An Assay for Lateral Line Regeneration in Adult Zebrafish

Summary

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.

Abstract

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 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.

Introduction

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 avoidance^1-5. 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 cells^7,8 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 analysis^6,9,10.

However, as many zebrafish models of neurological and non-neurological diseases^11-16 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) 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.

Protocol

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

1. Prepare gentamicin sulfate in normal saline at a final concentration of 0.004% (4.32 mM).
2. Place adult fish (D. rerio, 4-6 months of age) in a container containing the 0.004% (4.32 mM) gentamicin solution. Any container can be used, but we use a fish container from a Pharmacal Aquatic System which is 7 in wide, 6 in high, and 7 in long. Place the container with fish in an incubator set at 28 °C for 24 hr. Set the total volume of fluid in the tank at a sufficient level to maintain fish in a viable state for the 24 hr period. Note: Aeration of the gentamicin fluid is not necessary if sufficient volume is used for the number of fish being treated.

2. Vital Staining of Hair Cells

1. Prepare a 0.08% concentration (in normal saline) of the fluorescent vital dye [4-4-diethylaminostyryl)-N-methylpyridinium iodide (485 nm excitation λ and 603 nm emission λ in methanol) from a working stock solution of 15 mg/ml in ethanol.
2. To determine if gentamicin treatment was effective a subset of control and gentamicin treated fish are stained immediately by placing fish in the well of a 6 well culture plate containing the vital dye. Use a sufficient number of fish (and culture plates as required for statistical significance to be achieved. Based on the examiner's speed of neuromast counting, place the fish in the plates in a staggered manner over time so that fish are not stained for over 75 min as described in step 2.3.
3. Place the plates from step 2.2 in a bench drawer by the fluorescent microscope to be used for examination of stained neuromasts. Turn off the room lights to prevent quenching of the vital dye over the 1 hr staining period at room temperature.
4. Prepare both dye wash-out and anesthetic water tanks. Dye wash-out water is normal fish water and for anesthetic water, add sufficient 2-phenoxyethanol so that a 1:1,000 dilution in normal fish water is achieved.
5. Place fish in excess normal fish water to rinse excess vital dye and proceed to step 3.1 for observation of vital dye stained fish.
6. To examine regeneration of neuromasts, transfer gentamicin-treated fish that were washed in normal fish water to an incubator for between 8-16 hr at 28 °C.
7. At various times between 8-16 hr, fish are removed from the incubator, washed and stained as indicated in steps 2.1-2.4. Proceed to step 3.1 for observation of vital dye stained fish.

3. Anesthetizing Fish and Fluorescent Counting of Neuromasts

1. Blot each fish on a paper towel to remove excess fluid and then place it on a dampened piece of filter paper that is centered on the lid of a plastic Petri dish.
2. Place the lid on the stage of a fluorescent stereo microscope to obtain a digital image of the vital dye stained neuromasts of the mid body stitches.
3. Use a digital camera placed on the fluorescent stereo microscope set a magnification of 2X to capture images for subsequent quantitative analysis. Note: The magnification setting of the stereo microscope may depend on the brand of microscope used, but the setting should allow easy viewing and counting of individual neuromasts within the mid body stitches.
4. Determine the amount of regeneration by counting the number of visible neuromasts within the four designated stitches on the bottom-most ventral side of the fish just proximal to the right pectoral fin (see Figure 1). For statistical analysis use an appropriate test such as ANOVA or the Student's T-test. Experiments should utilize a minimum of 5 fish per time point and all experiments should be repeated a minimum of 3x.
5. Based on the neuromast regeneration time curve (see Figure 3), count neuromasts between 8-16 hr post gentamicin wash-out to be within the linear phase of the regeneration curve. Note: Use of the linear time phase allows for proper quantitative analysis between the control and experimental groups.

4. Fluorescent Counting of Individual Hair Cells for Obtaining Higher Resolution of the Quantitative Analysis if the Neuromast Analysis is Not Statistically Significant

1. If the quantitative analysis at the level of neuromasts is not significant, analysis at the level of the individual hair cell can also be utilized to obtain a higher degree of resolution. Select fish at a particular time point post gentamicin wash-out (time point based on the earlier neuromast studies), vital dye stain the fish as described in Protocol 2, and then euthanize the fish using 2-phenoxyethanol at a 1:500 dilution for 1-5 min.
2. In subdued light to prevent quenching, make four incisions so that a square skin flap preparation is made as follows. Make an incision along the upper ribs of the fish until it is aligned with the anal fins, then make an incision across the belly, and finally, make two vertical incisions on each side of these incisions so the square skin flap is created. Note: This skin preparation will incorporate the mid body stitches used in the neuromast experiments.
3. Place the skin specimen on a glass slide and then place a circular glass cover slip over the excised skin specimen to help anchor and flatten the tissue for subsequent digital imaging.
4. Using the skin specimens from step 4.3, obtain digital images of the hair cells within each neuromast of the mid body stitches. Take images at a magnification of at least 60X and then count the hair cells within individual neuromasts for comparative quantitative analysis of the control and experimental groups (see Figure 4).

Results

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 difficult^6,17,19,20. As seen in Figure 1A, the head has a significantly higher number of neuromasts compared to either the mid-section or tail; with...

Discussion

Based on the extensive body of literature that has been established for analysis of lateral line (LL) regeneration in embryonic and larval zebrafish^8,24,25, 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:...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gentamicin sulfate solution (50 mg/ml)	Sigma Aldrich	G1397
2 Phenoxyethanol	Sigma Aldrich	P1126
4-4-Diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp) in methanol	Aldrich	D-3418	485 nm excitation λ and 603 nm emission λ
6-well Plates	Mid Sci	TP92006
Petri Dishes	Fisher Scientific	08-757-13
Glass Bottom Microwell Dishes	Matek Corporation	P35G-1.5-14-C
Sodium Chloride	Sigma Aldrich	S3014
Dissecting  Microscope	Nikon	TMZ-1500	Any dissecting microscope is fine.
Camera for Imaging	Nikon	Q imaging	Any camera is suitable.
ImageJ software	National Institutes of Health	NIH Image
NIS Elements	Nikon		Any imaging software is suitable.
Confocal microscope	Olympus	FV10i	Any high resolution fluorescent microscope is suitable
Aquatic System	KG Aquatics ZFS Rack System		Any aquatic system can be used