Fishes:+An+Aid+to+Systematic+Studies.">Fluorescent Dye Staining of Neuromasts in Live Fishes: An Aid to Systematic Studies.</a> Ichthyol Res. , 286-290 (2012).</li><li>Magrassi, L., Purves, D., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Probes+That+Stain+Living+Nerve+Terminals.">Fluorescent Probes That Stain Living Nerve Terminals.</a> The J. Neurosci. 7 (4), 1207-1214 (1987).</li><li>Owens, K. N., Coffin, A. B., Hong, L. S., Bennett, K. O., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Mechanosensory+Hair+Cells+of+the+Zebrafish+Lateral+Line+to+Aminoglycosides+Reveals+Distinct+Cell+Death+Pathways.">Response of Mechanosensory Hair Cells of the Zebrafish Lateral Line to Aminoglycosides Reveals Distinct Cell Death Pathways.</a> Hear. Res. 253 (1-2), 1-2 (2009).</li><li>Namdaran, P., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Identification of Modulators of Hair Cell Regeneration in the Zebrafish Lateral. 32 (10), 3516-3528 (2012).</li><li>Herrera, A. A., Banner, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+and+Effects+of+Vital+Fluorescent+Dyes:+Observation+of+Motor+Nerve+Terminals+and+Satellite+Cells+in+Living+Frog+Muscles.">The Use and Effects of Vital Fluorescent Dyes: Observation of Motor Nerve Terminals and Satellite Cells in Living Frog Muscles.</a> J. Neurocytol. 19 (1), 67-83 (1990).</li><li>Hickey, P. C., Jacobson, D., Read, N. 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Based on the extensive body of literature that has been established for analysis of lateral line (LL) regeneration in embryonic and larval zebrafish8,24,25, the goal of our study was to develop a quantitative assay for lateral line regeneration in zebrafish that could be applied to disease models that are best studied in the adult fish. We found that certain critical points are important when applying procedures developed for embryonic/larval fish to the adult fish. The most important of these points regarded:...

The lateral line (LL) system is a mechanosensory organ found in both fish and amphibians that is responsible for hearing, balance, rheotaxis and mediating behaviors such as schooling and predator avoidance1-5. It is composed of clusters of hair cells surrounded by supporting cells, both of which are positioned in structures called neuromasts6. These neuromasts are typically organized into vertical lines (called stitches) along the longitudinal axis of the body and tail with some horizontal stitches observed in the head of the fish. In the adult, neuromasts are significantly greater in number within the stitches as compared to embryonic or larval fish6. Biomedical studies in zebrafish have focused on the effect of antibiotic treatment, noise-induced trauma, chronic infection, etc. on hair cells7,8 in an attempt to better understand their effects in humans.
Unlike most vertebrates, teleosts, such as the zebrafish (Danio rerio), have the ability to regenerate lost hair cells. Zebrafish are particularly useful because of their rapid development time and high regenerative capacity. To date, however; zebrafish studies on lateral line development and/or regeneration have mainly utilized the embryonic and larval stage fish due to the reduced number of lateral line neuromasts which allows for easier counting and analysis6,9,10.
However, as many zebrafish models of neurological and non-neurological diseases11-16 are studied in the adult fish and not the larvae, we focused on developing a lateral line regenerative assay in adult zebrafish using gentamicin (an aminoglycoside previously used in zebrafish larvae and more recently used with adult fish17)&#160;so that an assay was available that could be applied to current adult zebrafish disease models. While previously published procedures by Van Trump et al.17 established the conditions for hair cell ablation in the adult fish, they did not establish a standard curve for neuromast regeneration which is required for quantitative comparison between control and experimental groups such as when using transgenic zebrafish lines or pharmacologically-induced disease states in zebrafish18. We therefore followed the procedures of Van Trump et al.17 for hair cell ablation, but built on their work to establish a standard curve of neuromast regeneration to enable investigators to use our data when comparing control and experimental groups such as with adult zebrafish disease models. The assay was also designed to allow extension of the analysis to the individual hair cell when a higher level of resolution is required.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Optimization of the procedures for quantifying neuromast regeneration of the lateral line in adult zebrafish.
The neuromasts of larval zebrafish are readily quantifiable; however, the lateral line of the adult zebrafish has a much greater number of neuromasts per stitch making quantitative analyses more difficult6,17,19,20. As seen in Figure 1A, the head has a significantly higher number of neuromasts compared to either the mid-section or tail; with...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

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