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 ....

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....

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