Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorate aminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5 days' post fertilization after neomycin (20 µM) and gentamicin (20 mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100 µM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300 µM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20 µM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake of neomycin into hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production and aminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Naringin attenuates cisplatin- and aminoglycoside-induced hair cell injury in the zebrafish lateral line via multiple pathways.

Exposure to ototoxic drugs is a significant cause of hearing loss that affects about 30 thousand children with potentially serious physical, social and psychological dysfunctions every year. Cisplatin (CP) and aminoglycosides are effective antineoplastic or bactericidal drugs, and their application has a high probability of ototoxicity which results from the death of hair cells (HCs). Here, we describe the therapeutic effect of the flavonoid compound naringin (Nar) against ototoxic effects of cisplatin and aminoglycosides include gentamicin (GM) and neomycin (Neo) in zebrafish HCs. Animals incubated with Nar (100-400 µmol/L) were protected against the pernicious effects of CP (150-250 µmol/L), GM (50-150 µmol/L) and Neo (50-150 µmol/L). We also provide evidence for the potential mechanism of Nar against ototoxicity, including antioxidation, anti-apoptosis, promoting proliferation and hair cell regeneration. We found that mRNA levels of the apoptotic- and pyroptosis-related genes are regulated by Nar both in vivo and in vitro. Finally, by proving that Nar does not affect the anti-tumour efficacy of CP and antibacterial activity of aminoglycosides in vitro, we highlight its value in clinical application. In conclusion, these results unravel a novel therapeutic role for Nar as an otoprotective drug against the adverse effects of CP and aminoglycosides.

Cxcl12a induces snail1b expression to initiate collective migration and sequential Fgf-dependent neuromast formation in the zebrafish posterior lateral line primordium.

The zebrafish posterior lateral line primordium migrates along a path defined by the chemokine Cxcl12a, periodically depositing neuromasts, to pioneer formation of the zebrafish posterior lateral line system. snail1b, known for its role in promoting cell migration, is expressed in leading cells of the primordium in response to Cxcl12a, whereas its expression in trailing cells is inhibited by Fgf signaling. snail1b knockdown delays initiation of primordium migration. This delay is associated with aberrant expansion of epithelial cell adhesion molecule (epcam) and reduction of cadherin 2 expression in the leading part of the primordium. Co-injection of snail1b morpholino with snail1b mRNA prevents the initial delay in migration and restores normal expression of epcam and cadherin 2 The delay in initiating primordium migration in snail1b morphants is accompanied by a delay in sequential formation of trailing Fgf signaling centers and associated protoneuromasts. This delay is not specifically associated with knockdown of snail1b but also with other manipulations that delay migration of the primordium. These observations reveal an unexpected link between the initiation of collective migration and sequential formation of protoneuromasts in the primordium.

Salvianolic acid B inhibits ototoxic drug-induced ototoxicity by suppression of the mitochondrial apoptosis pathway.

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti-apoptosis effects of Sal B in a cultured HEI-OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK-8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase-3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC-1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl-2. The expression pattern of p-PI3K and p-Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin- and cisplatin-induced apoptotic features, enhanced cell viability and accompanied with decreased caspase-3 activity in the HEI-OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl-2 expression and down-regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin- and cisplatin-injured HEI-OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin- and cisplatin-induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug-induced HC apoptosis, offering a potential use in the treatment of hearing loss.

Effects of the Essential Oil from Pistacia lentiscus Var. chia on the Lateral Line System and the Gene Expression Profile of Zebrafish (Danio rerio).

Mastic essential oil exhibits anti-bacterial, anti-inflammatory, and anti-oxidant properties. With the growing interest of the use of mastic oil in the food and pharmaceutical industry, systematic in vivo studies are needed to address controlled usage and safety issues. In the present work we evaluated the safety of mastic oil using as a model the zebrafish lateral line system. In addition, we studied the gene expression profile of zebrafish fed with mastic oil-supplemented diet using microarray analysis. Our results showed that the hair cells of lateral line neuromasts are functional upon exposure of zebrafish larvae up to 20 ppm of mastic essential oil, while treatment with higher concentrations, 100 and 200 ppm, resulted in increased larvae mortality. Dietary supplementation of zebrafish with mastic essential oil led to differential expression of interferon response-related genes as well as the immune responsive gene 1 (irg1) that links cellular metabolism with immune defense. Notably, mucin 5.2, a constituent of the mucus hydrogel that protects the host against invading pathogens, was up-regulated. Our in vivo work provides information concerning the safety of mastic essential oil use and suggests dietary effects on gene expression related with the physical and immunochemical properties of the gastrointestinal system.

Retinoic acid is required and Fgf, Wnt, and Bmp signaling inhibit posterior lateral line placode induction in zebrafish.

The lateral line system is a mechanosensory systems present in aquatic animals. The anterior and posterior lateral lines develop from anterior and posterior lateral line placodes (aLLp and pLLp), respectively. Although signaling molecules required for the induction of other cranial placodes have been well studied, the molecular mechanisms underlying formation of the lateral line placodes are unknown. In this study we tested the requirement of multiple signaling pathways, such as Wnt, Bmp Fgf, and Retinoic Acid for aLLp and pLLp induction. We determined that aLLp specification requires Fgf signaling, whilst pLLp specification requires retinoic acid which inhibits Fgf signaling. pLLp induction is also independent of Wnt and Bmp activities, even though these pathways limit the boundaries of the pLLp. This is the first report that the aLLp and pLLp depend on different inductive mechanisms and that pLLp induction requires the inhibition of Fgf, Wnt and Bmp signaling.

The effects of lateral line ablation and regeneration in schooling giant danios.

Fish use multiple sensory systems, including vision and their lateral line system, to maintain position and speed within a school. Although previous studies have shown that ablating the lateral line alters schooling behavior, no one has examined how the behavior recovers as the sensory system regenerates. We studied how schooling behavior changes in giant danios, Devario aequipinnatus, when their lateral line system is chemically ablated and after the sensory hair cells regenerate. We found that fish could school normally immediately after chemical ablation, but that they had trouble schooling 1-2 weeks after the chemical treatment, when the hair cells had fully regenerated. We filmed groups of giant danios with two high-speed cameras and reconstructed the three-dimensional positions of each fish within a group. One fish in the school was treated with gentamycin to ablate all hair cells. Both types of neuromasts (canal and superficial) were completely ablated after treatment, but fully regenerated after 1 week. We quantified the structure of the school using nearest neighbor distance, bearing, elevation, and the cross-correlation of velocity between each pair of fish. Treated fish maintained a normal position within the school immediately after the lateral line ablation, but could not school normally 1 or 2 weeks after treatment, even though the neuromasts had fully regenerated. By 4-8 weeks post-treatment, the treated fish could again school normally. These results demonstrate that the behavioral recovery after lateral line ablation is a longer process than the regeneration of the hair cells themselves.

A mitochondrial targeting tetrapeptide Bendavia protects lateral line hair cells from gentamicin exposure.

The hearing loss induced by aminoglycosides is caused by the permanent loss of mechanosensory hair cells of the inner ear. The aim of the present study is therefore to evaluate the protective effect of Bendavia, a novel antioxidant, on gentamicin-induced hair cell damage in zebrafish lateral lines. The results demonstrated the pretreatment of Bendavia exhibited dose-dependent protection against gentamicin in both acute and chronic exposure. We found that Bendavia at 150 µm conferred optimal protection from either acute or chronic exposure with ototoxin. Bendavia reduced uptake of fluorescent-tagged gentamicin via mechanoelectrical transduction channels, suggesting its protective effects may be partially due to decreasing ototoxic molecule uptake. The intracellular death pathways inhibition triggered by gentamicin might be also included as no blockage of gentamicin was observed. Our data suggest that Bendavia represents a novel otoprotective drug that might provide a therapeutic alternative for patients receiving aminoglycoside treatment.

The alcohol-sensitive period during early octavolateral organ development in zebrafish (Danio rerio).

Fetal alcohol exposure can cause Fetal Alcohol Spectrum Disorders (FASD), completely preventable developmental disabilities characterized by permanent birth defects. However, specific gestational timing when developing organs are most sensitive to alcohol exposure is unclear. In this study, we examined the temporal effects of embryonic alcohol exposure on octavolateral organs in zebrafish (Danio rerio), including inner ears and lateral line neuromasts that function in hearing, balance, and hydrodynamic detection, respectively. To determine an alcohol-sensitive period in the first 24 hours post fertilization (hpf), Et(krt4:EGFP)sqet4 zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours. Octavolateral organs of control and alcohol-exposed larvae were examined at 3, 5, and 7 days post fertilization (dpf). Using confocal and light microscopy, we found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at 3 dpf. Only alcohol-exposed larvae from 12-17 hpf had smaller otic vesicles at 5 dpf, smaller saccular otoliths at 7 dpf and fewer saccular hair cells, neuromasts and hair cells per neuromast at 3 dpf. In addition, auditory function was assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. Our results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. This study implies that embryonic alcohol exposure timing during early development can influence severity of hearing deficits. © 2017 Wiley Periodicals, Inc.

Tetrabromobisphenol-A induces apoptotic death of auditory cells and hearing loss.

Phenolic tetrabromobisphenol-A (TBBPA) and its derivatives are commonly used flame-retardants, in spite of reported toxic effects including neurotoxicity, immunotoxicity, nephrotoxicity, and hepatotoxicity. However, the effects of TBBPA on ototoxicity have not yet been reported. In this study, we investigated the effect of TBBPA on hearing function in vivo and in vitro. Auditory Brainstem Response (ABR) threshold was markedly increased in mice after oral administration of TBBPA, indicating that TBBPA causes hearing loss. In addition, TBBPA induced the loss of both zebrafish neuromasts and hair cells in the rat cochlea in a dose-dependent manner. Mechanistically, hearing loss is largely attributed to apoptotic cell death, as TBBPA increased the expression of pro-apoptotic genes but decreased the expression of anti-apoptotic genes. We also found that TBBPA induced oxidative stress, and importantly, pretreatment with NAC, an anti-oxidant reagent, reduced TBBPA-induced reactive oxygen species (ROS) generation and partially prevented cell death. Our results show that TBBPA-mediated ROS generation induces ototoxicity and hearing loss. These findings implicate TBBPA as a potential environmental ototoxin by exerting its hazardous effects on the auditory system.

Chemical Ototoxicity of the Fish Inner Ear and Lateral Line.

Hair cell-driven mechanosensory systems are crucial for successful execution of a number of behaviors in fishes, and have emerged as good models for exploring questions relevant to human hearing. This review focuses on ototoxic effects in the inner ear and lateral line system of fishes. We specifically examine studies where chemical ototoxins such as aminoglycoside antibiotics have been employed as tools to disable the lateral line. Lateral line ablation results in alterations to feeding behavior and orientation to water current in a variety of species. However, neither behavior is abolished in the presence of additional sensory cues, supporting the hypothesis that many fish behaviors are driven by multisensory integration. Within biomedical research, the larval zebrafish lateral line has become an important model system for understanding signaling mechanisms that contribute to hair cell death and for developing novel pharmacological therapies that protect hair cells from ototoxic damage. Furthermore, given that fishes robustly regenerate damaged hair cells, ototoxin studies in fishes have broadened our understanding of the molecular and genetic events in an innately regenerative system, offering potential targets for mammalian hair cell regeneration. Collectively, studies of fish mechanosensory systems have yielded insight into fish behavior and in mechanisms of hair cell death, protection, and regeneration.

Neurotoxicity of a Biopesticide Analog on Zebrafish Larvae at Nanomolar Concentrations.

Despite the ever-increasing role of pesticides in modern agriculture, their deleterious effects are still underexplored. Here we examine the effect of A6, a pesticide derived from the naturally-occurring α-terthienyl, and structurally related to the endocrine disrupting pesticides anilinopyrimidines, on living zebrafish larvae. We show that both A6 and an anilinopyrimidine, cyprodinyl, decrease larval survival and affect central neurons at micromolar concentrations. Focusing on a superficial and easily observable sensory system, the lateral line system, we found that defects in axonal and sensory cell regeneration can be observed at much lower doses, in the nanomolar range. We also show that A6 accumulates preferentially in lateral line neurons and hair cells. We examined whether A6 affects the expression of putative target genes, and found that genes involved in apoptosis/cell proliferation are down-regulated, as well as genes reflecting estrogen receptor activation, consistent with previous reports that anilinopyrimidines act as endocrine disruptors. On the other hand, canonical targets of endocrine signaling are not affected, suggesting that the neurotoxic effect of A6 may be due to the binding of this compound to a recently identified, neuron-specific estrogen receptor.

The influence of turbulence on the sensory basis of rheotaxis.

Rheotaxis is a widespread behavior with many potential benefits for fish and other aquatic animals, yet the sensory basis of rheotaxis under different fluvial conditions is still poorly understood. Here, we examine the role that vision and the lateral line play in the rheotactic behavior of a stream-dwelling species (Mexican tetra, Astyanax mexicanus) under both rectilinear and turbulent flow conditions. Turbulence lowered the flow speed at which threshold levels of rheotactic performance were elicited, an effect that was independent of sensory condition. Compared to fish with access to visual information, fish without access exhibited cross-stream casting behaviors and a decrease in the accuracy with which they oriented upstream. Visual deprivation effects were independent of availability of lateral line information and whether flow was rectilinear or turbulent. Fish deprived of lateral line information exhibited no measureable deficits under any of the conditions of this study. This study indicates that rheotactic abilities persist in the absence of both vision and lateral line under both turbulent and non-turbulent conditions, but that turbulence enhances either the motivation or ability of fish to orient to slow currents.

Flow sensing in developing Xenopus laevis is disrupted by visual cues and ototoxin exposure.

We explored how lateral line cues interact with visual cues to mediate flow sensing behaviors in the nocturnal developing frog, Xenopus laevis, by exposing animals to current flows under different lighting conditions and after exposure to the ototoxin gentamicin. Under dark conditions, Xenopus tadpoles move downstream at the onset of current flow, then turn, and orient toward the direction of the flow with high accuracy. Postmetamorphic froglets also exhibit positive rheotaxis but with less accuracy and longer latency. The addition of discrete light cues to an otherwise dark environment disrupts rheotaxis and positioning. Orientation is less accurate, latency to orient is longer, and animals do not move as far downstream in the presence of light. Compared with untreated tadpoles tested in the dark, tadpoles exposed to gentamicin show less accurate rheotaxis with longer latency and do not move as far downstream in response to flow. These effects are compounded by the presence of light cues. The disruptive effects of light on flow sensing in Xenopus emphasize the disturbances to natural behaviors that may be produced by anthropogenic illumination in nocturnal habitats.

The lateral line is necessary for blind cavefish rheotaxis in non-uniform flow.

When encountering a unidirectional flow, many fish exhibit an unconditioned orienting response known as rheotaxis. This multisensory behavior can reportedly involve visual, vestibular, tactile and lateral line cues. However, the precise circumstances under which different senses contribute are still unclear and there is considerable debate, in particular, about the contributions of the lateral line. In this study, we investigate the rheotactic behavior of blind cavefish under conditions of spatially non-uniform flow (a jet stream), which in theory, should promote reliance on lateral line cues. The behavior of individual lateral line enabled and disabled fish was videorecorded under IR light in a square arena that prevented streamwise biases and that contained a narrow jet stream in the center of the tank. Whereas the stream's peak velocity (8 cm s(-1)) declined very little in the streamwise direction, it declined steeply in the cross-stream direction (∼3-4.5 cm s(-1) cm(-1)). Lateral line enabled fish showed higher levels of orientation to the stream and its source (a 1-cm-wide nozzle) when in the central (jet stream) region of the tank compared with surrounding regions, whereas lateral line disabled fish showed random orientations in all regions of the tank. The results of this study indicate that the spatial characteristics of flow play a role in determining the sensory basis of rheotaxis.

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.

Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death.

Intracellular Ca(2+) is a key regulator of life or death decisions in cultured neurons and sensory cells. The role of Ca(2+) in these processes is less clear in vivo, as the location of these cells often impedes visualization of intracellular Ca(2+) dynamics. We generated transgenic zebrafish lines that express the genetically encoded Ca(2+) indicator GCaMP in mechanosensory hair cells of the lateral line. These lines allow us to monitor intracellular Ca(2+) dynamics in real time during aminoglycoside-induced hair cell death. After exposure of live larvae to aminoglycosides, dying hair cells undergo a transient increase in intracellular Ca(2+) that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca(2+) elevation through either caged chelators or pharmacological inhibitors of Ca(2+) effectors mitigates toxic effects of aminoglycoside exposure. Conversely, artificial elevation of intracellular Ca(2+) by caged Ca(2+) release agents sensitizes hair cells to the toxic effects of aminoglycosides. These data suggest that alterations in intracellular Ca(2+) homeostasis play an essential role in aminoglycoside-induced hair cell death, and indicate several potential therapeutic targets to stem ototoxicity.

Identification of modulators of hair cell regeneration in the zebrafish lateral line.

The external location of the zebrafish lateral line makes it a powerful model for studying mechanosensory hair cell regeneration. We have developed a chemical screen to identify FDA-approved drugs and biologically active compounds that modulate hair cell regeneration in zebrafish. Of the 1680 compounds evaluated, we identified two enhancers and six inhibitors of regeneration. The two enhancers, dexamethasone and prednisolone, are synthetic glucocorticoids that potentiated hair cell numbers during regeneration and also induced hair cell addition in the absence of damage. BrdU analysis confirmed that the extra hair cells arose from mitotic activity. We found that dexamethasone and prednisolone, like other glucocorticoids, suppress zebrafish caudal fin regeneration, indicating that hair cell regeneration occurs by a distinctly different process. Further analyses of the regeneration inhibitors revealed that two of the six, flubendazole and topotecan, significantly suppress hair cell regeneration by preventing proliferation of hair cell precursors. Flubendazole halted support cell division in M-phase, possibly by interfering with normal microtubule activity. Topotecan, a topoisomerase inhibitor, killed both hair cells and proliferating hair cell precursors. A third inhibitor, fulvestrant, moderately delayed hair cell regeneration by reducing support cell proliferation. Our observation that hair cells do not regenerate when support cell proliferation is impeded confirms previous observations that cell division is the primary route for hair cell regeneration after neomycin treatment in zebrafish.

The stat3/socs3a pathway is a key regulator of hair cell regeneration in zebrafish. [corrected].

All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors (i.e., hair cells) (Corwin and Cotanche, 1988; Lombarte et al., 1993; Baird et al., 1996), but mammals possess only a very limited capacity for regeneration after birth (Roberson and Rubel, 1994). As a result, mammals experience permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates (Fritzsch et al., 2007), we chose to study hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique digital gene expression, which leverages the power of next-generation sequencing ('t Hoen et al., 2008). We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological inhibition of stat3 signaling accelerated hair cell regeneration without overproducing cells. Taking other published datasets into account (Sano et al., 1999; Schebesta et al., 2006; Dierssen et al., 2008; Riehle et al., 2008; Zhu et al., 2008; Qin et al., 2009), we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target for a broad application in tissue repair and injury healing.

Profiling drug-induced cell death pathways in the zebrafish lateral line.

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while D-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.