Sound shock response in larval zebrafish: A convenient and high-throughput assessment of auditory function.

Given that hearing ability can be challenged in diverse ways, it is necessary to develop an easily conducted, high-throughput method for assessing potential auditory risks. Measuring the acoustic startle response (ASR) has become a critical behavioral method in hearing research using zebrafish (Danio rerio). In this study, changes in the activity of zebrafish larvae (10 days post fertilization (dpf)) due to exposure to a sudden easily-generated broad-band noise were automatically and objectively recorded and analyzed without building sophisticated equipments. A significant increase in activity was induced by the noise stimulation and the alterations were impaired by gentamicin. In addition, a clear dose-response trend was observed between gentamicin exposure and the impaired activity, and a similar phenomenon was observed between gentamicin exposure and damage to hair cells. Our results suggested that alterations in the activity induced by a broad-band noise can potentially be used as an efficient assay for assessing hearing ability.

Automated High-Throughput Damage Scoring of Zebrafish Lateral Line Hair Cells After Ototoxin Exposure.

Zebrafish have emerged as a powerful biological system for drug development against hearing loss. Zebrafish hair cells, contained within neuromasts along the lateral line, can be damaged with exposure to ototoxins, and therefore, pre-exposure to potentially otoprotective compounds can be a means of identifying promising new drug candidates. Unfortunately, anatomical assays of hair cell damage are typically low-throughput and labor intensive, requiring trained experts to manually score hair cell damage in fluorescence or confocal images. To enhance throughput and consistency, our group has developed an automated damage-scoring algorithm based on machine-learning techniques that produce accurate damage scores, eliminate potential operator bias, provide more fidelity in determining damage scores that are between two levels, and deliver consistent results in a fraction of the time required for manual analysis. The system has been validated against trained experts using linear regression, hypothesis testing, and the Pearson's correlation coefficient. Furthermore, performance has been quantified by measuring mean absolute error for each image and the time taken to automatically compute damage scores. Coupling automated analysis of zebrafish hair cell damage to behavioral assays for ototoxicity produces a novel drug discovery platform for rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Quercetin protects against hair cell loss in the zebrafish lateral line and guinea pig cochlea.

Eighteen supplement drugs were screened using hair cells to determine a protective effect against the adverse effects of neomycin by using the zebrafish lateral line. The zebrafish were administered the supplement drugs 1 h before neomycin exposure. One hour later, animals were fixed in paraformaldehyde. Dose-response curves were generated to evaluate the protective effect on hair cells. The screen identified 3 supplements (quercetin, catechin and tannic acid). Three minutes after exposure to neomycin, increased antioxidant activity was found in the lateral line hair cells, as determined by the analysis of oxidative stress. Quercetin decreases antioxidant activity. The identified drugs were also investigated to determine whether they protect the cochlea against noise-induced hearing loss in guinea pigs. The drugs were administered via the intraperitoneal route in the guinea pigs 3 days before and 4 days after noise exposure. Seven days after noise exposure (130-dB sound pressure level for 3 h), the auditory brainstem response threshold shifts were assessed. We observed that the auditory brainstem response threshold shift was significantly less in the quercetin group than in the vehicle control group. The results of our study indicate that screening drugs using zebrafish can determine additional protective drugs for the inner ear.

Identification of small molecule inhibitors of cisplatin-induced hair cell death: results of a 10,000 compound screen in the zebrafish lateral line.

HYPOTHESIS: The zebrafish lateral line can be used to identify small molecules that protect against cisplatin-induced hair cell death. BACKGROUND: Cisplatin is a commonly used chemotherapeutic agent, which causes hearing loss by damaging hair cells of the inner ear. There are currently no FDA-approved pharmacologic strategies for preventing this side effect. The zebrafish lateral line has been used successfully in the past to study hair cell death and protection. METHODS: In this study, we used the zebrafish lateral line to screen a library of 10,000 small molecules for protection against cisplatin-induced hair cell death. Dose-response relationships for identified protectants were determined by quantifying hair cell protection. The effect of each protectant on uptake of a fluorescent cisplatin analog was also quantified. RESULTS: From this screen, we identified 2 compounds exhibiting dose-dependent protection: cisplatin hair cell protectant 1 and 2 (CHCP1 and 2). CHCP1 reduced the uptake of a fluorescent cisplatin analog, suggesting its protective effects may be due to decreased cisplatin uptake. CHCP2 did not affect uptake, which suggests an intracellular mechanism of action. Evaluation of analogs of CHCP2 revealed 3 additional compounds that significantly reduced cisplatin-induced hair cell death, although none exceed the effectiveness or potency of the parent compound. CONCLUSION: The zebrafish lateral line was used to identify 2 small molecules that protected against cisplatin-induced hair cell death.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Uranium-induced sensory alterations in the zebrafish Danio rerio.

The effect of chronic exposure to uranium ions (UO(2)(2+)) on sensory tissues including the olfactory and lateral line systems was investigated in zebrafish (Danio rerio) using scanning electron microscopy. The aim of this study was to determine whether exposure to uranium damaged sensory tissues in fish. The fish were exposed to uranium at the concentration of 250 µg l(-1) for 10 days followed by a depuration period of 23 days. Measurements of uranium uptake in different fish organs: olfactory rosettes and bulbs, brain, skin, and muscles, were also determined by ICP-AES and ICP-MS during the entire experimental period. The results showed that uranium displayed a strong affinity for sensory structures in direct contact with the surrounding medium, such as the olfactory and lateral line systems distributed on the skin. A decreasing gradient of uranium concentration was found: olfactory rosettes>olfactory bulbs>skin>muscles>brain. At the end of the experiment, uranium was present in non-negligible quantities in sensory tissues. In parallel, fish exposed to uranium showed severe sensory tissue alterations at the level of the olfactory and lateral line systems. In both sensory systems, the gross morphology was altered and the sensory hair cells were significantly damaged very early after the initiation of exposure (from the 3rd day). At the end of the experiment, after 23 days of depuration, the lateral line system still displayed slight tissue alterations, but approximately 80% of the neuromasts in this system had regenerated. In contrast, the olfactory system took more time to recover, as more than half of the olfactory rosettes observed remained destroyed at the end of the experiment. This study showed, for the first time, that uranium is able to damage fish sensory tissues to such an extent that tissue regeneration is delayed.

Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin.

Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death.

Ototoxin-induced cellular damage in neuromasts disrupts lateral line function in larval zebrafish.

The ototoxicity of a number of marketed drugs is well documented, and there is an absence of convenient techniques to identify and eliminate this unwanted effect at a pre-clinical stage. We have assessed the validity of the larval zebrafish, or more specifically its lateral line neuromast hair cells, as a microplate-scale in vivo surrogate model of mammalian inner ear hair cell responses to ototoxin exposure. Here we describe an investigation of the pathological and functional consequences of hair cell loss in lateral line neuromasts of larval zebrafish after exposure to a range of well known human and non-human mammalian ototoxins. Using a previously described histological assay, we show that hair cell damage occurs in a concentration-dependent fashion following exposure to representatives from a range of drug classes, including the aminoglycoside antibiotics, salicylates and platinum-based chemotherapeutics, as well as a heavy metal. Furthermore, we detail the optimisation of a semi-automated method to analyse the stereotypical startle response in larval zebrafish, and use this to assess the impact of hair cell damage on hearing function in these animals. Functional assessment revealed robust and significant attenuation of the innate startle, rheotactic and avoidance responses of 5 day old zebrafish larvae after treatment with a number of compounds previously shown to induce hair cell damage and loss. Interestingly, a startle reflex (albeit reduced) was still present even after the apparent complete loss of lateral line hair cell fluorescence, suggesting some involvement of the inner ear as well as the lateral line neuromast hair cells in this reflex response. Collectively, these data provide evidence to support the use of the zebrafish as a pre-clinical indicator of drug-induced histological and functional ototoxicity.

Vibratory sources as compound stimuli for the octavolateralis systems: dissection of specific stimulation channels using multiple behavioral approaches.

Underwater vibratory sources simultaneously present acoustic and hydrodynamic disturbances. Because vibratory dipole sources are poor sonic projectors, most researchers have assumed that such sources are of greatest relevance to the lateral line system. Both hydroacoustic principles and empirical studies have shown that dipole sources are also a potent stimulus to the inner ear. Responses to vibratory sources in mottled sculpin (Cottus bairdi) were assessed using unconditioned orienting, differential and nondifferential conditioning. Orienting responses are dominated by lateral line inputs and eliminated by lateral line inactivation. Simple conditioning depends on inputs from other systems and was not affected by lateral line inactivation. Differential conditioning alters behavioral control, and sculpin could be conditioned to ignore substrate-borne vibrations and respond only to hydroacoustic stimulation of the ear. The lateral line and inner ear of mottled sculpin do not necessarily exhibit range fractionation, as both systems operate over a similar distance (within 1.5 body lengths) and respond to many of the same sources. Vibratory dipole sources generate compound stimuli that simultaneously activate multiple octavolateralis systems, and sculpin make use of the channels differentially under different behavioral tasks.

Chemical screening for hair cell loss and protection in the zebrafish lateral line.

In humans, most hearing loss results from death of hair cells, the mechanosensory receptors of the inner ear. Two goals of current hearing research are to protect hair cells from degeneration and to regenerate new hair cells, replacing those that are lost due to aging, disease, or environmental challenges. One limitation of research in the auditory field has been the relative inaccessibility of the mechanosensory systems in the inner ear. Zebrafish possess hair cells in both their inner ear and their lateral line system that are morphologically and functionally similar to human hair cells. The external location of the mechanosensory hair cells in the lateral line and the ease of in vivo labeling and imaging make the zebrafish lateral line a unique system for the study of hair cell toxicity, protection, and regeneration. This review focuses on the lateral line system as a model for understanding loss and protection of mechanosensory hair cells. We discuss chemical screens to identify compounds that induce hair cell loss and others that protect hair cells from known toxins and the potential application of these screens to human medicine.

Physiological dimensions of ototoxic responses in a model fish species.

Pharmaceutical agents known to be toxic to the human auditory system also impair sensory hair cells of teleosts, and this supports the use of fish models for the screening of such compounds. However, previous investigations have focused almost exclusively on anatomical changes after drug administration without assessing macro-level physiological effects. Using the goldfish (Carassius auratus), we demonstrate that the acquisition of auditory evoked potentials offers a rapid and non-invasive means for tracking ototoxin-induced shifts in auditory thresholds. Gentamicin (100mg/mL) was the agent of choice as it is an extensively-studied human ototoxin. Significant shifts (p<0.05) in hearing sensitivity were observed between 300 Hz and 600 Hz and these shifts depended on acoustic pressure, but not particle motion. This differential elevation of auditory thresholds may be caused by impairment of specific populations of auditory sensory hair cells.

Extracellular divalent cations modulate aminoglycoside-induced hair cell death in the zebrafish lateral line.

Aminoglycoside antibiotics cause death of sensory hair cells. Research over the past decade has identified several key players in the intracellular cascade. However, the role of the extracellular environment in aminoglycoside ototoxicity has received comparatively little attention. The present study uses the zebrafish lateral line to demonstrate that extracellular calcium and magnesium ions modulate hair cell death from neomycin and gentamicin in vivo, with high levels of either divalent cation providing significant protection. Imaging experiments with fluorescently-tagged gentamicin show that drug uptake is reduced under high calcium conditions. Treating fish with the hair cell transduction blocker amiloride also reduces aminoglycoside uptake, preventing the toxicity, and experiments with variable calcium and amiloride concentrations suggest complementary effects between the two protectants. Elevated magnesium, in contrast, does not appear to significantly attenuate drug uptake, suggesting that the two divalent cations may protect hair cells from aminoglycoside damage through different mechanisms. These results provide additional evidence for calcium- and transduction-dependent aminoglycoside uptake. Divalent cations provided differential protection from neomycin and gentamicin, with high cation concentrations almost completely protecting hair cells from neomycin and acute gentamicin toxicity, but offering reduced protection from continuous (6 h) gentamicin exposure. These experiments lend further support to the hypothesis that aminoglycoside toxicity occurs via multiple pathways in a both a drug and time course-specific manner.

Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle.

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.

Using the zebrafish lateral line to screen for ototoxicity.

The zebrafish is a valuable model for studying hair cell development, structure, genetics, and behavior. Zebrafish and other aquatic vertebrates have hair cells on their body surface organized into a sensory system called the lateral line. These hair cells are highly accessible and easily visualized using fluorescent dyes. Morphological and functional similarities to mammalian hair cells of the inner ear make the zebrafish a powerful preparation for studying hair cell toxicity. The ototoxic potential of drugs has historically been uncovered by anecdotal reports that have led to more formal investigation. Currently, no standard screen for ototoxicity exists in drug development. Thus, for the vast majority of Food and Drug Association (FDA)-approved drugs, the ototoxic potential remains unknown. In this study, we used 5-day-old zebrafish larvae to screen a library of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection II) for ototoxic effects in hair cells of the lateral line. Hair cell nuclei were selectively labeled using a fluorescent vital dye. For the initial screen, fish were exposed to drugs from the library at a 100-muM concentration for 1 h in 96-well tissue culture plates. Hair cell viability was assessed in vivo using fluorescence microscopy. One thousand forty drugs were rapidly screened for ototoxic effects. Seven known ototoxic drugs included in the library, including neomycin and cisplatin, were positively identified using these methods, as proof of concept. Fourteen compounds without previously known ototoxicity were discovered to be selectively toxic to hair cells. Dose-response curves for all 21 ototoxic compounds were determined by quantifying hair cell survival as a function of drug concentration. Dose-response relationships in the mammalian inner ear for two of the compounds without known ototoxicity, pentamidine isethionate and propantheline bromide, were then examined using in vitro preparations of the adult mouse utricle. Significant dose-dependent hair cell loss in the mouse utricle was demonstrated for both compounds. This study represents an important step in validating the use of the zebrafish lateral line as a screening tool for the identification of potentially ototoxic drugs.