Drosophila mechanical nociceptors preferentially sense localized poking

  1. Zhen Liu
  2. Meng-Hua Wu
  3. Qi-Xuan Wang
  4. Shao-Zhen Lin
  5. Xi-Qiao Feng
  6. Bo Li
  7. Xin Liang

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    Evaluation Summary:

    Liu et al present fascinating findings that significantly extend the understanding of molecular and cellular pathways of mechanical nociception in Drosophila larvae. This work is of very high interest to neuroscientists studying sensory function and its molecular underpinnings with implications for our understanding of acute sensation of painful stimuli. The approach and data are of very high quality and provide unprecedented insight into mechanosensory functions in an intact tissue environment.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Mechanical nociception is an evolutionarily conserved sensory process required for the survival of living organisms. Previous studies have revealed much about the neural circuits and sensory molecules in mechanical nociception, but the cellular mechanisms adopted by nociceptors in force detection remain elusive. To address this issue, we study the mechanosensation of a fly larval nociceptor (class IV da neurons, c4da) using a customized mechanical device. We find that c4da are sensitive to mN-scale forces and make uniform responses to the forces applied at different dendritic regions. Moreover, c4da showed a greater sensitivity to localized forces, consistent with them being able to detect the poking of sharp objects, such as wasp ovipositor. Further analysis reveals that high morphological complexity, mechanosensitivity to lateral tension and possibly also active signal propagation in dendrites contribute to the sensory features of c4da. In particular, we discover that Piezo and Ppk1/Ppk26, two key mechanosensory molecules, make differential but additive contributions to the mechanosensitivity of c4da. In all, our results provide updates into understanding how c4da process mechanical signals at the cellular level and reveal the contributions of key molecules.

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  1. Version published to 10.7554/elife.76574 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    1. Information is missing about the regions of interest in which calcium responses were measured. Judging from Fig. 1E, calcium signals were measured in the somata, and this should be specified. Also judging from this figure, calcium signals seem to be largely confined to the somata and virtually absent from dendritic arbors. Fig. 6a shows very faint signals in the dendrites, yet those signals seem to have been measured rather far from the point of force application (a scale bar is shown but undefined), and, for some unknown reason, not between soma and force application point). Should there be detectable calcium signals in the somata, respective image gains should be adjusted so that those signals can be appreciated by the reader. If there are no clear signals in the dendrites, this would affect interpretations concerning e.g. Ca-α1D.

    Calcium responses can be observed in the soma and dendrites, which was presented in the original manuscript (Figure 6). Inspired by the 2nd suggestion from this reviewer, we went through our data and refined our measurement of the dendritic signal in the revised manuscript (see revised Figure 6). In addition, we also showed that the dendritic response was dependent on Ca-α1D (see revised Figure 6 and Figure 6-figure supplement 1). Finally, in the revised manuscript, we made it clear that all F/F0 were measured from the soma unless otherwise stated (see Figure 2, legend).

    1. Along this line, analyzing also the spacial distribution of dendritic calcium responses to the pokes would provide a much more detailed picture about how the dendritic tree responds to the various pokes. The beauty of the imaging approach chosen here is that it provides such information. Rather than ignoring this possibility, it should be exploited in this study, especially as respective data might provide much deeper insights into the relation between the mechanosensory function of the cell and its dendritic tree (and bolster the modelling results in Fig. 4 experimentally).

    In the original manuscript, we included the data on the dendritic calcium signal and showed that the dendritic signal was reduced when the activity of VGCCs were inhibited or in the Ca-α1D knockdown mutant (see Fig. 6 A-B in the original manuscript). Inspired by the suggestion from the reviewers, we had a closer look at our data and performed additional experiments. In the revised Figure 6 A-B, we showed that the mechanical stimuli could evoke calcium responses not only in the soma, but also in the homolateral (i.e. between the soma and the force probe) and contralateral (i.e. opposite side of the force probe) dendrites, suggesting that the dendritic signals are propagating within the dendritic arbors. Moreover, in the revised Figure 6 A-B and Figure 6-figure supplement 1, we showed that these dendritic signals were reduced in the mutant strains of Ca-α1D or if the fillet preparation was treated with nimodipine, demonstrating a clear dependence on the activity of VGCCs. However, because our imaging speed is not fast enough to capture the dendritic flow of calcium signals, the dynamics of signal propagation remains undefined. This would be an interesting issue to study in the future. Along with the revised Figure 6, we also revised the text and legends accordingly.

    1. When showing response functions as in e.g. Figs. 2C, G, H, 3D, 5C-E, etc., the y-axis should have a logarithmic scaling; receptor potentials of receptor cells usually scale proportionally to the logarithm of the stimulus amplitude. Only then, the reader will be able to fully appreciate the sensitivity differences. This will also alter interpretation of response function slopes.

    We thank the reviewer for the suggestion. However, the stimulation force is actually a distal stimulus for the cell, while the proximal stimuli (e.g. local deformation) are difficult to measure/estimate. Therefore, we are not sure if the cellular responses scale necessarily to the logarithm of macroscopic forces (i.e. the distal stimuli). However, simply by looking at the data, we found that the response is proportional to the force and for conciseness, and thus we fitted the plot using a linear function.

    1. The knockdown and mutant data is interesting, yet important controls are missing. For the RNAi lines used, qPCR data on the knockdown-efficiency should be added. For the channel mutations, available genetic rescue lines should be used as controls. Data on protein localization is presented for the mechanosensitive channels, but not for voltage-gated calcium channel subunit. Should antibodies be available, respective stainings should be included. If not, the authors should at least check whether Ca-α1D is expressed in the cell using e.g. Mi{ET1}Ca-α1D[MB06807] that is available at Bloomington.

    First, we did not use RNAi mutant for Piezo. The PiezoKO line is a genomic mutant strain.

    Second, for Ca-α1D, because there are only a small number of c4da in each animal and Ca-α1D has a quite broad expression in various types of neurons (see our revised Figure 6-figure supplement 2), we expected that the reduction in the expression level of Ca-α1D in c4da would be very difficult to detect. Therefore, we knocked down the expression of Ca-α1D in the whole animal using the same uas-Ca-α1Di strain and the tub-gal4 strain. Using RT-PCR, we showed that the expression level of Ca-α1D was significantly reduced (revised Figure 6-figure supplement 2). In fact, the same RNAi strain was also used in other functional studies.

    1. The statistics used is not entirely convincing. T-test are used throughout, though I do not feel that all the data is distributed normally. Moreover, some figures include multiple comparisons, apparently without statistical correction. The data should be re-analyzed using appropriate statistical procedures.

    We thank the reviewer for this suggestion. We have now used Mann-Whitney U test or Kruskal Wallis test for all the data that were not proven to follow a normal distribution. For multiple comparisons, we used One-way ANOVA. We have now included the relevant information in the revised figure legends.

  3. eLife

    Evaluation Summary:

    Liu et al present fascinating findings that significantly extend the understanding of molecular and cellular pathways of mechanical nociception in Drosophila larvae. This work is of very high interest to neuroscientists studying sensory function and its molecular underpinnings with implications for our understanding of acute sensation of painful stimuli. The approach and data are of very high quality and provide unprecedented insight into mechanosensory functions in an intact tissue environment.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Liu et al present findings that significantly extend the understanding of molecular and cellular pathways of mechanical nociception in Drosophila larvae. They present a detailed analysis of the mechanical response properties of the nociceptors that is performed using a newly developed preparation for optical recordings from these cells. Using mechanical probes of varying tip diameters they are able to investigate the responses as they relate to force and pressure. Mutants in the cut gene, which show reduced branching of the nociceptors, are used to interrogate how the dendritic morphology structure might relate to their physiological responses. As well, the response profiles of the neurons that are mutant for mechanosensory channels ppk-26 and piezo are investigated. The ppk-26 mutant shows a more strongly impaired deficit in comparison to piezo mutants and double mutants show a nearly abolished mechanical response. The mechanosensory neurons are also found to respond to mechanical forces that are outside of the main dendritic fields which suggests that they are able to detect forces that are viscoelatically coupled through the overlying epidermis. Finally, a voltage activated calcium channel is found to be important which is consistent with findings from prior studies (Terada et al 2016) that the dendrites of these nociceptive neurons may be "active" (as opposed to passive). Overall, the study significantly advances our understanding of the response properties of the mechanical nociceptors of the Drosophila larva.

    Although the overall findings are quite interesting, the conclusions of the study could be strengthened according to the following points:

    The duration of the calcium responses long outlasts the force application. It has been previously proposed that dendritic breakage could be a contributing factor in the transduction mechanism in the Drosophila nociceptive neurons (Tracey, 2017). What is the relationship of dendritic breakage occur with the various force stimuli and probe sizes in the imaging setup? Sharp probes (smaller probes) may be more likely to break dendrites. Similarly, do the sharp probes that trigger rolling in Figure 2J break dendrites?

    A very nice paper from Joe Howards lab has found that axonal activation of the cIVda neurons can happen even in the absence of any dendritic Calcium signals. This finding argues that any active properties of the dendrites only occur with very strong activation and/or direct stimulation of the dendrites. The authors must cite this paper and incorporate it into their discussion: "Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca 2+ signals:, Rajshekhar Basak , Sabyasachi Sutradhar, Jonathon Howard 2, DOI: 10.1016/j.bpj.2021.06.001

    Similarly, do forces applied outside the dendritic field activate calcium signals in dendrites or are these signals limited to the soma and axon?

    Although prior studies of Terada et al have demonstrated the importance of Ca-alpha1D channels in the dendritic Ca transients, the distribution of the Ca-alpha-1D channels has not yet been demonstrated in these cells. It will be important to know in future studies if these channels distribute distally in the dendrites or if they have a more proximal localizations.

    The results of experiments with cut are very interesting, and the authors are open and cautious in their interpretation when they state the caveats of unknown epistatic effects that may result from removal of a transcription factor. Even with the stated caveats, the unknown effects of cut removal causes the results to be very difficult to interpret. It cannot be strongly concluded that the deficits are a consequence of reduced branching. For example, perhaps calcium-alpha1d is a target of cut transcription?

    Where are the values for the elastic modulus for larval cuticle coming from in line 543? Experimental measurements of the stiffness of larval cuticle come in at 0.39 +/- 0.01 MPa which is an order of magnitude lower than the values input to the model (10Mpa). Kohane M, Daugela A, Kutomi H, Charlson L, Wyrobek A, Wyrobek J. Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development. J Biomed Mater Res A. 2003 Sep 1;66(3):633-42. doi: 10.1002/jbm.a.10028. PMID: 12918047. The modeling should be repeated with more realistic values of larval elastic modulus.

    Also in the model, the true value for instar larval cuticle is closer to 20 microns thick according to experimental measurements. (Christine E. Kaznowski, Howard A. Schneiderman, Peter J. Bryant, Cuticle secretion during larval growth in Drosophila melanogaster, Journal of Insect Physiology, Volume 31, Issue 10, 1985, Pages 801-813, https://doi.org/10.1016/0022-1910(85)90073-3).

    The reference provided for elastic modulus of muscle comes from a study on human muscles and is therefore not valid for a study performed on Drosophila larvae.

    Is the force probe is compressing the entire larval filet such that there is an indentation into the PDMS (as depicted in figure 1A)? If so, then isn't it true that the forces on the filet (dendrites) are not coming solely from the force probe itself? If the larva is being squished between the probe and the PDMS, then the larva is also being exposed to an opposing force that is coming from the PDMS.

  5. eLife

    Reviewer #2 (Public Review):

    The present work by the authors characterizes the mechano-sensitive properties of nociceptive neurons in Drosophila larvae (so called c4da neurons) in a very precise way and aims to show how their morphology and specifically expressed channels contribute to their functional responses.

    The authors developed a sophisticated piezo-driven probe to deliver precisely defined mechanical stimuli and combined it with functional imaging of Drosophila larval nociceptors in semi-intact preparations. Their clever setup allowed them to measure mechanical responses of these neurons to mN range stimuli. By using defined probes with different diameters, they showed that c4da neurons display almost uniform responses nearly independent of the stimulation site within their dendritic field. Moreover, the authors convincingly show that c4da neurons preferentially respond to small diameter probes (30 microns) and that their uniform dendritic coverage is also required for detection of distal mechanical stimuli. Stimulation of these neurons results in defensive rolling behavior, which has likely evolved in Drosophila larvae to defend against being stung by parasitoid wasps. The typical ovipositor of such wasps is 5-20um suggesting that c4da neuron responses are indeed optimized for detecting small diameter mechanical stimuli as shown by the authors. Elaborating on the broader biological significance of the authors' findings (and extrapolation to other species/higher organisms) might have been useful for a more general audience.

    Using off-dendrite stimulation and theoretical modeling of perpendicular and lateral pressure distribution of their mechanical probe, the authors nicely show that sensory neuron dendrites are sensitive to lateral tension thus featuring an expanded force-receptive field. They continue by showing that two of the expressed mechanosensory channels, ppk26 and piezo, differentially contribute to c4da neuron responses. The authors argue that ppk26 is important for overall and piezo for localized mechanosensitivity, with both participating in sensing lateral tension. The interpretation of the data seems simplified for multiple reasons: a) piezo has been shown to be widely expressed in different tissues (e.g. Kim et al. Nature 2012), thus it might contribute in different ways to c4da mechanosensitivity. b) piezo loss of function does not strongly affect responses to the 30/60 micron probe when applied proximal to the neuron, but does so only in distal regions. c) loss of ppk26 results in disproportionally stronger loss of responses to the 30 micron probe (i.e. more local force), which suggests it could be particularly relevant for perpendicular pressure sensing. Overall, this might reflect that these channels are differentially involved in maintaining similar cellular responses throughout the dendritic field (similar to the analysis performed later on showing the role of Ca-alpha1D in signal propagation). The behavioral differences are less informative and not necessarily consistent, as it is impossible to deliver the probe accurately enough in this assay to distinguish proximal vs. distal responses with the 30 micron probe, which however seems relevant for piezo_ko.

    Lastly, the authors investigate the contribution of the VGCC Ca-alpha1D, which has previously been implicated in c4da neuron function. They perform challenging dendritic calcium recordings and implicate Ca-alpha1D in signal propagation particularly for smaller diameter stimuli activating a smaller portion of the dendritic arbor. These data are of high quality and consistent with the authors' model. The authors could have considered if and how Ca-alpha1D might contribute to propagating piezo vs. ppk26 activation, which is particularly important for distal receptive field function in regard to small diameter stimuli.
    One limitation of the study is that it does not entirely represent the in vivo situation, as the animal had to be fileted to get access to the nociceptive neurons under the authors' experimental setup. Drosophila larvae are filled with hemolymph and display very different mechanical properties than the PDMS membrane used to pin the larval filet. This likely affects the absolute force/pressure needed to activate c4da neurons. In addition, neuronal responses depend on the physiological buffer used under these conditions, which do not necessarily mimic in vivo conditions (extracellular calcium concentration, pH, osmolality etc. will affect the evoked responses).

    Overall, by establishing a cutting edge method to deliver precise mechanical stimulation the authors provide data showing that mechanical stimuli are sensed by the entire receptive field of these neurons, preferentially detecting small diameter stimuli resembling sharp objects. The study thus provides a so far unprecedented level of detail how noxious mechanical stimuli are sensed by a respective sensory neuron within a native tissue environment and contributes significantly to our understanding of mechanical nociception. This study is a big step forward in the field, as mechanosensation and the channels involved are notoriously difficult to study. Despite some limitations outlined above, this is a very exciting study providing interesting insight into how mechanical stimuli are sensed by respective sensory/auxiliary channels at the subcellular level and translated into robust neuronal responses and behavior.

  6. eLife

    Reviewer #3 (Public Review):

    Liu et al. analyze the mechanosensory function of the nociceptive c4da neuron of Drosophila larvae by monitoring its calcium response while poking its dendritic tree. Using calibrated force probes, the authors find that the neuron is more sensitive to focal stimulation with sharp probes than to a more global stimulation with blunt probes, and that the receptive field of the neuron covers -and even extends beyond- its dendritic tree. Manipulating the complexity of this tree mainly affects the neuron's sensitivity to focal stimulation, especially when these stimuli are applied distally to the dendritic tree. A model is presented that suggests that neurons expand their receptive fields by monitoring both pressure and lateral tension. Moreover, mutant analysis suggests that whereas the global mechanosensitivity of the neuron requires PPK1/PPK26 channels, Piezo channels mainly mediate responses to focal pressure, with Ca-α1D, the pore-forming subunit of a voltage-gated channel, contributing to dendritic signal propagation/amplification. Relating dendritic tree morphology to mechanosensory function and mechanosensory ion channels, these findings are remarkable. There are, however, some issues that remain to be addressed.

    1. Information is missing about the regions of interest in which calcium responses were measured. Judging from Fig. 1E, calcium signals were measured in the somata, and this should be specified. Also judging from this figure, calcium signals seem to be largely confined to the somata and virtually absent from dendritic arbors. Fig. 6a shows very faint signals in the dendrites, yet those signals seem to have been measured rather far from the point of force application (a scale bar is shown but undefined), and, for some unknown reason, not between soma and force application point). Should there be detectable calcium signals in the somata, respective image gains should be adjusted so that those signals can be appreciated by the reader. If there are no clear signals in the dendrites, this would affect interpretations concerning e.g. Ca-α1D.

    2. Along this line, analyzing also the spacial distribution of dendritic calcium responses to the pokes would provide a much more detailed picture about how the dendritic tree responds to the various pokes. The beauty of the imaging approach chosen here is that it provides such information. Rather than ignoring this possibility, it should be exploited in this study, especially as respective data might provide much deeper insights into the relation between the mechanosensory function of the cell and its dendritic tree (and bolster the modelling results in Fig. 4 experimentally).

    3. When showing response functions as in e.g. Figs. 2C,G,H, 3D, 5C-E, etc., the y-axis should have a logarithmic scaling; receptor potentials of receptor cells usually scale proportionally to the logarithm of the stimulus amplitude. Only then, the reader will be able to fully appreciate the sensitivity differences. This will also alter interpretation of response function slopes.

    4. The knockdown and mutant data is interesting, yet important controls are missing. For the RNAi lines used, qPCR data on the knockdown-efficiency should be added. For the channel mutations, available genetic rescue lines should be used as controls. Data on protein localization is presented for the mechanosensitive channels, but not for voltage-gated calcium channel subunit. Should antibodies be available, respective stainings should be included. If not, the authors should at least check whether Ca-α1D is expressed in the cell using e.g. Mi{ET1}Ca-α1D[MB06807] that is available at Bloomington.

    5. The statistics used is not entirely convincing. T-test are used throughout, though I do not feel that all the data is distributed normally. Moreover, some figures include multiple comparisons, apparently without statistical correction. The data should be re-analyzed using appropriate statistical procedures.

  7. Version published to 10.1101/2022.01.04.474910v1 on bioRxiv