Retrograde Mitochondrial Transport Is Essential for Organelle Distribution and Health in Zebrafish Neurons.

In neurons, mitochondria are transported by molecular motors throughout the cell to form and maintain functional neural connections. These organelles have many critical functions in neurons and are of high interest as their dysfunction is associated with disease. While the mechanics and impact of anterograde mitochondrial movement toward axon terminals are beginning to be understood, the frequency and function of retrograde (cell body directed) mitochondrial transport in neurons are still largely unexplored. While existing evidence indicates that some mitochondria are retrogradely transported for degradation in the cell body, the precise impact of disrupting retrograde transport on the organelles and the axon was unknown. Using long-term, in vivo imaging, we examined mitochondrial motility in zebrafish sensory and motor axons. We show that retrograde transport of mitochondria from axon terminals allows replacement of the axon terminal population within a day. By tracking these organelles, we show that not all mitochondria that leave the axon terminal are degraded; rather, they persist over several days. Disrupting retrograde mitochondrial flux in neurons leads to accumulation of aged organelles in axon terminals and loss of cell body mitochondria. Assays of neural circuit activity demonstrated that disrupting mitochondrial transport and function has no effect on sensory axon terminal activity but does negatively impact motor neuron axons. Taken together, our work supports a previously unappreciated role for retrograde mitochondrial transport in the maintenance of a homeostatic distribution of mitochondria in neurons and illustrates the downstream effects of disrupting this process on sensory and motor circuits.SIGNIFICANCE STATEMENT Disrupted mitochondrial transport has been linked to neurodegenerative disease. Retrograde transport of this organelle has been implicated in turnover of aged organelles through lysosomal degradation in the cell body. Consistent with this, we provide evidence that retrograde mitochondrial transport is important for removing aged organelles from axons; however, we show that these organelles are not solely degraded, rather they persist in neurons for days. Disrupting retrograde mitochondrial transport impacts the homeostatic distribution of mitochondria throughout the neuron and the function of motor, but not sensory, axon synapses. Together, our work shows the conserved reliance on retrograde mitochondrial transport for maintaining a healthy mitochondrial pool in neurons and illustrates the disparate effects of disrupting this process on sensory versus motor circuits.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset.

In sensory hair cells of auditory and vestibular organs, the ribbon synapse is required for the precise encoding of a wide range of complex stimuli. Hair cells have a unique presynaptic structure, the synaptic ribbon, which organizes both synaptic vesicles and calcium channels at the active zone. Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To selectively assess the impact of ribbon size on synapse function, we examined hair cells in transgenic zebrafish that have enlarged ribbons, without postsynaptic alterations. Morphologically, we found that enlarged ribbons had more associated vesicles and reduced presynaptic calcium-channel clustering. Functionally, hair cells with enlarged ribbons had larger global and ribbon-localized calcium currents. Afferent neuron recordings revealed that hair cells with enlarged ribbons resulted in reduced spontaneous spike rates. Additionally, despite larger presynaptic calcium signals, we observed fewer evoked spikes with longer latencies from stimulus onset. Together, our work indicates that hair-cell ribbon size influences the spontaneous spiking and the precise encoding of stimulus onset in afferent neurons.SIGNIFICANCE STATEMENT Numerous studies support that hair-cell ribbon size corresponds with functional sensitivity differences in afferent neurons and, in the case of inner hair cells of the cochlea, vulnerability to damage from noise trauma. Yet it is unclear whether ribbon size directly influences sensory encoding. Our study reveals that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onset, a distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli.

Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2.

The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells.

Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This sensory transmission necessitates rapid and sustained neurotransmitter release, which relies on a large pool of synaptic vesicles at the hair-cell presynapse. Work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, but how new synaptic material reaches the presynapse in hair cells is not known. We show that the kinesin motor protein Kif1a and an intact microtubule network are necessary to enrich synaptic vesicles at the presynapse in hair cells. We use genetics and pharmacology to disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral-line system. We find that these manipulations decrease synaptic-vesicle populations at the presynapse in hair cells. Using electron microscopy, along with in vivo calcium imaging and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon-synapse function. Kif1a mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Additionally, we find that kif1a mutants exhibit impaired rheotaxis, a behavior reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a-based microtubule transport is critical to enrich synaptic vesicles at the active zone in hair cells, a process that is vital for proper ribbon-synapse function.

Spontaneous calcium transients in hair cell stereocilia.

The hair bundle of auditory and vestibular hair cells converts mechanical stimuli into electrical signals through mechanoelectrical transduction (MET). The MET apparatus is built around a tip link that connects neighboring stereocilia that are aligned in the direction of mechanosensitivity of the hair bundle. Upon stimulation, the MET channel complex responds to changes in tip-link tension and allows a cation influx into the cell. Ca2+ influx in stereocilia has been used as a signature of MET activity. Using genetically encoded Ca2+ sensors (GCaMP3, GCaMP6s) and high-performance fluorescence confocal microscopy, we detect spontaneous Ca2+ transients in individual stereocilia in developing and fully formed hair bundles. We demonstrate that this activity is abolished by MET channel blockers and thus likely originates from putative MET channels. We observe Ca2+ transients in the stereocilia of mice in tissue explants as well as in vivo in zebrafish hair cells, indicating this activity is functionally conserved. Within stereocilia, the origin of Ca2+ transients is not limited to the canonical MET site at the stereocilia tip but is also present along the stereocilia length. Remarkably, we also observe these Ca2+ transients in the microvilli-like structures on the hair cell surface in the early stages of bundle development, prior to the onset of MET. Ca2+ transients are also present in the tallest rows of stereocilia in auditory hair cells, structures not traditionally thought to contain MET channels. We hypothesize that this newly described activity may reflect stochastic and spontaneous MET channel opening. Localization of these transients to other regions of the stereocilia indicates the presence of a pool of channels or channel precursors. Our work provides insights into MET channel assembly, maturation, function, and turnover.

Presynaptic CaV1.3 channels regulate synaptic ribbon size and are required for synaptic maintenance in sensory hair cells.

L-type calcium channels (Ca(V)1) are involved in diverse processes, such as neurotransmission, hormone secretion, muscle contraction, and gene expression. In this study, we uncover a role for Ca(V)1.3a in regulating the architecture of a cellular structure, the ribbon synapse, in developing zebrafish sensory hair cells. By combining in vivo calcium imaging with confocal and super-resolution structured illumination microscopy, we found that genetic disruption or acute block of Ca(V)1.3a channels led to enlargement of synaptic ribbons in hair cells. Conversely, activating channels reduced both synaptic-ribbon size and the number of intact synapses. Along with enlarged presynaptic ribbons in ca(V)1.3a mutants, we observed a profound loss of juxtaposition between presynaptic and postsynaptic components. These synaptic defects are not attributable to loss of neurotransmission, because vglut3 mutants lacking neurotransmitter release develop relatively normal hair-cell synapses. Moreover, regulation of synaptic-ribbon size by Ca(2+) influx may be used by other cell types, because we observed similar pharmacological effects on pinealocyte synaptic ribbons. Our results indicate that Ca(2+) influx through Ca(V)1.3 fine tunes synaptic ribbon size during hair-cell maturation and that Ca(V)1.3 is required for synaptic maintenance.

Asymmetric mechanotransduction by hair cells of the zebrafish lateral line.

In the lateral line system, water motion is detected by neuromast organs, fundamental units that are arrayed on a fish's surface. Each neuromast contains hair cells, specialized mechanoreceptors that convert mechanical stimuli, in the form of water movement, into electrical signals. The orientation of hair cells' mechanosensitive structures ensures that the opening of mechanically gated channels is maximal when deflected in a single direction. In each neuromast organ, hair cells have two opposing orientations, enabling bi-directional detection of water movement. Interestingly, Tmc2b and Tmc2a proteins, which constitute the mechanotransduction channels in neuromasts, distribute asymmetrically so that Tmc2a is expressed in hair cells of only one orientation. Here, using both in vivo recording of extracellular potentials and calcium imaging of neuromasts, we demonstrate that hair cells of one orientation have larger mechanosensitive responses. The associated afferent neuron processes that innervate neuromast hair cells faithfully preserve this functional difference. Moreover, Emx2, a transcription factor required for the formation of hair cells with opposing orientations, is necessary to establish this functional asymmetry within neuromasts. Remarkably, loss of Tmc2a does not impact hair cell orientation but abolishes the functional asymmetry as measured by recording extracellular potentials and calcium imaging. Overall, our work indicates that oppositely oriented hair cells within a neuromast employ different proteins to alter mechanotransduction to sense the direction of water motion.

In vivo investigation of mitochondria in lateral line afferent neurons and hair cells.

To process sensory stimuli, intense energy demands are placed on hair cells and primary afferents. Hair cells must both mechanotransduce and maintain pools of synaptic vesicles for neurotransmission. Furthermore, both hair cells and afferent neurons must continually maintain a polarized membrane to propagate sensory information. These processes are energy demanding and therefore both cell types are critically reliant on mitochondrial health and function for their activity and maintenance. Based on these demands, it is not surprising that deficits in mitochondrial health can negatively impact the auditory and vestibular systems. In this review, we reflect on how mitochondrial function and dysfunction are implicated in hair cell-mediated sensory system biology. Specifically, we focus on live imaging approaches that have been applied to study mitochondria using the zebrafish lateral-line system. We highlight the fluorescent dyes and genetically encoded biosensors that have been used to study mitochondria in lateral-line hair cells and afferent neurons. We then describe the impact this in vivo work has had on the field of mitochondrial biology as well as the relationship between mitochondria and sensory system development, function, and survival. Finally, we delineate the areas in need of further exploration. This includes in vivo analyses of mitochondrial dynamics and biogenesis, which will round out our understanding of mitochondrial biology in this sensitive sensory system.

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechanoelectrical transduction (MET) apparatus. Whether these interactions are functionally relevant across mechanosensory organs and vertebrate species is unclear. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Using Light-Sheet Microscopy to Study Spontaneous Activity in the Developing Lateral-Line System.

Hair cells are the sensory receptors in the auditory and vestibular systems of all vertebrates, and in the lateral-line system of aquatic vertebrates. The purpose of this work is to explore the zebrafish lateral-line system as a model to study and understand spontaneous activity in vivo. Our work applies genetically encoded calcium indicators along with light-sheet fluorescence microscopy to visualize spontaneous calcium activity in the developing lateral-line system. Consistent with our previous work, we show that spontaneous calcium activity is present in developing lateral-line hair cells. We now show that supporting cells that surround hair cells, and cholinergic efferent terminals that directly contact hair cells are also spontaneously active. Using two-color functional imaging we demonstrate that spontaneous activity in hair cells does not correlate with activity in either supporting cells or cholinergic terminals. We find that during lateral-line development, hair cells autonomously generate spontaneous events. Using localized calcium indicators, we show that within hair cells, spontaneous calcium activity occurs in two distinct domains-the mechanosensory bundle and the presynapse. Further, spontaneous activity in the mechanosensory bundle ultimately drives spontaneous calcium influx at the presynapse. Comprehensively, our results indicate that in developing lateral-line hair cells, autonomously generated spontaneous activity originates with spontaneous mechanosensory events.

Prolonged Dexamethasone Exposure Enhances Zebrafish Lateral-Line Regeneration But Disrupts Mitochondrial Homeostasis and Hair Cell Function.

The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans.

Dopamine has been implicated in the modulation of diverse forms of behavioral plasticity, including appetitive learning and addiction. An important challenge is to understand how dopamine's effects at the cellular level alter the properties of neural circuits to modify behavior. In the nematode C. elegans, dopamine modulates habituation of an escape reflex triggered by body touch. In the absence of food, animals habituate more rapidly than in the presence of food; this contextual information about food availability is provided by dopaminergic mechanosensory neurons that sense the presence of bacteria. We find that dopamine alters habituation kinetics by selectively modulating the touch responses of the anterior-body mechanoreceptors; this modulation involves a D1-like dopamine receptor, a Gq/PLC-beta signaling pathway, and calcium release within the touch neurons. Interestingly, the body touch mechanoreceptors can themselves excite the dopamine neurons, forming a positive feedback loop capable of integrating context and experience to modulate mechanosensory attention.

Caenorhabditis elegans TRPA-1 functions in mechanosensation.

Members of the transient receptor potential (TRP) ion channel family mediate diverse sensory transduction processes in both vertebrates and invertebrates. In particular, members of the TRPA subfamily have distinct thermosensory roles in Drosophila, and mammalian TRPA1 is postulated to have a function in noxious cold sensation and mechanosensation. Here we show that mutations in trpa-1, the C. elegans ortholog of mouse Trpa1, confer specific defects in mechanosensory behaviors related to nose-touch responses and foraging. trpa-1 is expressed and functions in sensory neurons required for these mechanosensory behaviors, and contributes to neural responses of these cells to touch, particularly after repeated mechanical stimulation. Furthermore, mechanical pressure can activate C. elegans TRPA-1 heterologously expressed in mammalian cells. Collectively, these data demonstrate that trpa-1 encodes an ion channel that can be activated in response to mechanical pressure and is required for mechanosensory neuron function, suggesting a possible role in mechanosensory transduction or modulation.

How Zebrafish Can Drive the Future of Genetic-based Hearing and Balance Research.

Over the last several decades, studies in humans and animal models have successfully identified numerous molecules required for hearing and balance. Many of these studies relied on unbiased forward genetic screens based on behavior or morphology to identify these molecules. Alongside forward genetic screens, reverse genetics has further driven the exploration of candidate molecules. This review provides an overview of the genetic studies that have established zebrafish as a genetic model for hearing and balance research. Further, we discuss how the unique advantages of zebrafish can be leveraged in future genetic studies. We explore strategies to design novel forward genetic screens based on morphological alterations using transgenic lines or behavioral changes following mechanical or acoustic damage. We also outline how recent advances in CRISPR-Cas9 can be applied to perform reverse genetic screens to validate large sequencing datasets. Overall, this review describes how future genetic studies in zebrafish can continue to advance our understanding of inherited and acquired hearing and balance disorders.

EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia.

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

Spatial asymmetry in the mechanosensory phenotypes of the C. elegans DEG/ENaC gene mec-10.

DEG/ENaC channels have been broadly implicated in mechanosensory transduction, yet many questions remain about how these proteins contribute to complexes that sense mechanical stimuli. In C. elegans, two DEG/ENaC channel subunits are thought to contribute to a gentle touch transduction complex: MEC-4, which is essential for gentle touch sensation, and MEC-10, whose importance is less well defined. By characterizing a mec-10 deletion mutant, we have found that MEC-10 is important, but not essential, for gentle touch responses in the body touch neurons ALM, PLM, and PVM. Surprisingly, the requirement for MEC-10 in ALM and PLM is spatially asymmetric; mec-10 animals show significant behavioral and physiological responses to stimulation at the distal end of touch neuron dendrites, but respond poorly to stimuli applied near the neuronal cell body. The subcellular distribution of a rescuing MEC-10::GFP translational fusion was found to be restricted to the neuronal cell body and proximal dendrite, consistent with the hypothesis that MEC-10 protein is asymmetrically distributed within the touch neuron process. These results suggest that MEC-10 may contribute to only a subset of gentle touch mechanosensory complexes found preferentially at the proximal dendrite.

Berbamine Analogs Exhibit Differential Protective Effects From Aminoglycoside-Induced Hair Cell Death.

Hearing loss is the third most common chronic health condition in the United States and largely results from damage to sensory hair cells. Major causes of hair cell damage include aging, noise exposure, and medications such as aminoglycoside antibiotics. Due to their potent antibacterial properties and low cost, aminoglycosides are often used for the treatment of gram-negative bacterial infections, surpassing expensive antibiotics with fewer harmful side effects. However, their use is coupled with permanent hearing loss in over 20% of patients requiring these life-sustaining antibiotics. There are currently no FDA-approved drugs that prevent hearing loss from aminoglycosides. A previous study by our group identified the plant alkaloid berbamine as a strong protectant of zebrafish lateral line hair cells from aminoglycoside damage. This effect is likely due to a block of the mechanotransduction channel, thereby reducing aminoglycoside entry into hair cells. The present study builds on this previous work, investigating 16 synthetic berbamine analogs to determine the core structure underlying their protective mechanisms. We demonstrate that nearly all of these berbamine analogs robustly protect lateral line hair cells from ototoxic damage, with ED50 values nearing 20 nM for the most potent analogs. Of the 16 analogs tested, nine strongly protected hair cells from both neomycin and gentamicin damage, while one conferred strong protection only from gentamicin. These data are consistent with prior research demonstrating that different aminoglycosides activate somewhat distinct mechanisms of damage. Regardless of the mechanism, protection required the entire berbamine scaffold. Phenolic alkylation or acylation with lipophilic groups appeared to improve protection compared to berbamine, implying that these structures may be responsible for mitigating damage. While the majority of analogs confer protection by blocking aminoglycoside uptake, 18% of our analogs also confer protection via an uptake-independent mechanism; these analogs exhibited protection when delivered after aminoglycoside removal. Based on our studies, berbamine analogs represent a promising tool to further understand the pathology of aminoglycoside-induced hearing loss and can serve as lead compounds to develop otoprotective drugs.

Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish.

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otoferlin result in deafness and depending on the species, mild to strong vestibular deficits. While studies in mouse models suggest a role for otoferlin in synaptic vesicle exocytosis and endocytosis, it is unclear whether these functions are conserved across species. To address this question, we characterized the impact of otoferlin depletion in zebrafish larvae and found defects in synaptic vesicle recycling, abnormal synaptic ribbons, and higher resting calcium concentrations in hair cells. We also observed abnormal expression of the calcium binding hair cell genes s100s and parvalbumin, as well as the nogo related proteins rtn4rl2a and rtn4rl2b. Exogenous otoferlin partially restored expression of genes affected by endogenous otoferlin depletion. Our results suggest that in addition to vesicle recycling, depletion of otoferlin disrupts resting calcium levels, alters synaptic ribbon architecture, and perturbs transcription of hair cells specific genes during zebrafish development.

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.