Contributions of mirror-image hair cell orientation to mouse otolith organ and zebrafish neuromast function.

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically-evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered - swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

Presynaptic Nrxn3 is essential for ribbon-synapse maturation in hair cells.

Hair cells of the inner ear and lateral-line system rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is critical for ribbon-synapse maturation in hair cells. In both mouse and zebrafish models, the loss of Nrxn3 results in significantly fewer intact ribbon synapses. We show in zebrafish that initially, nrxn3 mutants have normal pre- and post-synapse numbers, but synapses fail to pair, leading to postsynapse loss. We also demonstrate that Nrxn3 subtly influences synapse selectivity in zebrafish lateral-line hair cells that detect anterior flow. A 60% loss of synapses in zebrafish nrxn3 mutants dramatically reduces pre- and post-synaptic responses. Despite fewer synapses, auditory responses in zebrafish and mice are unaffected. This work demonstrates that Nrxn3 is a critical and conserved molecule required for the maturation of ribbon synapses. Understanding how ribbon synapses mature is essential to generating novel therapies to treat synaptopathies linked to auditory or vestibular dysfunction.

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.

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.

Presynaptic Nrxn3 is essential for ribbon-synapse assembly in hair cells.

Hair cells of the inner ear rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is critical for ribbon-synapse assembly in hair cells. In both mouse and zebrafish models, loss of Nrxn3 results in significantly fewer intact ribbon synapses. In zebrafish we demonstrate that a 60% loss of synapses in nrxn3 mutants dramatically reduces both presynaptic responses in hair cells and postsynaptic responses in afferent neurons. Despite a reduction in synapse function in this model, we find no deficits in the acoustic startle response, a behavior reliant on these synapses. Overall, this work demonstrates that Nrxn3 is a critical and conserved molecule required to assemble ribbon synapses. Understanding how ribbon synapses assemble is a key step towards generating novel therapies to treat forms of age-related and noise-induced hearing loss that occur due to loss of ribbon synapses.

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.

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.

Transcription factors underlying photoreceptor diversity.

During development, retinal progenitors navigate a complex landscape of fate decisions to generate the major cell classes necessary for proper vision. Transcriptional regulation is critical to generate diversity within these major cell classes. Here, we aim to provide the resources and techniques required to identify transcription factors necessary to generate and maintain diversity in photoreceptor subtypes, which are critical for vision. First, we generate a key resource: a high-quality and deep transcriptomic profile of each photoreceptor subtype in adult zebrafish. We make this resource openly accessible, easy to explore, and have integrated it with other currently available photoreceptor transcriptomic datasets. Second, using our transcriptomic profiles, we derive an in-depth map of expression of transcription factors in photoreceptors. Third, we use efficient CRISPR-Cas9 based mutagenesis to screen for null phenotypes in F0 larvae (F0 screening) as a fast, efficient, and versatile technique to assess the involvement of candidate transcription factors in the generation of photoreceptor subtypes. We first show that known phenotypes can be easily replicated using this method: loss of S cones in foxq2 mutants and loss of rods in nr2e3 mutants. We then identify novel functions for the transcription factor Tbx2, demonstrating that it plays distinct roles in controlling the generation of all photoreceptor subtypes within the retina. Our study provides a roadmap to discover additional factors involved in this process. Additionally, we explore four transcription factors of unknown function (Skor1a, Sall1a, Lrrfip1a, and Xbp1), and find no evidence for their involvement in the generation of photoreceptor subtypes. This dataset and screening method will be a valuable way to explore the genes involved in many other essential aspects of photoreceptor biology.

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.

Chronic neurotransmission increases the susceptibility of lateral-line hair cells to ototoxic insults.

Sensory hair cells receive near constant stimulation by omnipresent auditory and vestibular stimuli. To detect and encode these stimuli, hair cells require steady ATP production, which can be accompanied by a buildup of mitochondrial byproducts called reactive oxygen species (ROS). ROS buildup is thought to sensitize hair cells to ototoxic insults, including the antibiotic neomycin. Work in neurons has shown that neurotransmission is a major driver of ATP production and ROS buildup. Therefore, we tested whether neurotransmission is a significant contributor to ROS buildup in hair cells. Using genetics and pharmacology, we disrupted two key aspects of neurotransmission in zebrafish hair cells: presynaptic calcium influx and the fusion of synaptic vesicles. We find that chronic block of neurotransmission enhances hair-cell survival when challenged with the ototoxin neomycin. This reduction in ototoxin susceptibility is accompanied by reduced mitochondrial activity, likely due to a reduced ATP demand. In addition, we show that mitochondrial oxidation and ROS buildup are reduced when neurotransmission is blocked. Mechanistically, we find that it is the synaptic vesicle cycle rather than presynaptic- or mitochondrial-calcium influx that contributes most significantly to this metabolic stress. Our results comprehensively indicate that, over time, neurotransmission causes ROS buildup that increases the susceptibility of hair cells to ototoxins.

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.

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.

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.

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.

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.

Synaptic mitochondria regulate hair-cell synapse size and function.

Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca2+ through CaV1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca2+ influx through CaV1.3 channels initiates mitochondrial-Ca2+ (mito-Ca2+) uptake adjacent to ribbons. Block of mito-Ca2+ uptake in mature cells depresses presynaptic-Ca2+ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca2+ uptake coincides with spontaneous rises in presynaptic-Ca2+ influx. Spontaneous mito-Ca2+ loading lowers cellular NAD+/NADH redox and downregulates ribbon size. Direct application of NAD+ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca2+ couple to confer proper presynaptic function and formation.

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.

Leveraging Zebrafish to Study Retinal Degenerations.

Retinal degenerations are a heterogeneous group of diseases characterized by death of photoreceptors and progressive loss of vision. Retinal degenerations are a major cause of blindness in developed countries (Bourne et al., 2017; De Bode, 2017) and currently have no cure. In this review, we will briefly review the latest advances in therapies for retinal degenerations, highlighting the current barriers to study and develop therapies that promote photoreceptor regeneration in mammals. In light of these barriers, we present zebrafish as a powerful model to study photoreceptor regeneration and their integration into retinal circuits after regeneration. We outline why zebrafish is well suited for these analyses and summarize the powerful tools available in zebrafish that could be used to further uncover the mechanisms underlying photoreceptor regeneration and rewiring. In particular, we highlight that it is critical to understand how rewiring occurs after regeneration and how it differs from development. Insights derived from photoreceptor regeneration and rewiring in zebrafish may provide leverage to develop therapeutic targets to treat retinal degenerations.