Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila

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1. 

   Version published to 10.7554/elife.76863 on eLife

   Jan 23, 2023
2. 

   eLife

   Oct 24, 2022

   Author Resonse

   Reviewer #1 (Public Review):

   The manuscript by Himmel et al is an interesting study representing a topic of substantial interest to the somatosensory neurobiology community. Here, the authors use CIII peripheral neurons to investigate polymodality of sensory neurons. From vertebrates to invertebrates, this is a long-standing question in the field: how is it that the same class of sensory neurons that express receptors for myriad sensory modalities encode different behavioral responses. This system in Drosophila seems to be an intriguing system to study this question, making use of the genetic toolkit in the fly and ease of behavioral assays. In this study, the authors identify a number of channels that are important for cold nociception, and they showed that some of these do not appear to also encode mechanosensation. …

   Author Resonse

   Reviewer #1 (Public Review):

   The manuscript by Himmel et al is an interesting study representing a topic of substantial interest to the somatosensory neurobiology community. Here, the authors use CIII peripheral neurons to investigate polymodality of sensory neurons. From vertebrates to invertebrates, this is a long-standing question in the field: how is it that the same class of sensory neurons that express receptors for myriad sensory modalities encode different behavioral responses. This system in Drosophila seems to be an intriguing system to study this question, making use of the genetic toolkit in the fly and ease of behavioral assays. In this study, the authors identify a number of channels that are important for cold nociception, and they showed that some of these do not appear to also encode mechanosensation. Despite my initial enthusiasm for this paper, halfway through, it felt as if I were reading two different papers that were loosely tied together. This lack of cohesion significantly reduced my enthusiasm for this work. Below are some of my criticisms:

   We thank Reviewer #1 for their feedback. In addition to the points below, and in accordance with the reviewer’s overall criticisms, we have revised the body text to make it more cohesive. Our main goal with this revision was to better explain to the reader the shift from anoctamins to SLC12 cotransporters.

   1. The first half of the paper is about a role for Anoctamins in cold nociception, but the second half switched somewhat abruptly to ncc69 and kcc. I assumed the authors would connect these genes in a genetic pathway, performing some kind of epistatic genetic interaction studies or even biochemical assays, and that this was the reason to switch the focus of the paper midway through. But this was not the case. Moreover, they performed a different constellation of experiments for the genes in the first half vs the second half of the paper (eg. Showed a role in cold nociception vs mechanosensation or showing phenotype from overexpression). This lack of cohesion made it difficult to follow the work.

   We have edited the text to better explain this shift. Two notable changes are: (1) moving the phylogenetics to Figure 1, to more immediately present and demonstrate that subdued is part of the ANO1/ANO2 family of calcium-activated chloride channels; and (2) a new cartoon schematic in Figure 6 to more strongly communicate to a reader that chloride is a hypothetical mechanism of cold discrimination.

   In short, previous work and our phylogenetic analyses indicate that subdued is a Cl- channel (we have moved the phylogeny earlier in the paper to make this clear from the onset). We were therefore surprised that knockdown/mutation resulted in reduced CT behavior, as neural Cl- currents are often inhibitory. Thus, we looked to known mechanisms of Cl- homeostasis to try to formulate an informed hypothesis about the function of anoctamins in this system; hence the shift in focus to SLC12.

   In response to the second half of the comment: We have in fact performed cold nociception and mechanosensation experiments for both the anoctamins and the SLC12 cotransporters, although the SLC12 mechanosensation results were in a supplemental figure. We have moved the mechanaosensation results to the main Figure 6 to make this clearer. With respect to simple overexpression, the goal of the anoctamin experiments was to test the necessity of anoctamins to cold-evoked behavior, whereas the goal of the SLC12 experiments was to differentially modulate Cl- homeostasis, and this could hypothetically be accomplished by both knockdown and overexpression (hence we performed both knockdown and overexpression).

   1. In Fig1B,C how does one confirm a CIII neuron is being analyzed. It might help the reader if there were at least some zoomed out photos where all the cell types are labeled and potentially compared to a schematic. Moreover, is there a CIII specific marker to use to co-stain for confirmation of neuron type?

   Our CIII fusion is a specific marker for CIII neurons. To better demonstrate this, we have added images of the new CIII fusion expression patterns overlapping with a previously described CIII GAL4 driver (i.e. nompC-GAL4), and provided text describing how the CIII fusion transgene was discovered and generated. Please see the new Figure 1-Figure supplement 1.

   1. As this paper is predicated on detecting differences by behavioral phenotype, the scoring analysis is not as robust as it could be, especially considering the wealth of tools in Drosophila for mapping behaviors. The "CT" phenotype is begging for a richer behavioral quantification. This critique becomes relevant here when considering the optogenetic induced CT behavior in Fig5. If the authors were to use unbiased quantitative metrics to measure behavior, they could show how similar the opto behavior is to the natural cold evoked behavior. Perhaps the two are not the same, although loosely fitting under the umbrella of "CT".

   In accordance with our response above to necessary revisions, we have added one additional metric and reorganized the figures to better demonstrate the complexity of the behavior. We have no further data or new tools at this time.

   To improve our optogenetic analyses, we have added data for Channelrhodopsin-dependent CIII activation, which has been previously shown to induce cold-like behaviors at high levels of activation and innocuous touch-like behaviors at low levels of activation (Turner, Armengol et al 2016). Further, we have added videos (Figure 5—videos 1-3) showing behavior in response to both Channelrhodopsin and Aurora activation.

   With respect to differences in behavior, we have pointed out some differences in the Aurora-evoked behavior from the cold-evoked behavior: chloride optogenetics induces innocuous touch-like behaviors following CT. Please see lines 296-299.

   1. Following on from the last comment, the touch assays in Fig3 have a different measurement system from the other figures. Perhaps touch deficits would be identified with richer behavioral quantification. Moreover, do these RNAi larvae show any responses to noxious mechanical stimulation?

   The touch assays necessarily have different metrics from cold assays, as the touch-evoked behaviors are quite different from cold-evoked change in length (which are relatively simple, prima facie).

   With respect to noxious mechanical stimulation, while Class III neurons have been shown to facilitate this modality and be connected to relevant circuitry (please see Hu et al 2017 https://doi.org/10.1038/nn.4580 and Takagi et al 2017 https://doi.org/10.1016/j.neuron.2017.10.030), Class IV neurons are the primary sensory neuron which initiate the noxious mechanical-induced rolling response. Although this is an interesting question, we believe it is outside the scope of this study.

   Reviewer #2 (Public Review):

   Himmel and colleagues study how individual sensory neurons can be tuned to detect noxious vs. gentle touch stimuli. Functional studies of Drosophila class III dendritic arborization neurons characterized roles in gentle touch and identified a receptor, NompC, and other factors that mediate these responses. Subsequent work primarily from the authors of the current study focused on roles for the same sensory neurons in cold nociception. The two proposed sensory inputs lead to quite distinct sets of behaviors, with touch leading to halting, head turning and reverse peristalsis, and noxious cold leading to whole body contraction. How activity of one type of sensory neuron could lead to such different responses remains an outstanding question, both at the levels of reception and circuitry.

   The cIII responses to noxious cold and innocuous touch raises questions that the authors address here, proposing that studies of this system could advance the understanding of chronic neuropathic pain. A candidate approach inspired by studies in vertebrate nociceptors led the authors to study anoctamin/TMEM16 channels subdued, and CG15270, termed wwk by the authors. The authors focus on a pathway for gentle touch vs. cold nociception discrimination through anoctamins. Several of the experiments in this manuscript are well done, in particular, the electrophysiological recordings provide a substantial advance. However, the genetic and expression analysis has several gaps and should be strengthened. The data also do not provide strong support for some key aspects of the proposed model, namely the importance of relative levels of Cl co-transporters.

   Major comments:

   1. Knockout studies are accomplished using two MiMIC insertions whose effects on subdued or CG15270/wwk are not characterized by the authors. This needs to be established. The MiMIC system is also not well explained in the text for readers.

   We have modified the text to better explain MiMICs (Lines 137-140) and we have verified the mutagenic effects of these MiMIC insertions via RT-PCR (Figure 2 – supplement 1). We believe these data, in conjunction with other converging lines of evidence (e.g. rescue) demonstrate necessity of these genes in cold nociception.

   1. Subdued expression is inferred by a Gal4 enhancer trap. This can be a hazardous way of determining expression patterns given the uncertain relevance of the local enhancers driving the expression. According to microarray analysis subdued is strongly expressed in cIII neurons, but c240-Gal4 is barely present compared to nearby neurons, raising questions about whether this line reflects the expression pattern, including levels, even though the authors suggest that the line is previously validated (line 95; it is unclear what previously validated means). Figure 1B should not be labeled "subdued > GFP" since it is not clear that this is the case. Another more direct method of assessing expression in cIII is necessary. Confidence is higher for wwk using a T2A-Gal4 line, however, Figure 1C might be misleading to readers and indicate that wwk-T2A-Gal4 is cIII specific whereas in supplemental data the authors show how it is much more broadly expressed. The expression pattern in the supplemental figures should be moved to the main figures.

   We have removed the phrase “previously validated” and we have modified Figure 1 to change how we refer to the GFP expression (removed “subdued > GFP”).

   In accordance with the response to necessary revisions above, we make use of several converging lines of evidence to infer expression, including GAL4 expression patterns, microarray, and qPCR (the two latter experiments from isolated CIII samples). That subdued and wwk are expressed in CIII is clearly the most parsimonious hypothesis.

   We have also carefully reviewed our body text to be certain we do not make claims of differential expression between different neural subtypes based on differences in fluorescence in the GAL4-driven GFP imaging. We do not believe that this would be a reasonable way to infer differences in expression levels in any instance.

   With respect to the design of Figure 1, the intent is not to mislead the reader, and we state in the text that wwk is not solely expressed in CIII (lines 120-125). As eLife makes supplemental figures available directly alongside the main figures, we have left the relevant supplemental figures as supplements – we simply think this makes more sense from a standpoint of readability and style.

   1. In figure 8 the authors propose a model in which the relative levels of K-Cl cotransporters Kcc (outward) and Ncc69 (inward) in cIII neurons determine high intracellular Cl- levels and a Cl- dependent depolarizing current in cIII neurons. They test this model using overexpression and loss of function data, but the results do not support their model since for most of the overexpression and LOF of kcc and ncc69 do not significantly affect cold nociception, the exception being ncc69 RNAi. The authors suggest that this could be due to Cl homeostasis regulated by other cotransporters. Nonetheless, it leaves a significant unexplained gap in the model that needs to be addressed.

   We respectfully disagree that our results are not consistent with the stated hypothesis. In fact, it is the lack of change under certain conditions which lend evidence against the alternative hypothesis that CIII neurons maintain relatively low intracellular Cl-. The hypothesis we are testing is that ncc69 expression is driving relatively high intracellular Cl- concentrations, thus resulting in depolarizing Cl- currents.

   Under this hypothesis, we would predict that knockdown of ncc69 and overexpression of kcc would reduce cold sensitivity at 5˚C. That knockdown of ncc69 and overexpression of kcc reduces cold sensitivity is consistent with this hypothesis (and we point out in text that the evidence for kcc is less convincing) – at the least, these results do not disprove it.

   Under this hypothesis, we would also predict that knockdown of kcc and overexpression of ncc69 would not result in reduced cold sensitivity at 5˚C. As there was no phenotype at 5C, our results are likewise consistent with the hypothesis (at the least, they do not disprove it).

   We did find it curious that ncc69 RNAi did not affect neural activity at 10˚C, but speculate that our inability to detect physiological effects for ncc69 knockdown are limitations of our electrophysiology methodology (and we discuss this in the manuscript).

   The only piece of data inconsistent with the hypothesis may be that kcc overexpression may not have affected cold nociception at 5˚C – the data aren’t overwhelmingly convincing. However, this is only one experiment among many, and we believe the preponderance of evidence is consistent with the hypothesis. That is not to say we believe this hypothesis has complete explanatory power, however, as noted by our discussion of both the ncc69 electrophysiological and kcc behavioral data, and by our suggestion that there may be other regulatory mechanisms at work. This latter suggestion is wholly speculative, and we believe appropriate for the discussion section. We agree (and state in the discussion) that this would require further experimentation.

   1. Related to the #3, the authors should verify the microarray data that form the basis for their differential expression model.

   We have performed qPCR for ncc69 and kcc. Although qPCR is semiquantitative when comparing between genes, the Ct value for ncc69 was lower than for kcc, indicating more transcripts were present at the onset (assuming identical efficacy). These data (although semi-quantitative), the microarray, and our behavioral and electrophysiological data are consistent with the stated hypothesis.

   Reviewer #3 (Public Review):

   There are also several modest weaknesses in the paper:

   1. A notable gap remains in the evidence for the hypothesized mechanisms that enhance electrical activity during cold stimulation and the proposed role of anoctamins (Fig. 8) - the lack of evidence for Ca2+-dependent activation of Cl- current. The recording methods used in the fillet preparation should enable direct tests of this important part of the model.

   We have performed an additional experiment at the reviewer’s suggestion. Please see above (in essential revisions) and below (in recommendations for authors).

   1. The behavioral and electrophysiological consequences of knocking down either of the two anoctamins are incomplete (Fig.2), raising the significant question of whether combined knock-down of both anoctamins in the CIII neurons would largely eliminate the cold-specific responses.

   While the results of this experiment would certainly be interesting, we are unsure of how it would be acutely informative in this context and are not convinced that any possible outcomes would disprove any particular hypothesis. In part, this is because we know that blocking synaptic transmission in CIII neurons (via tetanus toxin) does not completely ablate cold-evoked behavior (Turner & Armengol et al 2016 https://doi.org/10.1016/j.cub.2016.09.038). This is also the case for combinatorial mutation of other genes associated with cold nociception (please see Turner & Armengol et al 2016; and more recently, Patel et al 2022 https://doi.org/10.3389/fnmol.2022.942548). Further, the husbandry required to generate the double knockdowns would be quite challenging and might result in GAL4 titration (hypothetically less strongly knocking down each gene). For these reasons, we have not performed this suggested experiment.

   1. Blind procedures were not used to minimize unconscious bias in the analyses of video-recorded behavior, although some of the analyses were partially automated.

   This is correct and a relative weakness of the study. We note it in our methods section. The use of semi-automated data analyses of the behavioral videos is designed to minimize experimenter-specific variability.

   1. The term "hypersensitization" is confusing. Pain physiologists typically use "sensitization" when behavioral or neural responses are increased from normal. In the case of increased neuronal sensitivity, if the mechanism involves an increase in responsiveness to depolarizing inputs or an increased probability of spontaneous discharge, the term "hyperexcitability" is appropriate. Hypersensitization connotes an extreme sensitization state compared to a known normal sensitization state (which already signifies increased sensitivity). In contrast, the effects of ncc69 overexpression in this manuscript are best described simply as sensitization (increased reflexive and neuronal sensitivity to cooling) and hyperexcitability (expressed as increased spontaneous activity at room temperature).

   We have modified the text in accordance with the reviewer’s suggestions (see recommendations for authors section). We have also changed the title of the paper to “Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila”

   Read the original source
3. 

   eLife

   Oct 24, 2022

   eLife assessment

   This paper is of interest for somatosensory neurobiologists studying how polymodality is achieved in peripheral sensory neurons. The work identifies roles in cold nociception and not mechanosensation in chloride transport for a number of ion channels.

   Read the original source
4. 

   eLife

   Oct 24, 2022

   Reviewer #1 (Public Review):

   The manuscript by Himmel et al is an interesting study representing a topic of substantial interest to the somatosensory neurobiology community. Here, the authors use CIII peripheral neurons to investigate polymodality of sensory neurons. From vertebrates to invertebrates, this is a long-standing question in the field: how is it that the same class of sensory neurons that express receptors for myriad sensory modalities encode different behavioral responses. This system in Drosophila seems to be an intriguing system to study this question, making use of the genetic toolkit in the fly and ease of behavioral assays. In this study, the authors identify a number of channels that are important for cold nociception, and they showed that some of these do not appear to also encode mechanosensation. Despite my initial …

   Reviewer #1 (Public Review):

   The manuscript by Himmel et al is an interesting study representing a topic of substantial interest to the somatosensory neurobiology community. Here, the authors use CIII peripheral neurons to investigate polymodality of sensory neurons. From vertebrates to invertebrates, this is a long-standing question in the field: how is it that the same class of sensory neurons that express receptors for myriad sensory modalities encode different behavioral responses. This system in Drosophila seems to be an intriguing system to study this question, making use of the genetic toolkit in the fly and ease of behavioral assays. In this study, the authors identify a number of channels that are important for cold nociception, and they showed that some of these do not appear to also encode mechanosensation. Despite my initial enthusiasm for this paper, halfway through, it felt as if I were reading two different papers that were loosely tied together. This lack of cohesion significantly reduced my enthusiasm for this work. Below are some of my criticisms:

   1. The first half of the paper is about a role for Anoctamins in cold nociception, but the second half switched somewhat abruptly to ncc69 and kcc. I assumed the authors would connect these genes in a genetic pathway, performing some kind of epistatic genetic interaction studies or even biochemical assays, and that this was the reason to switch the focus of the paper midway through. But this was not the case. Moreover, they performed a different constellation of experiments for the genes in the first half vs the second half of the paper (eg. Showed a role in cold nociception vs mechanosensation or showing phenotype from overexpression). This lack of cohesion made it difficult to follow the work.

   2. In Fig1B,C how does one confirm a CIII neuron is being analyzed. It might help the reader if there were at least some zoomed out photos where all the cell types are labeled and potentially compared to a schematic. Moreover, is there a CIII specific marker to use to co-stain for confirmation of neuron type?

   3. As this paper is predicated on detecting differences by behavioral phenotype, the scoring analysis is not as robust as it could be, especially considering the wealth of tools in Drosophila for mapping behaviors. The "CT" phenotype is begging for a richer behavioral quantification. This critique becomes relevant here when considering the optogenetic induced CT behavior in Fig5. If the authors were to use unbiased quantitative metrics to measure behavior, they could show how similar the opto behavior is to the natural cold evoked behavior. Perhaps the two are not the same, although loosely fitting under the umbrella of "CT".

   4. Following on from the last comment, the touch assays in Fig3 have a different measurement system from the other figures. Perhaps touch deficits would be identified with richer behavioral quantification. Moreover, do these RNAi larvae show any responses to noxious mechanical stimulation?

   Read the original source
5. 

   eLife

   Oct 24, 2022

   Reviewer #2 (Public Review):

   Himmel and colleagues study how individual sensory neurons can be tuned to detect noxious vs. gentle touch stimuli. Functional studies of Drosophila class III dendritic arborization neurons characterized roles in gentle touch and identified a receptor, NompC, and other factors that mediate these responses. Subsequent work primarily from the authors of the current study focused on roles for the same sensory neurons in cold nociception. The two proposed sensory inputs lead to quite distinct sets of behaviors, with touch leading to halting, head turning and reverse peristalsis, and noxious cold leading to whole body contraction. How activity of one type of sensory neuron could lead to such different responses remains an outstanding question, both at the levels of reception and circuitry.

   The cIII responses to …

   Reviewer #2 (Public Review):

   Himmel and colleagues study how individual sensory neurons can be tuned to detect noxious vs. gentle touch stimuli. Functional studies of Drosophila class III dendritic arborization neurons characterized roles in gentle touch and identified a receptor, NompC, and other factors that mediate these responses. Subsequent work primarily from the authors of the current study focused on roles for the same sensory neurons in cold nociception. The two proposed sensory inputs lead to quite distinct sets of behaviors, with touch leading to halting, head turning and reverse peristalsis, and noxious cold leading to whole body contraction. How activity of one type of sensory neuron could lead to such different responses remains an outstanding question, both at the levels of reception and circuitry.

   The cIII responses to noxious cold and innocuous touch raises questions that the authors address here, proposing that studies of this system could advance the understanding of chronic neuropathic pain. A candidate approach inspired by studies in vertebrate nociceptors led the authors to study anoctamin/TMEM16 channels subdued, and CG15270, termed wwk by the authors. The authors focus on a pathway for gentle touch vs. cold nociception discrimination through anoctamins. Several of the experiments in this manuscript are well done, in particular, the electrophysiological recordings provide a substantial advance. However, the genetic and expression analysis has several gaps and should be strengthened. The data also do not provide strong support for some key aspects of the proposed model, namely the importance of relative levels of Cl co-transporters.

   Major comments:

   1. Knockout studies are accomplished using two MiMIC insertions whose effects on subdued or CG15270/wwk are not characterized by the authors. This needs to be established. The MiMIC system is also not well explained in the text for readers.

   2. Subdued expression is inferred by a Gal4 enhancer trap. This can be a hazardous way of determining expression patterns given the uncertain relevance of the local enhancers driving the expression. According to microarray analysis subdued is strongly expressed in cIII neurons, but c240-Gal4 is barely present compared to nearby neurons, raising questions about whether this line reflects the expression pattern, including levels, even though the authors suggest that the line is previously validated (line 95; it is unclear what previously validated means). Figure 1B should not be labeled "subdued > GFP" since it is not clear that this is the case. Another more direct method of assessing expression in cIII is necessary. Confidence is higher for wwk using a T2A-Gal4 line, however, Figure 1C might be misleading to readers and indicate that wwk-T2A-Gal4 is cIII specific whereas in supplemental data the authors show how it is much more broadly expressed. The expression pattern in the supplemental figures should be moved to the main figures.

   3. In figure 8 the authors propose a model in which the relative levels of K-Cl cotransporters Kcc (outward) and Ncc69 (inward) in cIII neurons determine high intracellular Cl- levels and a Cl- dependent depolarizing current in cIII neurons. They test this model using overexpression and loss of function data, but the results do not support their model since for most of the overexpression and LOF of kcc and ncc69 do not significantly affect cold nociception, the exception being ncc69 RNAi. The authors suggest that this could be due to Cl homeostasis regulated by other cotransporters. Nonetheless, it leaves a significant unexplained gap in the model that needs to be addressed.

   4. Related to the #3, the authors should verify the microarray data that form the basis for their differential expression model.

   Read the original source
6. 

   eLife

   Oct 24, 2022

   Reviewer #3 (Public Review):

   1. The described studies seek to test a plausible hypothesis having important biological implications: that Ca2+ coming through TRP channels and/or from intracellular stores during cold stimulation activates anoctamin Cl- channels, which further depolarize the CIII neuron via inward Cl- current (outward Cl- diffusion) resulting from high intracellular Cl- concentration caused by high expression of the outwardly directed Cl- transporter ncc69, thereby driving the intense electrical activity in CIII neurons needed to trigger cold-specific behavioral responses.

   2. Elegant phylogenetic analysis is provided to show that Drosophila subdued and white walker are orthologous to human TMEM16/anoctamins ANO1/2 and ANO8, respectively, to go along with ncc69 already known to be orthologous to human NKCC1.

   3. Strong …

   Reviewer #3 (Public Review):

   1. The described studies seek to test a plausible hypothesis having important biological implications: that Ca2+ coming through TRP channels and/or from intracellular stores during cold stimulation activates anoctamin Cl- channels, which further depolarize the CIII neuron via inward Cl- current (outward Cl- diffusion) resulting from high intracellular Cl- concentration caused by high expression of the outwardly directed Cl- transporter ncc69, thereby driving the intense electrical activity in CIII neurons needed to trigger cold-specific behavioral responses.

   2. Elegant phylogenetic analysis is provided to show that Drosophila subdued and white walker are orthologous to human TMEM16/anoctamins ANO1/2 and ANO8, respectively, to go along with ncc69 already known to be orthologous to human NKCC1.

   3. Strong genetic and behavioral evidence shows that knocking down the expression of subdued or white walker globally or selectively in CIII neurons reduces the incidence and magnitude of a cold-specific contraction response ("CT") to 5 degree C stimulation but not responses to gentle touch.

   4. These knock-downs also reduce electrical activity recorded in cell bodies of CIII neurons induced by cooling to 15 or 10 degrees C in a semi-intact ("fillet") preparation.

   5. CIII-specific knock-down of ncc69 reduces CT responses while overexpression of kcc (which should have the opposite effect on intracellular Cl- concentration) also tends to reduce these responses, indicating that the balance of Cl- pump activity in these neurons favors excitation when Cl- channels are opened (e.g., during cold stimulation).

   6. Optogenetic activation of an exogenously expressed Cl- channel (Aurora) in CIII neurons evokes CT responses, showing that Cl- currents are sufficient to produce these responses, presumably by strongly activating the CIII neurons.

   7. Reducing extracellular Cl- enhances ongoing electrical activity of CIII neurons, strengthening the conclusion that opening Cl- channels excites these neurons.

   8. Overexpressing ncc69 in CIII neurons enhances basal and evoked electrical activity, and sensitizes larvae CT responses to cooling to 10 degrees C, further strengthening the conclusion that opening Cl- channels excites CIII neurons and suggesting that this specific genetic manipulation could provide a model in Drosophila for detailed investigations into a potentially general mechanism contributing to neuropathic sensitization and pain.

   9. The authors integrate findings from the present study with those from their recent cold acclimation paper to make the speculative but interesting suggestion that mechanisms selected during evolution to enable cold acclimation might also be recruited in neuropathic contexts to produce maladaptive sensitization.

   There are also several modest weaknesses in the paper:

   1. A notable gap remains in the evidence for the hypothesized mechanisms that enhance electrical activity during cold stimulation and the proposed role of anoctamins (Fig. 8) - the lack of evidence for Ca2+-dependent activation of Cl- current. The recording methods used in the fillet preparation should enable direct tests of this important part of the model.

   2. The behavioral and electrophysiological consequences of knocking down either of the two anoctamins are incomplete (Fig.2), raising the significant question of whether combined knock-down of both anoctamins in the CIII neurons would largely eliminate the cold-specific responses.

   3. Blind procedures were not used to minimize unconscious bias in the analyses of video-recorded behavior, although some of the analyses were partially automated.

   4. The term "hypersensitization" is confusing. Pain physiologists typically use "sensitization" when behavioral or neural responses are increased from normal. In the case of increased neuronal sensitivity, if the mechanism involves an increase in responsiveness to depolarizing inputs or an increased probability of spontaneous discharge, the term "hyperexcitability" is appropriate. Hypersensitization connotes an extreme sensitization state compared to a known normal sensitization state (which already signifies increased sensitivity). In contrast, the effects of ncc69 overexpression in this manuscript are best described simply as sensitization (increased reflexive and neuronal sensitivity to cooling) and hyperexcitability (expressed as increased spontaneous activity at room temperature).

   Read the original source
7. 

   Version published to 10.1101/2021.12.10.472093v1 on bioRxiv

   Dec 11, 2021