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. 2003 May 6;4(2):219–234. doi: 10.1007/s10162-002-3022-x

Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio rerio)

Julie A Harris 1,2, Alan G Cheng 1, Lisa L Cunningham 1, Glen MacDonald 1,4, David W Raible 2,3, Edwin W Rubel 1,2,✉
PMCID: PMC3202713  PMID: 12943374

Abstract

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Keywords: hair cell death, lateral line, aminoglycosides, hair cell regeneration, zebrafish, genetic screening

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Acknowledgements

The authors thank Dale Cunningham for his help with scanning electron microscopy, Laurie Johnson for manuscript preparation assistance, and Melinda Modrell and Tiffany Kao for their excellent technical support. We also thank Dr. Eric Bauer and Setsuko Murakami for additional histology and valuable help. This work was supported by NIDCD grants DC04661, DC00018, and DC02854, and the University of Washington RRF.

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