Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish

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1. 

   Version published to 10.7554/elife.69264 on eLife

   Oct 19, 2021
2. 

   eLife

   Apr 15, 2021

   Author Response:

   We would like to thank the reviewers for their thoughtful and thorough critique of our manuscript. In our revised preprint, we added important additional data and restructured our manuscript to reflect as many of the recommendations as possible. Additionally, we have added experiments to define the cellular mechanisms underlying observed damage following mechanical injury. The most significant additions of new data include:

   • Further experiments demonstrating block of glutamate clearance exacerbates stimulus-induced hair-cell synapse loss.
   • Analysis of neuromast disruption in lhfpl5b mutant null larvae showing mechanical displacement. Lhfpl5b mediates mechanosensitivity in lateral-line hair cells, allowing us to determine whether mechanotransduction is required for mechanical disruption of neuromasts.
   • Testing the vibratory …

   Author Response:

   We would like to thank the reviewers for their thoughtful and thorough critique of our manuscript. In our revised preprint, we added important additional data and restructured our manuscript to reflect as many of the recommendations as possible. Additionally, we have added experiments to define the cellular mechanisms underlying observed damage following mechanical injury. The most significant additions of new data include:

   • Further experiments demonstrating block of glutamate clearance exacerbates stimulus-induced hair-cell synapse loss.
   • Analysis of neuromast disruption in lhfpl5b mutant null larvae showing mechanical displacement. Lhfpl5b mediates mechanosensitivity in lateral-line hair cells, allowing us to determine whether mechanotransduction is required for mechanical disruption of neuromasts.
   • Testing the vibratory stimulus at various frequencies to confirm the optimal frequency to induce acute, generally sub-lethal damage to lateral-line hair cells is 60 Hz.
   • Assessment of neuromast supporting cell and hair cell proliferation following mechanical overstimulation.
   • Quantitative analysis of kinocilia SEM and confocal images of hair bundles in control and stimulus exposed fish. Individual comments are addressed as outlined below.

   Reviewer #1:

   1. The authors use a vertically-oriented Brüel+Kjær LDS Vibrator to deliver a 60 Hz vibratory stimulus to damage lateral line hair cells. It is not made clear on why this frequency was selected. Did the authors choose this frequency because they screened a number of frequencies and this is the one that did the most damage to hair cells or was it chosen for another reason? Or, do all frequencies do the same amount of damage? The authors should screen a number of frequencies and choose the stimulus that does the most damage to hair cells. This would set the field in the best direction, should members of the community attempt this new technique. It is not necessary to repeat all of the experiments, but the authors should show which frequencies are best for inducing damage.

   The frequency selected for mechanical overexposure of lateral-line organs was based on previous studies showing 60 Hz to be within the optimal upper frequency range of mechanical sensitivity of superficial posterior lateral-line neuromasts, with maximal response between 10-60 Hz, but a suboptimal frequency for hair cells of the anterior macula in the ear (Weeg and Bass 2002, Trapani et al, 2009, Levi et al, 2015). To confirm that 60 Hz was the optimal frequency to induce damage, we tested 45, 60, and 75 Hz at comparable intensities. We observed at 75 Hz no apparent damage to lateral line neuromasts while 45 Hz at a comparable intensity proved toxic i.e. it was lethal to the fish. We have updated the Results and Method Details to include our rationale for choosing 60 Hz.

   1. The SEM images of the hair bundle are beautiful and do show damage to the hair bundle, but historically speaking older studies in mammals have shown that the actin core of the stereocilia is damaged. It would be critical to know if this was the case. Showing damage to the kinocilium and stereocilia splaying is a start, but readers would need to know if the actin cores are damaged. So, TEM should be used to find damage to the actin cores of stereocilia.

   Our main goal of this initial manuscript was to survey morphological and functional changes in mechanically injured lateral line organs with an emphasis on inflammation and synapse loss. We agree TEM studies showing damage to the actin core of the stereocilia will be important to determine whether mechanical damage to neuromast hair bundles fully mimics mammalian stereocilia damage, but these experiments will require significant time to perform and optimize. We have expanded our analysis of hair-bundle morphology in this study and intend to pursue deeper analysis of hair bundle damage, i.e. examination of the stereocilia actin core, in future follow-up studies.

   1. I think the use of "Noise-exposed lateral line" as a term for mechanically overstimulated lateral line hair cells is not correct and could be misleading. The lateral line senses water motion not sound as the word noise would imply. Calling the stimulus "noise" should be removed throughout.

   We have removed the term “noise” throughout the manuscript and replaced it with either “strong water current stimulus” or “mechanical overstimulation” where appropriate.

   1. Decreases in mechanotransduction are shown by dye entry. These results should be strengthened using microphonic potentials to determine the extent of damage. This experiment is not necessary but would improve the quality of the document.

   While we agree that microphonic recordings would provide further support for reduced mechanotransduction, quantitative FM1-43 uptake in zebrafish lateral line hair cells is a well-established proxy for microphonic measurements. In a previous study using the same protocol utilized in our manuscript, FM1-43 labeling intensity was shown to directly correspond with microphonic amplitude (Toro et al, 2015). Moreover, the fixable analogue of FM1-43 (FM1-43FX) gave us comparable relative measurements of uptake as live FM1-43 and provided the additional advantage of high temporal resolution and the ability to simultaneously assay entire cohorts of control and overstimulated fish (which is not possible with microphonic measurements or live FM1-43 imaging), as we could expose groups of fish briefly to the dye at determined time intervals following overstimulation, then immediately place in fixative.

   1. In figure 2, PSD labeling is not clear.

   We assume the reviewer meant PSD labeling in Figure 4 and we agree it is difficult to discern. We have changed the hair-cell label from gray to blue in the images so that the green PSD labeling is clear.

   Reviewer #2:

   1. While the findings are carefully measured and described, the effects of insult on hair cells are relatively minor, with a change in hair cell number, extent of innervation or synapses per hair cell (Figs 3 and 4) in the range of 10% reduction compared to control. One potential value of the model would be to use it to discover underlying pathways of damage or screen for potential therapeutics. However with these modest changes it is not clear that there will be enough power to determine effects of potential interventions.

   One advantage of the zebrafish model is the ability to overstimulate large cohorts of larvae, thereby providing enough power to uncover modest but significant changes resulting from moderate damage to hair cells. While not as well suited for unbiased large-scale screens of therapeutics, our overexposure protocol provides the opportunity to determine the role of specific cellular pathways (e.g. metabolic stress, inflammation, and glutamate excitotoxicity) in hair-cell damage and synapse loss following mechanically-induced damage via genetic or pharmacological manipulation of these pathways. Additionally, as the hair cell synapses fully repair following stimulus-induced loss, the zebrafish model has the potential for identifying novel pathways for repair through transcriptomic profiling (for an example, see Mattern et al, Front. Cell Dev. Biol., 2018). Cumulatively, these future experimental directions will provide important mechanistic information that could be used toward the development of targeted therapeutic interventions.

   1. The most dramatic phenotype after shaking is a physical displacement of hair cells, described as disrupted morphology. However it is not clear what the underlying cause of this change. Are only posterior neuromasts damaged in this way? Is it a wounding response as animals are exposed to an air interface during shaking? It is also not clear to what extent this displacement reveals more general principles of the effects of noise on hair cells. Additional discussion of underlying causes would be welcome.

   We agree that the underlying causes of the physical displacement of posterior lateral-line neuromasts warranted further investigation and we have expanded appropriate sections of the results. To determine if excessive hair-cell activity plays a role in the displacement of neuromasts we have exposed lhfpl5b mutant—fish that have intact hair cell function in the ear, but no mechanotransduction in hair cells of the lateral line—to mechanical overstimulation. We observed comparable disruption of neuromasts lacking mechanotransduction, supporting that displacement of lateral-line hair cells is due to mechanical damage and does not require intact mechnotransduction. Further, when examining the adjacent supporting cells in disrupted neuromasts, we observed they are similarly displaced and elongated. We conclude that observed disruption of hair cells is a consequence of mechanical displacement of the entire neuromast organ. We have added additional discussion of this phenomenon to the Results and Discussion sections of the manuscript.

   1. Because afferent neurons innervate more than one neuromast and more than one hair cell per neuromast, measurements of innervation of neuromasts (Figure 3) or synapses per hair cell (Fig 4) cannot be assumed to be independent events. That is, changes in a single postsynaptic neuron may be reflected across multiple synapses, hair cells, and even neuromasts. This needs to be accounted for in experimental design for statistical analysis.

   We agree that changes in single postsynaptic neurons, which innervate groups of hair cells of the same polarity within a neuromast, could be reflected across multiple synapses. Additionally, it is plausable that excitotoxic events at the postsynapse, while not contributing to apparent neurite retraction, could be contributing to synapse loss across multiple innervated hair cells. We have updated the manuscript to reflect the potential contribution of postsynaptic signaling to synapse loss and added experiments pharmacologically blocking glutamate uptake.

   1. The SEM analysis provides compelling snapshots of apical damage, but could be supplemented by quantitative analysis with antibody staining or transgenic lines where kinocilia are labeled. The amount of reduced FM1-43 labeling is one of the more dramatic effects of the shaking insult, suggesting widespread disruption to mechanotransduction that could be related to this apical damage. Further examination of the recovery of mechanotransduction would be interesting.

   To supplement the SEM snapshots of severe apical damage, we have expanded the SEM image analysis with quantitative data on kinocilia morphology. We have also added confocal images of hair bundles using antibody labeling of acetylated tubulin in a transgenic line expressing β-actin-GFP in hair cells. We agree that correlative studies of mechanotransduction recovery relative to hair-bundle morphology would be interesting, and we intend to examine this question in a future follow-up study.

   1. A previous publication by Uribe et al.2018 describes a somewhat similar shaking protocol with somewhat different results - more long-lasting changes in hair cell number, presynaptic changes in synapses, etc. It would be worth discussing potential differences across the two studies.

   We agree we did not adequately address the considerable differences between our mechanical damage protocol for the zebrafish lateral line and the damage protocol described by Uribe et al, 2018. We have provided a more direct comparison in the Results section and addressed the differences in our protocols in-depth in the Discussion section.

   Our damage protocol uses a stimulus within the known frequency range of lateral-line hair cells (60 Hz) that is applied to free-swimming larvae and evokes a behaviorally relevant response (fast start response). The damage is observable immediately following noise exposure, is specific to posterior lateral-line neuromasts, and appears to be rapidly repaired. Some features of the damage we observe—reduced mechanotransduction and hair-cell synapse loss—may correspond to mechanically induced damage of hair cell organs in other species. Notably, hair cell synapse loss in seemingly intact neuromasts is exacerbated by pharmacologically blocking synaptic glutamate clearance, supporting that the 60 Hz frequency stimulus is overstimulating neuromast hair cells directly and suggesting that the mechanism of synapse loss may be similar to inner hair cell synapse loss reported in mice following moderate noise exposures.

   By contrast, the damage protocol published by Uribe et al used ultrasonic transducers (40-kHz) to generate small, localized shock waves rather than directly stimulate neuromast hair cells. The damaged they reported—delayed hair-cell death and modest synapse loss with no effect on hair-cell mechanotransduction—was not apparent until 48 hours following exposure and not specific to the lateral-line organ. Some of the features of the damage they observed—delayed onset apoptosis and hair-cell death—may correspond to damage reported in mice following blast injuries.

   Reviewer #3:

   1. As the authors point out, zebrafish hair cells can be regenerated. With that in mind, and to make the relevance for mammalian hair cell repair clear, a clear distinction between mechanisms mediated by "repair" or "regeneration" needs to be made. The authors discuss that proliferative hair cell generation can be excluded based on the short time period, but suggest that transdifferentiation might be involved. Recovery of NM hair cell number occurs within the same 2 hour period in which NM morphology and hair cell function improved, making it difficult to determine the extent to which "regeneration" contributed to the recovery. The amount of transdifferentiation has to be shown experimentally (lineage tracing?).

   We agree that the distinction between "repair" and "regeneration" needs to be made when discussing this model of mechanical damage to zebrafish hair cell organs. We have tried to clarify that most of what we observe regarding recovery—restoration of neuromast shape, mechanostransduction, afferent contacts, and synapse number —reflect mechanisms of repair following mechanical damage (and, in the case of synapse loss, overstimulation) rather than regeneration. However, one feature of damage that may reflect rapid regeneration is restoration of hair cells number following mechanical injury. To experimentally determine whether proliferation contributed to hair cell generation, we assessed the incorporation of the thymidine analog EdU during a 4 hour recovery following mechanical overexposure in a transgenic line expressing GFP in neuromast supporting cells and observe a modest but not statistically significant increase in the number of proliferating supporting cells in neuromasts exposed to strong current stimulus, suggesting recovery of lost hair cells is not primarily due to renewed proliferation.

   The number of hair cells that are lost and recover within several hours are low, i.e., typically ~1 hair cell/neuromast. We observed this consistently in all of our experiments, but the mechanisms responsible are not clear. Based on previous studies of hair cell regeneration in the lateral line, the recovery time appears too rapid to be caused by renewed proliferation, a notion that is further supported by our Edu studies. On the other hand, it is possible that a few supporting cells may undergo the initial phases of phenotypic change into hair cells during this short time period, and we speculate that such transdifferentiation may be responsible for the observed recovery. We should emphasize that this is a new observation and, at present, we do not fully understand the underlying mechanism. However, the focus of the present study is on mechanical damage, synaptic loss, and subsequent repair. We believe that it is important to report our consistent findings of low level hair cell loss and recovery, but a detailed characterization of the mechanism would require considerable effort and would best be the topic of a future study.

   1. The classification of "normal" vs "disrupted" is vague and not quantitative. The examples shown in the paper seem to be quite clear-cut, but this reviewer doubts that was the case throughout all analyzed samples. Formulate clear benchmarks and criteria for the disrupted phenotype (even when blind analysis is performed).

   We have defined measurable criteria for "normal" vs "disrupted" neuromasts that we have added to the Method Details section: “We defined exposed neuromast morphology as “normal” when hair cells appeared radially organized with a relatively uniform shape and size, with ≤7 μm difference observed when comparing the lengths from apex to base of an opposing pair of anterior/posterior hair cells. Length was measured from a fixed point at the center of the hair bundle to the basolateral end of each opposing hair cell. We defined neuromasts as “disrupted” when hair cells appeared elongated and displaced to one side, with >7 μm difference observed when comparing the lengths of an opposing pair of anterior/posterior hair cells. Generally, the apical ends of the hair cells were displaced posteriorly, with the basolateral ends oriented anteriorly.”

   1. Sustained and periodic exposure: These two exposure protocols not only differ with respect to sustained vs periodic, they also differ in total exposure time (Fig 2B). This complicates the interpretation, especially considering the authors own finding that a pre-exposure is protective.

   To clarify—pre-exposure was not protective to hair-cell survival. Rather, in preliminary experiments, pre-exposure appeared to reduce larval mortality, and we have clarified that observation in the text of the Results and the Methods Details sections. We agree with the reviewer that comparing the two protocols based on differences in time distribution is complicated in that they also differ in total exposure time. For the purpose of clarity, we now focus on the sustained exposure in the main figures and created supplemental figures for the reduced damage still observed using periodic exposure, specifying that reduced damage may be the result of periodic time distribution of stimulus and/or less cumulative time exposed to the stimulus.

   1. The data on the mitochondrial ROS aspect seems not well integrated into the overall story.

   We agree that the ROS story was not well integrated and incomplete. We have removed the data describing mpv17-/- mutants and mitochondrial disfunction from this manuscript. A more comprehensive report of mpv17-/- mutant mitochondrial function and morphological analysis of neuromasts following noise exposure is now described in a follow-up manuscript (“Influence of Mpv17 on hair-cell mitochondrial homeostasis, synapse integrity, and vulnerability to damage in the zebrafish lateral line”).

   1. It is surprising that the hair bundle morphology was not assessed after recovery. This is crucial. Overall, it would be good to see some quantification of the SEM data, e.g. kinocilia length and number of splayed bundles.

   We have expanded the SEM image analysis to quantitatively access kinocilia morphology following exposure. We agree that assessment of recovery using live imaging of hair bundles paired with subsequent SEM analysis will be informative, and we intend to perform those experiments in a future study.

   1. Behavioral recovery (measured as number of "fast start" responses) was also not assessed. This is essential for determining the functional relevance of the recovery.

   We attempted to measure behavior recovery of lateral-line function by measuring “fast-start” responses immediately and several hours after recovery, and discovered that i) strong water current provided stimulation that was too intense to reveal subtle behavioral changes following lateral-line damage and recovery, and ii) when testing larvae immediately following sustained strong current exposures, it was difficult to discern if fewer “fast-start” responses were due to lateral-line organ damage or larval fatigue. We agree that behavioral recovery is important to assay but acknowledge assessing lateral-line mediated behavior following mechanical damage will require a more sensitive testing paradigm that stimulates the lateral-line sensory organ with a relatively gentile, calibrated water flow stimulus. We are currently performing a follow-up study to this paper using a testing paradigm developed by a postdoctoral associate in our lab that analyses subtle changes in larval orientation to water flow (rheotaxis) mediated by the lateral-line organ. Using this behavior paradigm, we will directly correlate morphological and functional recovery over time.

   1. This reviewer is not yet convinced that this damage model displays enough commonalities to mammalian noise damage to justify the ubiquitous use of the term "noise" throughout the manuscript. It would be more prudent to use a more careful term along the lines of "mechanical overstimulation-induced damage".

   We have removed the term “noise” throughout the manuscript and replaced it with either “strong water current stimulus” or “mechanical overstimulation” where appropriate.

   1. Overall, there was a lack of experimental and analysis detail in the results section. For example, how was afferent innervation quantified? Just counting GFP labeled contacts to hair cells?

   Innervation of neuromast hair cells was quantified during blinded analysis by scrolling through confocal z-stacks of each neuromast (step size 0.3 μm) containing hair cell and afferent labeling and identifying hair cells that were not directly contacted by an afferent neuron i.e. no discernable space between the hair cell and the neurite. Hair cells that were identified as no longer innervated showed measurable neurite retraction; there was generally >0.5 μm distance between a retracted neurite and hair cell. We have added this information to the Methods Detail section.

   There was also inconsistency in the use of two variations of the mechanical damage protocol, the time points at which repair was assessed, and whether the damage was quantified in all neuromasts or in normal vs. disrupted neuromasts separately, making the data difficult to interpret.

   We have revised our figure legends to clearly indicate when we are assessing damage in all exposed neuromasts (pooled) to control vs. comparative analysis of normal vs. disrupted neuromasts relative to control. In addition, we now focus on the sustained exposure in the main figures, which was the exposure protocol used for the time points in which repair and recovery were assessed.

   Read the original source
3. 

   eLife

   Sep 25, 2020

   ###Reviewer #3:

   Holmgren et al describe a novel model of reversible mechanical damage to zebrafish neuromast hair cells. The authors demonstrate that when zebrafish are exposed to strong currents, neuromast morphology, hair cell number, innervation, and MET function suffer various types and degrees of damage, from which the NMs recover within 2 days. Additionally, they show macrophage recruitment to damaged neuromasts, where they may be phagocytosing synaptic debris. Based on various mechanistic and phenotypic commonalities (involvement of ROS, stereocilia and synapse phenotype), the authors argue that this model is a good approximation of noise-induced hair cell damage in mammals.

   Overall impact:

   This reviewer agrees that a "noise" damage model in the zebrafish would be a powerful tool to better understand the mechanisms underlying …

   ###Reviewer #3:

   Holmgren et al describe a novel model of reversible mechanical damage to zebrafish neuromast hair cells. The authors demonstrate that when zebrafish are exposed to strong currents, neuromast morphology, hair cell number, innervation, and MET function suffer various types and degrees of damage, from which the NMs recover within 2 days. Additionally, they show macrophage recruitment to damaged neuromasts, where they may be phagocytosing synaptic debris. Based on various mechanistic and phenotypic commonalities (involvement of ROS, stereocilia and synapse phenotype), the authors argue that this model is a good approximation of noise-induced hair cell damage in mammals.

   Overall impact:

   This reviewer agrees that a "noise" damage model in the zebrafish would be a powerful tool to better understand the mechanisms underlying noise-induced hearing loss. However, due to various weaknesses of the data (detailed below), the main claims of the paper are not sufficiently supported. In addition, noise-induced hearing loss has been previously modeled in the zebrafish model. The present model, therefore, does not provide a significant methodological innovation.

   Major concerns:

   1. As the authors point out, zebrafish hair cells can be regenerated. With that in mind, and to make the relevance for mammalian hair cell repair clear, a clear distinction between mechanisms mediated by "repair" or "regeneration" needs to be made. The authors discuss that proliferative hair cell generation can be excluded based on the short time period, but suggest that transdifferentiation might be involved. Recovery of NM hair cell number occurs within the same 2 hour period in which NM morphology and hair cell function improved, making it difficult to determine the extent to which "regeneration" contributed to the recovery. The amount of transdifferentiation has to be shown experimentally (lineage tracing?).

   2. The classification of "normal" vs "disrupted" is vague and not quantitative. The examples shown in the paper seem to be quite clear-cut, but this reviewer doubts that was the case throughout all analyzed samples. Formulate clear benchmarks and criteria for the disrupted phenotype (even when blind analysis is performed).

   3. Sustained and periodic exposure: These two exposure protocols not only differ with respect to sustained vs periodic, they also differ in total exposure time (Fig 2B). This complicates the interpretation, especially considering the authors own finding that a pre-exposure is protective.

   4. The data on the mitochondrial ROS aspect seems not well integrated into the overall story.

   5. It is surprising that the hair bundle morphology was not assessed after recovery. This is crucial. Overall, it would be good to see some quantification of the SEM data, e.g. kinocilia length and number of splayed bundles.

   6. Behavioral recovery (measured as number of "fast start" responses) was also not assessed. This is essential for determining the functional relevance of the recovery.

   7. This reviewer is not yet convinced that this damage model displays enough commonalities to mammalian noise damage to justify the ubiquitous use of the term "noise" throughout the manuscript. It would be more prudent to use a more careful term along the lines of "mechanical overstimulation-induced damage".

   8. Overall, there was a lack of experimental and analysis detail in the results section. For example, how was afferent innervation quantified? Just counting GFP labeled contacts to hair cells? There was also inconsistency in the use of two variations of the mechanical damage protocol, the time points at which repair was assessed, and whether the damage was quantified in all neuromasts or in normal vs. disrupted neuromasts separately, making the data difficult to interpret.

   Read the original source
4. 

   eLife

   Sep 25, 2020

   ###Reviewer #2:

   Holmgren et al. describe the development of a model for hair cell noise damage using the zebrafish lateral line line system. Using an electrodynamic shaker, the authors induce quantifiable damage and death of hair cells after a two-hour treatment. They describe gross morphological changes of hair cells, changes in innervation and synapse distribution. In addition they describe disruption of stereocilia and kinocilia, as well as reduced mechanotransduction-dependent uptake of FM1-43 dye. Damage is no longer detectable several hours after insult, demonstrating recovery.

   1. While the findings are carefully measured and described, the effects of insult on hair cells are relatively minor, with a change in hair cell number, extent of innervation or synapses per hair cell (Figs 3 and 4) in the range of 10% reduction compared to …

   ###Reviewer #2:

   Holmgren et al. describe the development of a model for hair cell noise damage using the zebrafish lateral line line system. Using an electrodynamic shaker, the authors induce quantifiable damage and death of hair cells after a two-hour treatment. They describe gross morphological changes of hair cells, changes in innervation and synapse distribution. In addition they describe disruption of stereocilia and kinocilia, as well as reduced mechanotransduction-dependent uptake of FM1-43 dye. Damage is no longer detectable several hours after insult, demonstrating recovery.

   1. While the findings are carefully measured and described, the effects of insult on hair cells are relatively minor, with a change in hair cell number, extent of innervation or synapses per hair cell (Figs 3 and 4) in the range of 10% reduction compared to control. One potential value of the model would be to use it to discover underlying pathways of damage or screen for potential therapeutics. However with these modest changes it is not clear that there will be enough power to determine effects of potential interventions.

   2. The most dramatic phenotype after shaking is a physical displacement of hair cells, described as disrupted morphology. However it is not clear what the underlying cause of this change. Are only posterior neuromasts damaged in this way? Is it a wounding response as animals are exposed to an air interface during shaking? It is also not clear to what extent this displacement reveals more general principles of the effects of noise on hair cells. Additional discussion of underlying causes would be welcome.

   3. Because afferent neurons innervate more than one neuromast and more than one hair cell per neuromast, measurements of innervation of neuromasts (Figure 3) or synapses per hair cell (Fig 4) cannot be assumed to be independent events. That is, changes in a single postsynaptic neuron may be reflected across multiple synapses, hair cells, and even neuromasts. This needs to be accounted for in experimental design for statistical analysis.

   4. The SEM analysis provides compelling snapshots of apical damage, but could be supplemented by quantitative analysis with antibody staining or transgenic lines where kinocilia are labeled. The amount of reduced FM1-43 labeling is one of the more dramatic effects of the shaking insult, suggesting widespread disruption to mechanotransduction that could be related to this apical damage. Further examination of the recovery of mechanotransduction would be interesting.

   5. A previous publication by Uribe et al.2018 describes a somewhat similar shaking protocol with somewhat different results - more long-lasting changes in hair cell number, presynaptic changes in synapses, etc. It would be worth discussing potential differences across the two studies.

   Read the original source
5. 

   eLife

   Sep 25, 2020

   ###Reviewer #1:

   In the manuscript titled "Mechanical overstimulation causes acute injury followed by fast recovery in lateral-line neuromasts of larval zebrafish" by Holmgren et al., the authors develop a method to overstimulate hair cells and determine some of the consequences of this overstimulation. The overarching goal of this work is to develop a model for noise-induced hair-cell damage in the zebrafish. The authors use the lateral line for their studies and stimulate hair cells using an electrodynamic shaker which generate significant aqueous agitation. The authors demonstrate physical damage to hair cells of the lateral line that are dependent on the position of the neuromast. The damage includes alteration of afferent synapses, afferent neurite retraction, limited damage to hair bundles and a decrease in mechanotransduction. …

   ###Reviewer #1:

   In the manuscript titled "Mechanical overstimulation causes acute injury followed by fast recovery in lateral-line neuromasts of larval zebrafish" by Holmgren et al., the authors develop a method to overstimulate hair cells and determine some of the consequences of this overstimulation. The overarching goal of this work is to develop a model for noise-induced hair-cell damage in the zebrafish. The authors use the lateral line for their studies and stimulate hair cells using an electrodynamic shaker which generate significant aqueous agitation. The authors demonstrate physical damage to hair cells of the lateral line that are dependent on the position of the neuromast. The damage includes alteration of afferent synapses, afferent neurite retraction, limited damage to hair bundles and a decrease in mechanotransduction. After damage, they show macrophage recruitment and quick recovery of hair cell neuromasts, which is surprising.

   The paper is interesting in that it brings a new capacity to the zebrafish animal model: mechanical overstimulation of the hair cell. Tempering this is a general feeling that the authors do not dig deep enough in the current form of the manuscript, but this could be remedied. More specifically, the authors are making a model in zebrafish for noise-induced damage, so they need to show that this model is similar to mammals in the way hair cells are damaged. This is done in the manuscript, but it is limited and should be expanded as suggested below.

   Major comments

   1. The authors use a vertically-oriented Brüel+Kjær LDS Vibrator to deliver a 60 Hz vibratory stimulus to damage lateral line hair cells. It is not made clear on why this frequency was selected. Did the authors choose this frequency because they screened a number of frequencies and this is the one that did the most damage to hair cells or was it chosen for another reason? Or, do all frequencies do the same amount of damage? The authors should screen a number of frequencies and choose the stimulus that does the most damage to hair cells. This would set the field in the best direction, should members of the community attempt this new technique. It is not necessary to repeat all of the experiments, but the authors should show which frequencies are best for inducing damage.

   2. The SEM images of the hair bundle are beautiful and do show damage to the hair bundle, but historically speaking older studies in mammals have shown that the actin core of the stereocilia is damaged. It would be critical to know if this was the case. Showing damage to the kinocilium and stereocilia splaying is a start, but readers would need to know if the actin cores are damaged. So, TEM should be used to find damage to the actin cores of stereocilia.

   3. I think the use of "Noise-exposed lateral line" as a term for mechanically overstimulated lateral line hair cells is not correct and could be misleading. The lateral line senses water motion not sound as the word noise would imply. Calling the stimulus "noise" should be removed throughout.

   4. Decreases in mechanotransduction are shown by dye entry. These results should be strengthened using microphonic potentials to determine the extent of damage. This experiment is not necessary but would improve the quality of the document.

   5. In figure 2, PSD labeling is not clear.

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6. 

   eLife

   Sep 25, 2020

   ##Preprint Review

   This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. Doris K Wu (NIDCD, NIH) served as the Reviewing Editor.

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7. 

   Version published to 10.1101/2020.07.15.205492 on bioRxiv

   Jul 17, 2020