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

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 lM 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.

Bax, Bcl2, and p53 Differentially Regulate Neomycin- and Gentamicin-Induced Hair Cell Death in the Zebrafish Lateral Line

“…As this mutation occurs on an AB background, age-matched wildtype *AB fish, which are genetically similar to AB fish, were used as controls. Similar sensitivity to aminoglycoside-induced hair cell death has been demonstrated in multiple fish strains, providing confidence that small genetic differences between wild-type lines will not confound our results (e.g., Williams and Holder 2000; Harris et al 2003).…”

“…For vital dye labeling, fish were incubated for 15 min in 0.005 % 2-(4-(dimethylamino)styryl)-Nethylpyridinium iodide (DASPEI) (Life Technologies, Grand Island, NY, USA), a mitochondrial dye that is specific for lateral line hair cells using our incubation protocol (Harris et al 2003;Owens et al 2007). Excess DASPEI was removed with two rinses in fresh EM and the fish were then anesthetized with 0.001 % MS-222 (Sigma-Aldrich).…”

“…Fish were viewed with a Leica MZFLIII fluorescent stereomicroscope and ×50 magnification. We quantified hair cell survival using a scoring system based on the relative fluorescent intensity of DASPEI labeling for each of 10 head neuromasts per fish (Harris et al 2003). A neuromast with intense labeling was scored "2," one with dim labeling was scored "1," and one with no labeling was scored "0," yielding a score of 0-20 for each fish.…”

“…The zebrafish lateral line system has been established as a useful preparation for studies of hair cell death (e.g., Williams and Holder 2000; Harris et al 2003;Ton and Parng 2005;Coffin et al 2010). Lateral line hair cells cluster in sensory organs called neuromasts that are externally located on the head and body of the animal (Metcalf et al 1985;Coombs et al 1989;Raible and Kruse 2000).…”

“…This sensory system is used to detect nearfield water movement and mediates a number of important behaviors including predator avoidance, prey capture, and orientation to flow (Dijkgraaf 1963;Montgomery and MacDonald 1987;Montgomery et al 1997;Coombs et al 2001;New et al 2001;Suli et al 2012). Lateral line hair cells and mammalian inner ear hair cells exhibit similar responses to ototoxic drugs, making the lateral line a suitable preparation for ototoxicity studies (Harris et al 2003;Ou et al 2007;Owens et al 2007;Hirose et al 2011). In larval zebrafish, hair cells in the lateral line are relatively mature by 5 days post-fertilization (dpf) and they respond robustly to aminoglycoside exposure (Murakami et al 2003;Santos et al 2006).…”